# *Chapter I: *

# Review of Animal Studies

# Relevant to Silicone Toxicity

# ^Principal\ Author:\ ^

# ^Nancy\ I.\ Kerkvliet,\ MS,\ PhD^

# ^Professor\ of\ Toxicology\ and\ Extension\ Toxicology\ Specialist^

# ^Department\ of\ Environmental\ and\ Molecular\ Toxicology^

# ^Oregon\ State\ University^

# ^Corvallis,\ Oregon^

# 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^

^\ Silanols\ \ \ \ \ \ \ \ \ \ I-23^

^\ Platinum\ \ \ \ \ \ \ \ \ \ I-23^

**^XI.\ Conclusions^**^\ \ \ \ \ \ \ \ \ \ I-24^

**^References^**^\ \ \ \ \ \ \ \ \ \ \ I-26^

**^Tables^^\ ^^\ ^^\ ^^\ ^^\ ^^\ ^^\ ^^\ ^^\ ^^\ ^^\ ^**^I-33^

# Chapter I

# 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\ (-CH^~^3^~^)\ 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).\ ^

# ^VI.\ Silicone\ and\ "Adjuvant\ Disease"^

^In\ the\ late\ 1980\'s,\ a\ number\ of\ articles\ began\ to\ appear\ suggesting\ a\ possible\ link\ between\ SBIs\ and\ autoimmune\ disorders\ in\ women.\ The\ concern\ seems\ to\ have\ been\ initiated\ by\ reports\ of\ delayed\ adverse\ reactions\ in\ some\ Japanese\ women\ that\ had\ been\ injected\ with\ silicone\ fluid\ admixed\ with\ other\ substances\ such\ as\ paraffin\ in\ the\ breast\ tissue\ many\ years\ previously\ (see\ citations\ in\ Picha\ and\ Goldstein,\ 1996).\ The\ connective\ tissue\ disease\ that\ was\ observed\ in\ these\ women\ was\ termed\ "human\ adjuvant\ disease"\ based\ on\ the\ theory\ that\ silicone\ could\ act\ like\ the\ experimental\ adjuvant\ known\ as\ "Complete\ Freund's\ Adjuvant"\ (CFA).\ CFA\ is\ an\ emulsified\ preparation\ of\ heat-killed\ mycobacteria\ in\ mineral\ oil.\ When\ an\ antigen\ is\ incorporated\ into\ CFA,\ the\ immune\ response\ to\ that\ antigen\ is\ increased\ and\ prolonged,\ a\ desirable\ situation\ for\ vaccine\ delivery.\ However,\ CFA\ itself\ is\ not\ used\ clinically\ because\ of\ severe\ local\ inflammatory\ reactions\ as\ well\ as\ possible\ sensitization\ to\ the\ mycobacterium.\ The\ mineral\ oil\ component\ of\ CFA\ functions\ to\ provide\ a\ depot\ effect\ for\ the\ water-in-oil\ emulsified\ antigen;\ the\ mycobacterial\ component\ induces\ an\ inflammatory\ response\ that\ facilitates\ the\ immune\ response\ to\ the\ antigen\ (Broderson,\ 1989).\ ^

^\ \ "Adjuvant\ arthritis"\ is\ an\ experimental\ inflammatory\ joint\ disease\ in\ rats\ that\ is\ induced\ by\ a\ single\ injection\ of\ CFA\ (Glenn\ and\ Gray,\ 1965;\ Pearson,\ 1956).\ The\ clinical\ manifestations\ of\ the\ disease\ resemble\ some\ inflammatory\ rheumatic\ diseases\ in\ humans\ such\ as\ rheumatoid\ arthritis,\ ankylosing\ spondylitis,\ and\ Reiter's\ disease\ (Muir\ and\ Dumonde,\ 1982).\ However,\ "adjuvant\ arthritis"\ appears\ to\ be\ a\ disease\ unique\ to\ rats.\ Among\ experimental\ animals\ tested,\ including\ mice,\ guinea\ pigs,\ rabbits,\ sheep\ and\ monkeys,\ CFA\ induces\ the\ disease\ only\ in\ the\ ^

^rat,\ and\ only\ in\ certain\ strains\ of\ rat,\ indicating\ that\ a\ specific\ genetic\ predisposition\ is\ required.\ In\ some\ rats\ (e.g.,\ Dark\ Agouti\ \[DA\]),\ arthritis\ can\ even\ be\ induced\ by\ the\ injection\ of\ mineral\ oil\ alone\ in\ the\ absence\ of\ mycobacteria\ (i.e.,\ Incomplete\ Freund's\ Adjuvant\ \[IFA\]).\ ^

^\ The\ mineral\ oil\ component\ of\ CFA\ functions\ nonspecifically,\ and\ many\ different\ types\ of\ oils\ were\ shown\ to\ be\ effective\ in\ inducing\ "adjuvant\ disease"\ in\ rats\ when\ emulsified\ with\ killed\ mycobacteria\ (Whitehouse\ et.\ al.,\ 1974).\ In\ these\ studies,\ a\ commercial\ silicone\ oil\ was\ also\ shown\ to\ be\ a\ "potent\ arthritogen."\ However,\ the\ silicone\ oil\ was\ described\ as\ "a\ lubricating\ oil\ of\ unknown\ composition"\ sold\ as\ a\ lock\ lubricant,\ which\ caused\ severe\ weight\ loss\ in\ the\ rats.\ Thus,\ this\ oil\ is\ not\ representative\ of\ the\ medical\ grade\ of\ silicone\ found\ in\ SBIs.^

^\ \ As\ summarized\ in\ Table\ 1,\ more\ recent\ studies\ have\ shown\ that\ neither\ silicone\ gel\ nor\ silicone\ oil\ (PDMS)\ was\ capable\ of\ eliciting\ arthritis\ in\ Lewis\ rats\ when\ injected\ alone\ or\ emulsified\ with\ mycobacteria\ (Chang,\ 1993;\ Picha\ and\ Goldstein,\ 1997).\ Similarly,\ in\ the\ DA\ rat,\ a\ mixture\ of\ silicone\ gel\ and\ oil\ was\ not\ effective\ in\ inducing\ arthritis\ (Naim\ et\ al.,1995)\ unless\ injected\ directly\ into\ the\ joint\ (Yoshino,\ 1994).\ Thus,\ there\ are\ no\ experimental\ data\ to\ support\ the\ labeling\ of\ silicone-associated\ disorders\ as\ "human\ adjuvant\ disease."^

# **^VII.\ Adjuvant\ Activity\ of\ Silicone^**^\ ^

^The\ term\ ^*^adjuvant^*^\ is\ a\ label\ more\ widely\ applied\ in\ recent\ years\ to\ describe\ "any\ substance\ that\ enhances\ the\ immune\ response\ to\ an\ antigen\ with\ which\ it\ is\ mixed"\ (Janeway\ and\ Travers,\ 1994).\ Effective\ vaccines\ for\ human\ diseases\ often\ depend\ on\ the\ incorporation\ of\ an\ adjuvant\ in\ order\ to\ generate\ and\ enhance\ the\ development\ of\ protective\ immunity.\ Under\ this\ broad\ definition,\ many\ diverse\ substances,\ acting\ by\ diverse\ mechanisms,\ have\ been\ shown\ to\ function\ as\ adjuvants.\ Oily\ substances\ that\ prolong\ antigen\ half-life\ in\ tissues\ and\ may\ enhance\ cellular\ uptake\ are\ especially\ effective\ adjuvants.\ ^

^\ As\ summarized\ in\ Table\ 2,\ several\ studies\ have\ examined\ the\ ability\ of\ silicones\ to\ function\ as\ adjuvants\ to\ increase\ antibody\ production\ or\ cell-mediated\ immune\ responses\ when\ injected\ as\ an\ emulsified\ preparation\ with\ the\ antigen.\ In\ general,\ it\ appears\ that\ adjuvant\ activity\ is\ seen\ more\ often\ with\ silicone\ gel\ than\ with\ silicone\ oil.\ The\ low\ molecular\ weight\ cyclosiloxane\ D4\ has\ also\ been\ shown\ to\ possess\ adjuvant\ activity\ in\ terms\ of\ enhancing\ antibody\ production\ to\ some\ antigens.\ ^

^\ Adjuvants\ have\ also\ been\ used\ experimentally\ to\ induce\ autoimmune\ disease\ in\ animals\ ^

^following\ immunization\ with\ autoantigens\ or\ cross-reacting\ foreign\ antigens.\ For\ example,\ an\ arthritic\ disease\ can\ be\ induced\ in\ animals\ following\ immunization\ with\ either\ homologous\ or\ heterologous\ type\ II\ collagen\ (Ellis\ et\ al.,\ 1992).\ In\ this\ collagen-induced\ arthritis\ model,\ collagen\ protein\ is\ emulsified\ in\ CFA\ or\ IFA\ and\ injected\ into\ rats\ or\ mice.\ After\ one\ or\ more\ immunizations,\ the\ onset\ of\ the\ disease\ is\ identified\ by\ severe\ swelling\ and\ erythema\ in\ the\ paws,\ which\ is\ associated\ with\ a\ massive\ inflammatory\ infiltrate\ into\ the\ synovium\ (Ellis\ et\ al.,\ 1992).\ The\ histological\ changes\ in\ the\ joints\ of\ these\ animals\ resemble\ those\ observed\ in\ rheumatoid\ arthritis\ patients.^

^\ The\ ability\ of\ silicone\ to\ substitute\ for\ mineral\ oil\ in\ the\ induction\ of\ collagen-induced\ arthritis\ has\ been\ examined\ in\ both\ rats\ and\ mice\ (see\ Table\ 2).\ Following\ the\ injection\ of\ bovine\ collagen\ emulsified\ in\ a\ silicone\ oil:gel\ mixture,\ Naim\ et\ al.\ (1995)\ reported\ that\ arthritis\ was\ induced\ in\ 4/10\ rats\ compared\ to\ 8/9\ rats\ injected\ with\ collagen\ in\ IFA.\ When\ silicone\ oil\ was\ tested\ independently\ from\ silicone\ gel,\ the\ incidence\ of\ arthritis\ was\ higher\ with\ the\ gel(7/10)\ than\ the\ oil\ (3/10).\ In\ contrast,\ D4\ was\ not\ an\ effective\ adjuvant\ for\ induction\ of\ arthritis\ (Naim\ et\ al.,\ 1995).\ Using\ the\ DBA/1\ mouse\ model,\ Schaefer\ (1997)\ reported\ that\ silicone\ oil\ was\ not\ an\ effective\ adjuvant\ for\ the\ induction\ of\ collagen-induced\ arthritis,\ even\ if\ the\ inoculum\ included\ killed\ mycobacteria,\ whereas\ 80%\ of\ the\ mice\ injected\ with\ collagen\ in\ CFA\ developed\ arthritis.^

^\ It\ is\ important\ to\ recognize\ that\ the\ successful\ induction\ of\ arthritis\ in\ rats\ with\ collagen\ emulsified\ in\ silicone\ does\ not\ reflect\ silicone-induced\ "adjuvant\ arthritis",\ which\ develops\ in\ the\ absence\ of\ active\ immunization.\ Rather,\ these\ results\ point\ to\ the\ successful\ immunization\ of\ the\ rat\ to\ the\ foreign\ collagen\ protein\ when\ silicone\ gel\ or\ oil\ was\ used\ as\ the\ adjuvant.\ On\ the\ other\ hand,\ in\ an\ animal\ model\ of\ experimental\ autoimmune\ thyroiditis,\ the\ injection\ of\ rat\ thyroglobulin\ (Tg)\ emulsfied\ in\ a\ silicone\ oil:gel\ mixture\ was\ unable\ to\ induce\ thyroiditis,\ while\ 100%\ of\ rats\ injected\ with\ Tg\ in\ CFA\ developed\ thyroid\ disease\ (Naim\ et\ al.,\ 1993).^

# ^VIII.\ Effects\ of\ Silicone\ in\ Animal\ Models\ of\ Autoimmune\ Disease^

^The\ question\ of\ whether\ or\ not\ silicone\ is\ capable\ of\ causing\ or\ aggravating\ autoimmune\ disease\ can\ be\ addressed\ most\ directly\ by\ laboratory\ animal\ studies\ using\ different\ experimental\ models\ of\ autoimmune\ disease.\ These\ established\ models\ of\ autoimmune\ disease\ have\ been\ developed\ to\ study\ the\ biological\ processes\ responsible\ for\ the\ symptoms\ associated\ with\ the\ disease,\ the\ ^

^predisposing\ genetic\ and\ environmental\ factors\ that\ influence\ the\ disease\ process,\ and\ the\ effectiveness\ of\ potential\ therapies.\ While\ no\ single\ animal\ model\ perfectly\ matches\ human\ disease,\ there\ are\ usually\ many\ parallels,\ and\ data\ obtained\ from\ different\ animal\ models\ can\ provide\ insight\ into\ the\ disease\ process\ in\ humans.\ ^

^\ Animal\ models\ in\ which\ autoimmune\ disease\ develops\ spontaneously\ are\ most\ relevant\ for\ the\ evaluation\ of\ the\ ability\ of\ silicone\ to\ exacerbate\ (promote)\ autoimmune\ disease.\ Promotion\ could\ be\ associated\ with\ the\ early\ appearance,\ increased\ severity,\ and/or\ increased\ incidence\ of\ autoimmune\ disease\ in\ animals\ that\ were\ destined\ to\ develop\ autoimmune\ disease\ due\ to\ genetic\ predisposition.\ Animal\ models\ in\ which\ autoimmune\ disease\ is\ induced\ by\ specific\ antigen\ injection\ are\ also\ useful\ to\ evaluate\ promotion\ when\ the\ severity\ of\ the\ induced\ disease\ in\ controls\ can\ be\ minimized.\ In\ contrast,\ it\ is\ much\ more\ difficult\ to\ evaluate\ whether\ or\ not\ silicone\ ^*^causes\ ^*^an\ autoimmune\ disease\ because\ of\ the\ multifactorial\ nature\ of\ disease\ induction.\ Causation\ could\ perhaps\ be\ deduced\ if\ a\ novel\ disease\ developed\ in\ an\ autoimmune-\ prone\ strain\ or\ if\ autoimmune\ disease\ was\ induced\ in\ a\ non-susceptible\ strain.\ As\ summarized\ in\ Table\ 3,\ the\ effects\ of\ silicone\ have\ been\ assessed\ in\ several\ experimental\ paradigms\ of\ autoimmunity.^

# ***^Arthritis-prone\ DBA/1\ Mice^***^\ \ \ \ ^

^\ Using\ arthritis-prone\ DBA/1\ mice,\ Schaefer\ et\ al\ (1997)\ examined\ the\ ability\ of\ silicones\ to\ induce\ arthritis.\ The\ results\ of\ these\ studies\ showed\ that\ mice\ implanted\ with\ silicone\ oil,\ gel\ or\ elastomer\ for\ as\ long\ at\ 12\ months\ did\ not\ develop\ arthritis.\ However,\ it\ should\ be\ noted\ that\ DBA/1\ mice\ injected\ with\ CFA\ also\ failed\ to\ develop\ arthritis\ in\ this\ study;\ thus\ the\ positive\ control\ group\ failed\ to\ document\ the\ sensitivity\ of\ the\ model.\ ^

^\ In\ a\ different\ animal\ model,\ genetically\ susceptible\ BALB/cAnPt\ mice\ injected\ in\ the\ peritoneal\ cavity\ with\ various\ silicone\ gels\ did\ not\ develop\ the\ arthritis\ that\ is\ frequently\ found\ in\ this\ strain\ when\ they\ are\ treated\ with\ pristane\ oil\ (Potter\ et\ al.,\ 1994).\ Similarly,\ single\ or\ multiple\ sc\ injections\ of\ silicone\ gel\ failed\ to\ induce\ arthritis\ in\ BALB/cAnPt\ mice\ even\ if\ the\ implant\ site\ was\ co-injected\ with\ Staphylococcus\ bacteria\ (MacDonald\ et\ al.,\ 1998).\ ^

^\ Taken\ together\ with\ the\ previously\ described\ ineffectiveness\ of\ silicone\ in\ rat\ adjuvant\ arthritis\ models,\ these\ findings\ indicate\ that\ silicones\ do\ not\ directly\ induce\ arthritis\ in\ arthritis-prone\ mice\ or\ rats.^

# ***^MRL\ ^**^lpr/lpr^**^\ Model\ of\ Lupus^***^\ \ \ \ \ \ \ \ \ ^

^MRL/lpr\ mice\ carry\ a\ spontaneous\ lymphoproliferative\ mutation\ (^*^lpr/lpr^*^)\ that\ results\ in\ the\ development\ of\ an\ autoimmune\ syndrome\ at\ approximately\ eight\ weeks\ of\ age.\ The\ disease\ progresses\ over\ 16--24\ weeks\ and\ is\ characterized\ by\ high\ levels\ of\ circulating\ autoantibodies\ leading\ to\ an\ immune-complex\ mediated\ glomerulonephritis,\ diffuse\ vasculitis\ and\ arthritis\ (Hang\ et\ al.,\ 1982).\ Approximately\ 50%\ of\ the\ mice\ die\ by\ 24\ weeks\ of\ age\ due\ to\ renal\ failure.\ The\ clinical\ symptoms\ in\ MRL\ ^*^lpr/lpr^*^\ mice\ closely\ resemble\ systemic\ lupus\ erythematosus\ (SLE)\ in\ humans.\ The\ arthritis\ that\ develops\ in\ MRL\ ^*^lpr/lpr^*^\ mice\ is\ similar\ to\ RA\ in\ humans.\ The\ mice\ also\ develop\ a\ Sjögren's-\ like\ inflammation\ of\ the\ conjunctiva.\ MRL+/+\ mice,\ which\ lack\ the\ ^*^lpr\ ^*^gene\ mutation,\ develop\ a\ milder\ autoimmune\ disease\ later\ in\ life\ as\ compared\ to\ MRL\ ^*^lpr/lpr^*^\ mice.\ \ Schaefer\ (1997)\ investigated\ the\ ability\ of\ silicones\ to\ modify\ disease\ in\ the\ MRL\ strain.\ At\ five\ weeks\ of\ age,\ prior\ to\ the\ onset\ of\ autoimmune\ symptoms,\ MRL\ ^*^lpr/lpr^*^\ and\ MRL+/+\ mice\ received\ sc\ implants\ of\ silicone\ gel,\ silicone\ oil\ or\ a\ sham\ implant.\ During\ the\ next\ 12\ weeks,\ clinical\ parameters\ of\ disease\ were\ measured\ by\ palpation\ of\ lymph\ nodes,\ urinary\ protein,\ and\ serum\ titers\ of\ collagen\ and\ DNA\ antibodies.\ Serum\ levels\ of\ several\ cytokines\ were\ also\ monitored.\ At\ sacrifice,\ kidneys\ were\ fixed\ and\ stained\ for\ immune\ complex\ deposition.^

^\ All\ MRL\ ^*^lpr/lpr^*^\ mice\ had\ severe\ glomerulonephritis\ by\ the\ time\ they\ were\ sacrificed,\ and\ silicone\ exposure\ did\ not\ influence\ the\ severity\ of\ the\ disease.\ MRL+/+\ mice\ showed\ minimal\ renal\ changes,\ and\ this\ too\ was\ unaffected\ by\ the\ silicone\ implants.\ Lymph\ node\ enlargement\ was\ also\ not\ influenced\ by\ silicone.\ Anti-DNA\ antibody\ titers\ were\ significantly\ higher\ in\ MRL\ ^*^lpr/lpr^*^\ mice\ that\ received\ silicone\ gel\ and\ in\ MRL+/+\ mice\ that\ received\ gel\ or\ oil\ implants\ as\ compared\ to\ sham\ controls.\ Some\ differences\ in\ the\ levels\ of\ certain\ cytokines\ were\ noted\ at\ various\ times\ during\ the\ experimental\ time\ period,\ but\ no\ pattern\ of\ change\ was\ revealed\ that\ would\ suggest\ that\ silicone\ altered\ disease\ by\ modifying\ cytokine\ production.\ ^

^\ \ In\ these\ and\ other\ experiments,\ Schaefer\ (1997)\ presents\ data\ that\ purport\ to\ demonstrate\ the\ presence\ of\ autoantibodies\ to\ silicone-bound\ proteins.\ However,\ the\ unorthodox\ procedures\ that\ were\ used\ to\ quantify\ the\ proteins,\ the\ lack\ of\ positive\ controls,\ and\ the\ manner\ in\ which\ the\ data\ were\ presented,\ do\ not\ allow\ such\ conclusions\ to\ be\ made.\ The\ data\ are\ not\ convincing\ of\ anything\ more\ than\ nonspecific\ binding\ of\ protein\ to\ the\ implant.^

^\ In\ similar\ studies\ using\ a\ different\ strain\ of\ mouse\ with\ the\ ^*^lpr^*^\ mutation,\ Osborn\ et\ al.\ (1995)\ reported\ that\ silicone\ oil\ containing\ 5%\ D4\ did\ not\ alter\ the\ incidence\ of\ mortality\ at\ 48\ ^

^weeks\ of\ age\ when\ compared\ to\ saline-injected\ mice.\ The\ frequency\ and\ latency\ of\ other\ disease\ symptoms\ also\ did\ not\ differ\ between\ the\ groups.^

# ^New\ Zealand\ Black\ (NZB)\ x\ New\ Zealand\ White\ (NZW)\ F1\ Murine\ Model\ of\ SLE^

^NZB/W\ mice\ spontaneously\ develop\ severe\ systemic\ autoimmune\ disease\ characterized\ by\ elevated\ titers\ of\ anti-nuclear\ antibodies\ (ANA),\ increased\ levels\ of\ serum\ IgG,\ polyclonal\ activation\ of\ B\ cells\ and\ the\ subsequent\ development\ of\ a\ fatal\ immune-complex\ mediated\ glomerulonephritis\ (Rose\ and\ Bhatia,\ 1995).\ The\ disease\ symptoms\ resemble\ human\ SLE.\ ^

^\ White\ et\ al.\ (1998)\ evaluated\ the\ ability\ of\ silicone\ gel\ implanted\ in\ the\ mammary\ region\ of\ female\ NZB/W\ mice\ to\ alter\ the\ course\ of\ the\ disease\ over\ a\ 78-day\ period.\ The\ effects\ of\ silicone\ were\ compared\ to\ two\ known\ inducers\ of\ autoimmune\ disease,\ mercuric\ chloride\ and\ D-penicillamine.\ These\ positive\ control\ groups\ are\ helpful\ in\ demonstrating\ the\ sensitivity\ of\ the\ model\ to\ exogenous\ autoimmune-inducing\ substances.\ The\ results\ of\ these\ studies\ showed\ that\ silicone\ gel-implanted\ mice\ did\ not\ differ\ from\ their\ sham\ controls\ in\ terms\ of\ total\ IgG\ levels\ or\ antibody\ titers\ to\ dsDNA,\ laminin,\ DNP-HSA\ or\ SRBC.\ Spleen\ weight\ was\ also\ not\ affected\ by\ silicone\ exposure.\ In\ contrast,\ all\ of\ these\ parameters\ were\ significantly\ elevated\ in\ the\ positive\ control\ groups\ when\ compared\ to\ their\ own\ controls.\ Although\ actual\ disease\ was\ not\ measured\ in\ this\ study,\ silicone\ gel\ exposure\ did\ not\ appear\ to\ be\ promoting\ the\ clinical\ signs\ that\ have\ been\ associated\ with\ development\ of\ the\ disease\ in\ this\ model.^

# ^Tight\ Skin\ Mouse\ Model\ of\ Scleroderma\ ^

^Mice\ bearing\ the\ TSK\ mutation\ (TSK/+)\ spontaneously\ develop\ skin\ fibrosis\ and\ characteristic\ autoantibodies\ which\ resemble\ human\ scleroderma.\ Frondoza\ et\ al.\ (1995)\ evaluated\ the\ influence\ of\ silicone\ on\ the\ pathogenesis\ of\ the\ disease\ in\ this\ mouse\ model.\ TSK/+\ mice\ as\ well\ as\ their\ phenotypically\ normal\ TSK/-\ litter\ mates\ were\ injected\ with\ low\ molecular\ weight\ silicone\ fluid,\ high\ molecular\ weight\ silicone\ gel,\ IFA\ as\ a\ positive\ control,\ or\ saline\ as\ a\ negative\ control.\ One\ month\ later,\ skin\ was\ examined\ histologically\ for\ development\ of\ hyperplasia\ and\ thickening.\ Other\ tissues\ (kidney,\ liver,\ spleen)\ were\ examined\ for\ pathological\ changes.\ Circulating\ autoantibodies\ to\ RNA\ polymerase\ I\ ,\ topoisomerase\ and\ bovine\ serum\ albumin\ (BSA)\ were\ also\ measured.^

^\ The\ results\ indicated\ that\ the\ normal\ progression\ of\ the\ disease\ seen\ in\ saline-treated\ ^

^control\ TSK/+\ mice\ as\ revealed\ by\ histological\ examination\ was\ not\ altered\ by\ silicone\ exposure.\ It\ was\ also\ not\ altered\ by\ IFA.\ No\ evidence\ of\ hyperplasia\ or\ pathology\ was\ seen\ in\ the\ TSK/-\ mice\ with\ any\ of\ the\ treatments.\ None\ of\ the\ mice\ showed\ pathological\ changes\ in\ the\ other\ organs.\ Circulating\ antibodies\ to\ RNA\ polymerase\ I,\ topoisomerase\ or\ BSA\ were\ not\ altered\ in\ TSK/+\ mice\ treated\ with\ silicone\ or\ IFA.\ as\ compared\ to\ saline-treated\ controls.\ The\ lack\ of\ effect\ in\ the\ IFA-treated\ positive\ control\ group\ limits\ the\ ability\ to\ interpret\ the\ lack\ of\ effects\ with\ silicone.\ ^

# ^Type\ II\ Collagen-induced\ Arthritis^

^Because\ collagen\ provides\ the\ basic\ framework\ of\ cartilage,\ the\ experimental\ induction\ of\ an\ immune\ response\ to\ collagen\ can\ produce\ the\ symptoms\ of\ arthritis.\ As\ previously\ described\ in\ Section\ VII,\ animal\ models\ of\ collagen-induced\ arthritis\ have\ been\ characterized\ in\ which\ the\ intradermal\ injection\ of\ bovine\ type\ II\ collagen\ emulsified\ with\ CFA\ or\ IFA\ induces\ an\ arthritis\ in\ genetically\ susceptible\ rats\ or\ mice\ (Holmdahl\ et\ al.,\ 1989).\ The\ lesions\ found\ in\ the\ affected\ joints\ are\ quite\ similar\ to\ those\ found\ in\ humans\ with\ rheumatoid\ arthritis.\ Anti-collagen\ antibodies\ develop\ in\ immunized\ rats\ and\ mice\ and\ are\ also\ found\ in\ RA\ patients,\ but\ the\ specific\ role\ they\ play\ in\ the\ pathogenesis\ of\ the\ disease\ is\ controversial.\ Circulating\ levels\ of\ inflammatory\ cytokines\ such\ as\ IL-1\$,\ TNF-\",\ and\ IL-6\ are\ elevated\ during\ the\ disease\ process,\ and\ experimental\ manipulation\ of\ cytokine\ activity\ (production\ or\ receptor\ blockade)\ modifies\ the\ disease.\ Thus,\ proinflammatory\ substances\ might\ be\ expected\ to\ promote\ arthritic\ disease.^

^\ Schaefer\ et\ al.\ (1997)\ used\ the\ mouse\ model\ of\ collagen-induced\ arthritis\ to\ examine\ the\ ability\ of\ various\ forms\ of\ silicone\ implants\ to\ promote\ the\ disease\ process.\ Mice\ were\ injected\ with\ silicone\ oil,\ gel\ or\ elastomer\ for\ three\ days\ or\ nine\ months\ prior\ to\ immunization\ with\ collagen\ in\ CFA.\ The\ results\ showed\ that\ silicone\ in\ all\ forms\ had\ no\ influence\ on\ the\ incidence\ or\ severity\ of\ arthritis\ as\ compared\ to\ sham-\ treated\ mice.\ However,\ because\ the\ incidence\ and\ severity\ of\ disease\ was\ high\ in\ the\ controls,\ significant\ promotional\ effects\ would\ have\ been\ difficult\ to\ demonstrate.\ Time-to-onset\ of\ disease\ was\ not\ reported.\ ^

^\ \ In\ a\ second\ study,\ Schaefer\ (1997)\ used\ collagen\ immunization\ with\ IFA\ instead\ of\ CFA\ to\ induce\ a\ lower\ incidence\ of\ disease\ in\ the\ controls.\ In\ this\ study,\ 9/10\ mice\ implanted\ with\ silicone\ elastomer\ nine\ months\ prior\ to\ immunization\ exhibited\ disease\ compared\ to\ 3/10\ control\ mice.\ The\ severity\ of\ the\ disease\ was\ also\ increased\ in\ the\ silicone\ elastomer-treated\ mice.\ In\ ^

^mice\ treated\ with\ silicone\ gel\ or\ oil,\ 6/9\ mice\ in\ each\ group\ developed\ disease\ and\ their\ arthritic\ scores\ also\ tended\ to\ be\ higher\ than\ the\ controls,\ although\ these\ changes\ were\ not\ statistically\ different\ from\ controls.\ Taken\ together,\ the\ results\ suggest\ that\ exposure\ to\ different\ forms\ of\ silicone\ may\ promote\ the\ development\ of\ arthritis\ in\ this\ model\ of\ autoimmune\ disease.\ However,\ these\ results\ must\ be\ considered\ preliminary\ and\ interpreted\ cautiously\ in\ light\ of\ the\ small\ number\ of\ animals\ tested\ and\ the\ fact\ that\ only\ 3\ control\ mice\ developed\ disease.\ Furthermore,\ the\ findings\ are\ less\ than\ compelling\ based\ on\ the\ fact\ that\ anti-collagen\ antibody\ titers\ were\ not\ altered\ by\ silicone,\ and\ cytokine\ levels\ were\ not\ consistently\ altered\ in\ a\ manner\ that\ might\ explain\ the\ increased\ incidence\ of\ disease.\ ^

***^NZB\ Mouse\ Model\ of\ Autoimmune\ Hemolytic\ Anemia^***

^NZB\ mice\ develop\ a\ form\ of\ autoimmune\ hemolytic\ anemia\ that\ closely\ resembles\ the\ human\ disease.\ The\ disease\ begins\ at\ about\ three\ months\ of\ age\ and\ by\ nine\ months\ almost\ all\ mice\ show\ reduced\ hematocrits\ as\ evidence\ of\ the\ disease\ process\ (Howie\ and\ Helyer,\ 1968).\ MacDonald\ et\ al.(1998)\ used\ the\ NZB\ model\ to\ study\ the\ influence\ of\ silicone\ gel\ implants\ on\ this\ autoimmune\ disease\ process.\ Groups\ of\ mice\ were\ injected\ with\ saline\ as\ a\ negative\ control,\ pristane\ as\ a\ positive\ control,\ or\ silicone\ gel.\ Injections\ given\ one\ time\ were\ compared\ to\ injections\ given\ three\ times\ to\ examine\ the\ effect\ of\ multiple\ exposures.\ Some\ mice\ were\ given\ an\ injection\ of\ silicone\ followed\ three\ months\ later\ by\ a\ capsulotomy\ to\ evaluate\ the\ effect\ of\ "traumatic\ rupture."\ Other\ mice\ were\ given\ an\ injection\ of\ silicone\ followed\ three\ months\ later\ by\ a\ low\ dose\ of\ ^*^Staphylococcus\ epidermidis^*^,\ intended\ to\ mimic\ infection\ with\ "a\ common\ contaminant\ found\ on\ the\ surface\ of\ breast\ implants\ and\ hypothesized\ to\ be\ involved\ in\ capsular\ contracture."\ Appropriate\ controls\ were\ included\ for\ all\ of\ the\ procedures\ except\ the\ capsulotomy.\ Mice\ were\ examined\ daily\ for\ ten\ months\ after\ which\ time\ all\ surviving\ mice\ were\ sacrificed.\ Blood\ was\ used\ to\ measure\ hematocrits\ and\ serum\ was\ analyzed\ for\ autoantibody\ production.\ Urinary\ protein\ levels\ were\ monitored\ bi\ weekly\ during\ the\ course\ of\ the\ study\ for\ the\ possible\ development\ of\ glomerulonephritis.^[^1]

^\ By\ ten\ months\ of\ age,\ some\ mortality\ had\ occurred\ in\ all\ groups,\ including\ the\ controls\ (20-30%).\ Only\ multiple\ treatments\ with\ pristane,\ the\ positive\ control,\ significantly\ increased\ the\ ^

^mortality\ of\ the\ mice\ to\ 80%.\ Increased\ mortality\ (60%,\ P\ \<\ 0.085)\ was\ also\ noted\ after\ multiple\ treatments\ of\ silicone.\ Hematocrits\ were\ much\ lower\ in\ all\ NZB\ mice\ compared\ to\ normal\ BALB/c\ mice.\ Hematocrits\ were\ further\ reduced\ in\ mice\ given\ pristane\ three\ times,\ and\ in\ all\ mice\ injected\ with\ silicone,\ although\ it\ was\ not\ clear\ what\ pairwise\ comparisons\ were\ made\ to\ determine\ statistical\ significance.\ In\ contrast,\ hemagglutination\ titers\ were\ similar\ in\ all\ groups\ compared\ to\ controls.\ ANA\ titers\ were\ elevated\ in\ silicone-implanted\ mice\ that\ had\ undergone\ a\ capsulotomy\ at\ three\ months.\ Anti-collagen\ IgM\ titers\ were\ elevated\ in\ mice\ that\ were\ injected\ with\ silicone\ and\ infected\ with\ ^*^S.\ epidermidis^*^\ whereas\ anti-collagen\ IgG\ titers\ were\ similar\ in\ all\ groups.\ Multiple\ injections\ of\ pristane\ or\ silicone\ increased\ urinary\ protein\ levels.\ ^

^\ Based\ primarily\ on\ the\ mortality\ and\ hematocrits,\ the\ results\ of\ this\ study\ provide\ limited\ evidence\ for\ a\ promotion\ of\ autoimmune\ hemolytic\ anemia\ by\ silicone\ in\ NZB\ mice.\ However,\ the\ results\ would\ have\ to\ be\ repeated\ before\ such\ a\ conclusion\ could\ be\ drawn.\ The\ data\ would\ also\ be\ more\ compelling\ if\ additional\ endpoints\ relevant\ to\ the\ disease\ process\ had\ been\ measured,\ as\ it\ is\ not\ clear\ what\ caused\ the\ death\ of\ the\ animals.\ In\ addition,\ the\ clinical\ data\ might\ have\ been\ more\ insightful\ if\ it\ had\ been\ collected\ prior\ to\ the\ onset\ of\ mortality.\ On\ the\ other\ hand,\ the\ relevance\ of\ this\ disease\ model\ to\ women\ with\ SBIs\ is\ unclear.^

^\ Normal\ BALB/c\ mice\ were\ also\ tested\ in\ parallel\ with\ the\ NZB\ mice\ and\ given\ identical\ pristane\ and\ silicone\ treatments\ to\ determine\ if\ a\ non-genetically\ disposed\ mouse\ could\ be\ induced\ to\ develop\ disease\ by\ exposure\ to\ silicone.\ However\ there\ was\ no\ significant\ mortality\ in\ any\ treatment\ group,\ and\ all\ had\ normal\ hematocrits.\ These\ data\ indicate\ silicone\ was\ not\ able\ to\ induce\ autoimmune\ disease\ in\ genetically\ resistant\ mice.\ ^

# ^IX.\ Immunotoxicity\ of\ Silicone\ in\ Animals^

^Although\ the\ majority\ of\ results\ from\ the\ animals\ models\ of\ autoimmune\ disease\ do\ not\ support\ an\ enhancement\ of\ the\ disease\ process\ by\ silicone,\ which\ was\ tested\ in\ several\ different\ forms\ and\ for\ various\ periods\ of\ time,\ arguments\ can\ be\ put\ forth\ as\ to\ why\ the\ animal\ models\ are\ different\ from\ the\ human\ situation\ and\ therefore\ not\ reflective\ of\ the\ human\ response\ to\ silicone.\ Thus,\ it\ is\ appropriate\ to\ review\ the\ animal\ studies\ that\ have\ examined\ the\ ability\ of\ silicone\ to\ alter\ processes\ that\ are\ believed\ to\ contribute\ to\ the\ development\ of\ autoimmunity.\ Based\ on\ a\ general\ conceptual\ understanding\ of\ autoimmune\ disease\ pathogenesis,\ several\ hypothetical\ mechanisms\ have\ been\ proposed\ by\ which\ silicone\ could\ induce\ or\ exacerbate\ the\ process.\ These\ include:^

^1.\ Silicone\ causes\ immune\ system\ "dysregulation"\ resulting\ in\ abnormal\ T\ cell\ and/or\ B\ cell\ activity\ leading\ to\ the\ generation\ of\ the\ autoimmune\ response.\ For\ example,\ polyclonal\ B\ cell\ activation\ or\ loss\ of\ suppressor\ T\ cell\ functions\ have\ been\ associated\ with\ some\ autoimmune\ diseases.^

^2.\ Silicone\ induces\ specific\ T\ cell\ activation\ by\ modification\ of\ self-proteins\ resulting\ in\ a\ novel\ autoimmune\ disease.\ ^

^3.\ Silicone\ causes\ inflammation\ and\ the\ resulting\ inflammatory\ cytokine\ production\ initiates\ or\ exacerbates\ autoimmune\ disease\ development.\ ^

^The\ evidence\ available\ to\ support\ or\ refute\ these\ hypotheses\ will\ be\ summarized\ below.^

# ^Evidence\ that\ Silicone\ Alters\ Immune\ Responsiveness\ of\ Animals^

^Several\ studies\ have\ been\ conducted\ in\ laboratory\ animals\ in\ an\ effort\ to\ determine\ the\ influence\ of\ silicone\ exposure\ on\ immune\ function.\ Comprehensive\ immunotoxicology\ studies\ were\ carried\ out\ in\ the\ early1990s\ by\ the\ Medical\ College\ of\ Virginia\ under\ contract\ to\ the\ National\ Toxicology\ Program.\ A\ standard\ immunotoxicology\ screen\ was\ utilized\ in\ mouse\ studies\ to\ examine\ the\ immunomodulatory\ effects\ of\ various\ doses^[^2]^\ of\ silicone\ oil,\ silicone\ gel,\ or\ silicone\ elastomer\ disks\ implanted\ subcutaneously\ in\ the\ breast\ area\ of\ female\ B6C3F1\ mice.\ ^

^\ Polyurethane\ disks\ were\ also\ tested.\ Controls\ were\ injected\ with\ saline.\ Immunological\ testing\ was\ carried\ out\ ten\ days\ or\ 180\ days\ after\ initiation\ of\ silicone\ exposure\ (Bradley\ et\ al.,\ 1994a,b).\ The\ ten-day\ period\ was\ selected\ to\ represent\ the\ peak\ inflammatory\ response.\ The\ 180-day\ exposure\ was\ intended\ to\ reflect\ the\ chronic\ condition\ of\ SBIs\ wherein\ a\ well-developed\ fibrous\ capsule\ had\ formed.\ Endpoints\ assessed\ included\ body\ weight,\ histopathology,\ hematology,\ serum\ complement\ levels,\ bone\ marrow\ colony\ formation,\ spleen\ cell\ subpopulations\ (B\ cells,\ T\ cells\ and\ T\ cell\ subsets);\ primary\ antibody\ response\ to\ SRBC\ (IgM\ and\ IgG\ PFCs);\ proliferative\ responses\ to\ B\ and\ T\ cell\ mitogens\ and\ to\ allogeneic\ lymphocytes,\ cytotoxic\ T\ lymphocyte\ response,\ NK\ cell\ cytotoxicity,\ reticuloendothelial\ system\ clearance\ of\ ^

^antigenic\ particles,\ peritoneal\ macrophage\ phagocytosis\ and\ IFN-(\ production.\ The\ ability\ of\ the\ animals\ to\ resist\ infection\ by\ ^*^Streptococcus\ pneumonia^*^\ or\ ^*^Listeria\ monocytogenes^*^\ and\ to\ control\ B16\ melanoma\ metastases\ were\ assessed\ as\ holistic\ measurements\ of\ overall\ immune\ status.\ ^

^\ \ The\ results\ of\ these\ immunotoxicology\ studies\ showed\ that\ silicone\ in\ any\ form\ tested\ did\ not\ induce\ systemic\ toxic\ effects\ or\ alter\ immune\ function.\ The\ only\ noteworthy\ silicone-related\ treatment\ effect\ was\ a\ decrease\ in\ NK\ cell\ activity\ in\ the\ spleen\ of\ mice\ injected\ with\ silicone\ gel\ or\ implanted\ with\ the\ elastomer\ disk\ for\ 180\ days.\ However,\ this\ effect\ on\ NK\ activity\ did\ not\ translate\ into\ an\ increase\ in\ B16\ tumor\ metastasis,\ which\ is\ the\ host\ resistance\ model\ considered\ sensitive\ to\ changes\ in\ NK\ cell\ activity.\ ^

^\ \ Follow-up\ studies\ were\ carried\ out\ to\ validate\ the\ effect\ of\ silicone\ on\ NK\ activity\ in\ a\ dose-response\ study.\ The\ results\ of\ the\ study\ confirmed\ the\ suppression\ of\ NK\ cell\ activity\ but\ only\ at\ the\ highest\ dose\ level.\ Comparative\ studies\ using\ F344\ rats\ also\ showed\ a\ suppressive\ effect\ on\ NK\ cell\ activity\ from\ silicone\ treatment.\ In\ rats,\ NK\ activity\ could\ be\ boosted\ in\ silicone-implanted\ rats\ by\ polyI:C\ but\ not\ to\ levels\ observed\ in\ controls\ (Wilson\ and\ Munson,\ 1996).\ ^

^\ Taken\ together,\ these\ results\ indicate\ a\ modest\ and\ somewhat\ consistent\ effect\ of\ silicone\ exposure\ on\ NK\ cell\ activity.\ The\ importance\ of\ the\ finding\ may\ derive\ from\ the\ fact\ that\ any\ systemic\ alteration\ in\ an\ immunological\ endpoint\ could\ be\ induced\ by\ the\ presence\ of\ a\ silicone\ implant.\ However,\ the\ importance\ of\ the\ finding\ in\ terms\ of\ autoimmune\ disease\ is\ not\ known\ since\ a\ role\ for\ NK\ cells\ in\ autoimmune\ disease\ development\ is\ not\ widely\ recognized.\ The\ mechanism\ by\ which\ silicone\ alters\ NK\ cell\ activity\ was\ not\ elucidated.^

^\ Although\ the\ NTP-sponsored\ immunotoxicity\ assessment\ of\ silicone\ was\ thorough\ and\ of\ high\ quality,\ there\ were\ some\ shortcomings\ in\ the\ experimental\ design.\ For\ example,\ these\ studies\ utilized\ a\ standard\ screening\ procedure\ to\ identify\ immunotoxic\ substances.\ The\ assays\ were\ previously\ validated\ to\ detect\ immunosuppressive\ substances\ and\ were\ not\ specifically\ oriented\ to\ address\ autoimmune-relevant\ endpoints.\ Another\ potential\ shortcoming\ is\ that\ the\ animals\ used\ in\ the\ studies\ were\ not\ predisposed\ to\ develop\ autoimmune\ disease\ and\ may\ therefore\ be\ more\ resistant\ to\ the\ effects\ of\ silicone\ than\ an\ autoimmune-prone\ strain.\ ^

# ***^Evidence\ for\ Antigenicity\ of\ Silicone^***^\ \ \ \ ^

^The\ question\ of\ silicone\ antigenicity\ has\ been\ addressed\ in\ a\ limited\ number\ of\ animal\ studies\ ^

^that\ have\ attempted\ to\ demonstrate\ silicone-specific\ antibodies\ or\ silicone-specific\ T\ cell\ responses,\ including\ the\ possible\ development\ of\ specific\ immunologic\ memory.\ The\ data\ from\ these\ studies\ will\ be\ reviewed\ here.^

^\ Delayed-type\ hypersensitivity\ (DTH)\ responses\ are\ useful\ measures\ of\ T-cell\ mediated\ immunity\ in\ animals.\ A\ true\ DTH\ response\ follows\ delayed\ kinetics\ that\ reflect\ the\ response\ of\ pre-sensitized\ antigen-specific\ memory\ T\ cells.\ These\ T\ cells\ proliferate\ in\ response\ to\ their\ specific\ antigen\ and\ secrete\ cytokines\ that\ activate\ macrophages,\ causing\ tissue\ swelling\ that\ peaks\ 48--72\ hours\ after\ antigen\ injection.\ The\ kinetics\ of\ the\ DTH\ response\ is\ crucial\ for\ differentiating\ T\ cell\ immunity\ from\ nonspecific\ inflammatory\ responses\ that\ occur\ within\ a\ few\ hours\ of\ injection\ and\ are\ not\ antigen-specific.\ The\ DTH\ response\ is\ also\ distinguished\ by\ its\ kinetics\ from\ a\ rapid\ contact\ hypersensitivity\ response\ that\ is\ mediated\ by\ antigen-specific\ IgE\ antibodies.\ ^

^\ Brantley\ et\ al.(1990)\ used\ a\ creative\ approach\ to\ ask\ if\ there\ is\ a\ cell-mediated\ immune\ response\ to\ silicone.\ They\ "immunized"\ rats\ to\ silicone\ by\ injecting\ silicone\ in\ CFA.\ Four\ weeks\ later,\ the\ animals\ were\ given\ silicone\ implants.\ Reactions\ to\ the\ implants\ were\ measured\ by\ capsule\ formation\ and\ histology\ of\ the\ capsule.\ Based\ on\ the\ similar\ histology\ of\ the\ capsules\ of\ immunized\ (CFA\ +\ silicone)\ and\ nonimmunized\ (CFA\ only)\ rats,\ there\ was\ no\ evidence\ of\ an\ immune\ response\ to\ silicone.\ One\ would\ have\ expected\ a\ characteristic\ tissue\ reaction\ if\ the\ rats\ had\ been\ immune\ to\ the\ silicone.\ However,\ these\ negative\ results\ must\ also\ be\ interpreted\ cautiously\ given\ that\ a\ positive\ control\ was\ not\ included\ to\ demonstrate\ the\ sensitivity\ of\ the\ technique.^

^\ In\ related\ studies,\ Brantley\ et\ al.\ (1990)\ immunized\ rats\ with\ silicone\ gel\ sonicated\ in\ CFA.\ Four\ weeks\ later,\ lymphocytes\ from\ the\ spleen\ were\ obtained\ and\ tested\ for\ their\ ability\ to\ respond\ to\ silicone\ in\ vitro.\ There\ was\ no\ difference\ in\ the\ proliferative\ response\ from\ silicone-immunized\ mice\ compared\ to\ mice\ injected\ only\ with\ CFA.\ Thus,\ there\ was\ no\ measurable\ memory\ response\ to\ silicone,\ the\ most\ unambiguous\ measure\ of\ antigen-specific\ immunity.\ ^

^\ Smith\ et\ al.\ (1990)\ immunized\ rats\ with\ fine\ particles\ of\ solid\ silicone\ from\ a\ bone\ prostheses\ emulsified\ with\ CFA.\ After\ six\ sensitizing\ injections,\ they\ reported\ evidence\ for\ an\ immune\ response\ to\ the\ silicone\ in\ a\ positive\ skin\ reaction\ to\ challenge\ and\ IgG\ deposition\ around\ the\ silicone\ implant.\ However,\ the\ study\ lacked\ appropriate\ controls.\ \ ^

^\ LeBeau\ (1967)\ reported\ that\ silicone\ gel\ strips\ did\ not\ induce\ a\ hypersensitivity\ response\ in\ ^

^the\ skin\ of\ guinea\ pigs.\ Naim\ et\ al\ (1993)\ found\ no\ evidence\ for\ a\ DTH\ response\ to\ silicone\ in\ rabbits.^

^\ Silicone-specific\ antibody\ production\ by\ B\ lymphocytes\ has\ also\ been\ examined\ to\ determine\ if\ there\ is\ a\ specific\ immune\ response\ to\ silicone.\ In\ order\ to\ demonstrate\ antigen\ specificity,\ one\ must\ be\ able\ to\ show\ specific\ binding\ of\ the\ induced\ antibody\ to\ the\ antigen\ ^*^in\ vitro^*^.\ In\ regard\ to\ silicone\ antibodies,\ these\ ^*^in\ vitro^*^\ assays\ have\ been\ fraught\ with\ problems\ associated\ with\ nonspecific\ protein\ binding\ to\ the\ silicone\ (Butler\ et\ al.,\ 1996;\ Rosenau\ et\ al.,\ 1996).\ The\ studies\ are\ also\ complicated\ by\ the\ hydrophobic\ nature\ of\ silicone\ materials\ and\ the\ difficulties\ of\ working\ with\ them\ in\ antibody\ assays\ .\ Unfortunately,\ because\ of\ the\ unconventional\ methods\ that\ have\ been\ used\ in\ past\ studies\ in\ an\ attempt\ to\ circumvent\ these\ problems,\ along\ with\ a\ failure\ to\ provide\ adequate\ assay\ validation,\ the\ data\ currently\ available\ are\ not\ convincing\ of\ silicone-specific\ antibody\ production.\ ^

# ***^Evidence\ that\ Silicone\ Induces\ Inflammation^***^\ ^

^Several\ studies\ have\ reported\ that\ subcutaneous\ or\ intramuscular\ injection\ of\ various\ types\ of\ silicone\ in\ experimental\ animals\ induces\ an\ inflammatory\ response\ similar\ to\ a\ foreign\ body\ reaction\ leading\ to\ the\ formation\ of\ a\ fibrous\ capsule\ around\ the\ implant\ (Lilla\ and\ Vistnes,\ 1975;\ Goodman\ et\ al.,\ 1988;\ Lemen\ et\ al.,\ 1992).\ The\ extent\ of\ the\ inflammatory\ response\ in\ any\ particular\ study\ depends\ on\ the\ type\ of\ silicone\ material\ that\ is\ implanted,\ the\ size\ and\ shape\ of\ the\ implant,\ its\ location,\ as\ well\ as\ other\ unidentified\ factors\ (Picha\ and\ Goldstein,\ 1991).\ In\ addition,\ differences\ in\ surgical\ techniques\ cannot\ be\ minimized\ since\ surgical\ trauma\ alone\ can\ account\ for\ a\ part\ of\ the\ early\ inflammation\ noted,\ and\ inadvertent\ bacterial\ contamination\ would\ likely\ increase\ the\ severity\ of\ the\ inflammatory\ response.\ However,\ in\ most\ studies,\ the\ inflammatory\ response\ declines\ over\ time,\ and\ the\ implants\ are\ usually\ found\ surrounded\ by\ a\ mature\ connective\ tissue\ capsule\ of\ varying\ thickness\ containing\ minimal\ numbers\ of\ macrophages,\ neutrophils\ and\ lymphocytes\ (Grasso\ et\ al.,\ 1965;\ Malczewski,\ 1984;\ Mudgett\ et\ al.,\ 1990;\ Picha\ and\ Goldstein,\ 1991;\ Rasmussen,\ 1988).\ Recently,\ Fabre\ et\ al.\ (1998)\ used\ a\ novel\ technique\ to\ demonstrate\ this\ process.\ They\ measured\ the\ cellular\ response\ to\ a\ cylindrical\ elastomer\ implant\ using\ flow\ cytometry.\ Two\ days\ after\ implantation,\ a\ mixture\ of\ monocytes\ and\ neutrophils\ predominated\ in\ the\ exudate\ that\ formed\ inside\ the\ tube.\ At\ day\ nine,\ cell\ subpopulations\ could\ still\ be\ identified,\ whereas\ by\ day\ 23,\ the\ cellular\ components\ had\ declined\ ^

^to\ undetectable\ levels.\ Fibrinogen\ levels\ rose\ progressively\ during\ this\ time.\ These\ results\ indicate\ a\ resolution\ of\ the\ active\ inflammatory\ response\ to\ the\ silicone\ elastomer\ within\ the\ 23-day\ time\ frame.^

^\ Injection\ of\ silicone\ fluid\ directly\ into\ the\ joint\ of\ DA\ rats\ induced\ arthritis,\ suggesting\ that\ a\ local\ inflammatory\ reaction\ was\ induced\ by\ silicone\ (Yoshino,\ 1994).\ This\ response\ is\ not\ particularly\ surprising\ since\ the\ DA\ rat\ is\ highly\ susceptible\ to\ arthritis\ induction.\ However,\ there\ is\ no\ evidence\ that\ silicone\ from\ SBIs\ is\ transported\ to\ joints\ at\ any\ significant\ concentration\ to\ directly\ induce\ arthritis.\ This\ is\ supported\ by\ the\ animal\ studies\ discussed\ previously\ that\ show\ silicone\ injections\ outside\ of\ the\ joint\ do\ not\ induce\ arthritis,\ even\ in\ the\ DA\ rat\ (see\ Table\ 1).^

^\ While\ it\ is\ generally\ accepted\ that\ silicones\ elicit\ varying\ degrees\ of\ local\ inflammation\ at\ the\ site\ of\ the\ implant,\ there\ is\ little\ evidence\ from\ controlled\ animal\ studies\ that\ suggest\ silicone\ causes\ systemic\ inflammatory\ responses.\ The\ most\ extensive\ examination\ of\ this\ possibility\ was\ carried\ out\ by\ Schaefer\ (1997),\ who\ measured\ levels\ of\ circulating\ cytokines\ at\ various\ times\ after\ silicone\ implantation\ in\ relationship\ to\ the\ development\ of\ autoimmune\ disease.\ The\ results\ of\ these\ studies,\ documented\ in\ Table\ 3,\ failed\ to\ provide\ evidence\ that\ silicones\ induced\ a\ systemic\ alteration\ in\ inflammatory\ cytokine\ production.\ ^

# ^Evidence\ that\ Silicone\ Activates\ Macrophages^

^As\ previously\ discussed,\ studies\ carried\ out\ in\ the\ late\ 1960s\ had\ shown\ that\ silicone\ fluid\ injected\ ip\ or\ sc\ appeared\ to\ be\ phagocytosed\ and\ distributed\ systemically\ via\ the\ lymphatics\ (Ben\ Hur\ et\ al.,\ 1967;\ Rees\ et\ al.,\ 1966;\ Sparchu\ and\ Clashman,\ 1970).\ In\ more\ recent\ studies,\ macrophages\ containing\ silicone\ were\ also\ found\ in\ rats\ injected\ with\ silicone\ fluid\ (Hill\ et\ al.,1996;\ Malczewski\ et\ al.,\ 1994),\ but\ not\ in\ rats\ implanted\ with\ silicone\ gel\ or\ elastomer\ (Malczewski\ et\ al.,\ 1994).\ Likewise,\ several\ other\ studies\ on\ silicone\ gel\ have\ found\ no\ evidence\ for\ the\ phagocytosis\ or\ systemic\ distribution\ of\ silicone\ gel^**^\ ^**^in\ rats\ that\ had\ been\ implanted\ for\ as\ long\ as\ two\ years\ (Goodman\ et\ al.,1988;\ Raposo\ do\ Amaral\ et\ al.,1993;\ Tiziani\ et\ al.,\ 1995).\ Taken\ together,\ these\ results\ suggest\ that\ phagocytosis\ of\ silicone\ by\ macrophages\ and\ systemic\ distribution\ of\ silicone\ is\ a\ phenomenon\ primarily\ associated\ with\ the\ injection\ of\ free\ silicone\ fluid\ rather\ than\ gel.\ ^

^\ However,\ because\ macrophages\ have\ been\ shown\ to\ be\ capable\ of\ engulfing\ silicone\ in\ any\ form,\ many\ of\ the\ hypotheses\ related\ to\ silicone-induced\ disease\ invoke\ a\ role\ for\ silicone-^

^induced\ activation\ of\ macrophages.\ These\ activated\ macrophages\ are\ then\ hypothesized\ to\ secrete\ cytokines\ that\ lead\ to\ the\ disease\ symptomology.\ Unfortunately,\ there\ are\ no\ definitive\ data\ available\ that\ have\ characterized\ the\ influence\ of\ silicone\ on\ such\ macrophage\ activation\ or\ cytokine\ production.^

^\ Das\ et\ al.\ (1990)\ examined\ the\ ability\ of\ silicone\ sonicated\ with\ CFA\ to\ cause\ long-term\ activation\ of\ macrophages\ as\ measured\ by\ their\ secretion\ of\ IL-1.\ At\ eight\ months\ after\ silicone\ injection,\ there\ was\ no\ difference\ in\ IL-1\ production\ compared\ to\ mice\ injected\ with\ CFA\ or\ saline.\ However,\ the\ negative\ results\ must\ be\ tempered\ by\ the\ fact\ that\ no\ relevant\ positive\ control\ was\ included.\ The\ addition\ of\ LPS\ ^*^in\ vitro^*^\ to\ activate\ macrophages\ was\ not\ an\ appropriate\ positive\ control\ ^

^\ \ MacDonald\ et\ al.\ (1996)\ reported\ that\ silicone\ gel\ injected\ into\ the\ peritoneal\ cavity\ of\ certain\ strains\ of\ mice\ induced\ a\ population\ of\ predominately\ macrophages\ that\ was\ able\ to\ induce\ a\ small\ degree\ of\ proliferation\ in\ CD4+\ T\ cells\ in\ a\ nonantigen-specific\ manner.\ The\ authors\ suggested\ that\ these\ "silicone-laden\ macrophages"\ induce\ a\ proliferative\ response\ that\ is\ "unique\ to\ silicone"\ because\ macrophages\ from\ pristane-\ or\ thioglycollate-injected\ mice\ did\ not\ induce\ proliferation.\ However,\ the\ authors\ failed\ to\ speculate\ on\ what\ unique\ factor\ silicone-laden\ cells\ produce\ that\ other\ inflammatory\ macrophages\ do\ not,\ nor\ did\ they\ demonstrate\ by\ any\ other\ criteria\ that\ the\ macrophages\ were\ indeed\ activated.\ Furthermore,\ they\ did\ not\ demonstrate\ that\ the\ activity\ was\ mediated\ by\ the\ macrophage\ component\ of\ the\ peritoneal\ exudate\ cells,\ nor\ that\ the\ macrophages\ indeed\ contained\ silicone.\ Thus,\ the\ data\ do\ not\ support\ the\ conclusions\ of\ the\ authors\ and\ provide\ no\ insight\ into\ the\ effect\ of\ silicone\ on\ macrophage\ activity.\ ^

^\ The\ studies\ of\ Bradley\ et\ al.\ (1994a,b)\ addressed\ the\ potential\ for\ silicone\ to\ alter\ systemic\ macrophage\ activity\ in\ mice\ that\ received\ short-term(ten\ day)\ or\ long-term\ (180\ day)\ implants\ of\ silicone\ oil,\ gel,\ or\ elastomer.\ Reticuloendothelial\ clearance\ of\ particles\ by\ tissue\ macrophages\ present\ in\ the\ liver,\ spleen,\ lymph\ nodes\ and\ lungs\ was\ evaluated\ by\ measuring\ the\ vascular\ clearance\ and\ tissue\ uptake\ of\ radioactively\ labeled\ foreign\ particles\ (SRBC\ or\ Covaspheres).\ The\ functional\ activity\ of\ adherent\ peritoneal\ cells\ (primarily\ macrophages)\ was\ evaluated\ by\ their\ ability\ to\ phagocytose\ radiolabeled\ particles\ in\ vitro,\ and\ by\ their\ ability\ to\ be\ activated\ by\ IFN(\ and\ LPS\ to\ kill\ tumor\ cells\ ^*^in\ vitro^*^.\ ^

^\ The\ results\ of\ these\ studies\ revealed\ no\ change\ in\ any\ of\ these\ parameters\ ten\ days\ after\ the\ implant\ surgery\ except\ for\ a\ decrease\ in\ macrophage\ tumoricidal\ function\ under\ various\ ^*^in\ vitro^*^\ ^

^conditions.\ However,\ this\ decrease\ in\ tumoricidal\ function\ did\ not\ translate\ into\ a\ change\ in\ the\ resistance\ of\ the\ mice\ to\ tumor\ growth.\ On\ the\ other\ hand,\ in\ one\ experiment,\ all\ of\ the\ mice\ in\ the\ silicone\ groups\ were\ more\ resistant\ to\ infection\ by\ Listeria\ bacteria,\ which\ could\ possibly\ reflect\ enhanced\ phagocytic\ activity.\ ^

^\ In\ mice\ that\ had\ been\ implanted\ with\ silicone\ materials\ 180\ days\ previously,\ there\ was\ increased\ uptake\ of\ SRBC\ by\ the\ liver\ of\ mice\ exposed\ to\ silicone\ gel,\ but\ this\ effect\ was\ not\ confirmed\ in\ a\ subsequent\ dose-response\ study.\ Peritoneal\ cells\ from\ silicone\ fluid-\ implanted\ mice\ had\ significantly\ increased\ phagocytic\ activity\ for\ Covaspheres,\ but\ this\ too\ was\ not\ confirmed\ in\ a\ dose-response\ study.\ Finally,\ the\ growth\ of\ iv-injected\ tumor\ cells\ in\ the\ lung\ was\ decreased\ in\ all\ silicone-treated\ mice\ compared\ to\ vehicle\ controls,\ which\ could\ reflect\ increased\ macrophage\ tumoricidal\ activity.\ However,\ since\ macrophage\ tumoricidal\ activity\ was\ not\ evaluated,\ additional\ studies\ would\ be\ required\ to\ document\ this\ effect.^

^\ Recently,\ Rhie\ et\ al.\ (1998)\ published\ a\ study\ in\ which\ macrophages\ were\ cultured\ on\ silicone\ gel\ that\ was\ centrifuged\ onto\ the\ bottom\ of\ tissue\ culture\ plates,\ allowing\ for\ direct\ contact\ between\ the\ cells\ and\ the\ gel.\ Subsequent\ analysis\ of\ the\ function\ of\ these\ macrophages\ demonstrated\ a\ significantly\ enhanced\ responsiveness\ compared\ to\ macrophages\ cultured\ directly\ on\ the\ plastic\ plate.\ The\ authors\ conclude\ that\ silicone\ gel\ activated\ the\ macrophages\ to\ augment\ immune\ function.\ However,\ the\ authors\ do\ not\ consider\ an\ equally\ plausible\ explanation\ for\ the\ data;\ that\ is,\ that\ by\ preventing\ the\ adherence\ of\ the\ macrophages\ to\ the\ plastic\ plate\ with\ the\ gel\ coating,\ the\ suppressive\ effect\ of\ adherence-induced\ activation\ was\ prevented.\ This\ possibility\ arises\ from\ the\ well-known\ and\ widely\ used\ technique\ of\ coating\ tissue\ culture\ plates\ and\ tubes\ with\ silicone\ to\ prevent\ macrophage\ adherence,\ and\ the\ equally\ well-known\ fact\ that\ an\ excess\ of\ activated\ macrophages\ usually\ suppresses\ immune\ function\ ^*^in\ vitro^*^.\ Unfortunately,\ Rhie\ et\ al.\ failed\ to\ provide\ an\ important\ control\ group\ that\ would\ have\ characterized\ a\ normal\ immune\ response\ without\ any\ added\ macrophages.\ They\ also\ failed\ to\ provide\ any\ direct\ evidence\ for\ the\ state\ of\ activation\ of\ the\ macrophages\ cultured\ on\ gel\ vs\ plastic\ (e.g.,\ adhesion\ molecule\ expression\ or\ cytokine\ production).\ Thus,\ these\ studies\ do\ not\ provide\ convincing\ evidence\ that\ silicone\ gel\ induces\ the\ activation\ of\ macrophages.\ ^

# **^X.^**^\ ^**^Potential\ Contribution\ of\ Other\ Materials\ in\ SBIs\ to\ Toxicity^**

# ^Low\ Molecular\ Weight\ Cyclosiloxanes^

^D4\ and\ D5\ are\ low\ molecular\ weight,\ cyclic\ silicones\ that\ have\ been\ detected\ in\ SBIs.\ D4\ has\ been\ analyzed\ at\ levels\ of\ approximately\ 500\ ppm\ in\ the\ gel\ and\ in\ the\ elastomer\ at\ a\ level\ of\ 100-300\ ppm^[^3]^\ (Van\ Dyke\ et\ al.,\ 1993).\ D5\ levels\ are\ similar.\ There\ has\ been\ some\ speculation\ that\ these\ molecules\ play\ a\ part\ in\ the\ health\ effects\ of\ SBIs.\ ^

The toxicities of D4 and D5 have been tested independently of silicone
because they were formulated and are marketed for use in a variety of
products. Inhalation of relatively large doses of D4 and D5 have been
shown to produce few toxic effects other than liver enlargement and
induction of hepatic drug metabolizing enzymes (McKim et al., 1988;
Mehendale, 1989; Siddiqui, 1989). Such hepatic effects are not seen
following the implantation of silicone gel (Bradley et al., 1994b;
Selwyn and Danner, 1988). High doses of D4 have also been reported to
enhance NK cell activity (Wilson and Munson, 1997), whereas exposure to
silicone gel suppresses NK function (Bradley et al., 1994a,b). Thus,
based on current studies, there is no data to implicate these low
molecular weight silicones in any health effect associated with SBIs.

# ^Silanols^

^The\ toxicity\ of\ silanols\ is\ considered\ not\ relevant\ to\ the\ SBI\ issue.\ Silanols\ are\ highly\ toxic\ chemicals\ with\ a\ profile\ of\ toxicity\ in\ rats\ that\ includes\ liver,\ kidney,\ and\ neural\ damage,\ and\ bleeding\ (Dow\ Report\ No.\ 2964).\ However,\ none\ of\ these\ toxicities\ are\ seen\ in\ rats\ injected\ with\ large\ amounts\ of\ silicone\ fluid,\ gel\ or\ elastomer.^

# ^Platinum^

^Platinum\ (Pt)\ is\ used\ as\ a\ catalyst\ in\ the\ preparation\ of\ silicone\ gels\ and\ elastomers.\ According\ to\ Lykissa\ et\ al.\ (1997),\ Pt\ was\ detectable\ in\ silicone\ gel\ at\ a\ level\ of\ approximately\ 700\ µg/kg\ (parts\ per\ billion)\ using\ ICP-MS.\ Furthermore,\ they\ indicate\ that\ when\ the\ gel\ was\ incubated\ in\ lipid-rich\ media,\ Pt\ diffused\ into\ the\ media\ at\ a\ rate\ of\ approximately\ 20--25\ µg/day/250g\ implant.\ Since\ the\ whole\ implant\ used\ in\ these\ studies\ ^

^would\ only\ contain\ 175\ µg\ of\ Pt,\ it\ suggests\ that\ all\ of\ the\ Pt\ would\ diffuse\ from\ the\ gel\ into\ the\ media\ within\ seven\ days.\ This\ is\ not\ logical.\ There\ are\ no\ data\ available\ that\ address\ the\ level\ of\ Pt\ in\ blood\ or\ tissues\ of\ animals\ or\ humans\ who\ have\ SBIs.\ However,\ if\ a\ worst-case\ exposure\ scenario\ was\ calculated\ based\ on\ the\ value\ published\ by\ Lykissa,\ wherein\ all\ of\ the\ Pt\ in\ two\ 300-ml\ implants\ was\ released\ into\ the\ body\ of\ a\ 50-kg\ woman,\ the\ Pt\ dose\ would\ be\ 8.4\ µg/kg\ or\ 8.4\ ppb.\ This\ concentration\ of\ Pt\ is\ approximately\ equivalent\ to\ the\ five\ ppb\ level\ found\ naturally\ in\ the\ environment.\ Pt\ toxicity\ is\ dependent\ on\ its\ chemical\ form.\ Pt\ salts\ primarily\ cause\ liver\ and\ kidney\ toxicity,\ which\ have\ not\ been\ associated\ with\ SBI\ materials.\ Thus,\ there\ is\ no\ evidence\ that\ Pt\ plays\ any\ role\ in\ the\ health\ effects\ associated\ with\ SBIs.^

# ^XI.\ Conclusions^

^This\ chapter\ has\ reviewed\ the\ experimental\ animal\ data\ to\ evaluate\ the\ evidence\ that\ silicone\ breast\ implants\ have\ the\ potential\ to\ cause\ systemic\ disease\ in\ humans.\ The\ results\ of\ this\ review\ indicate\ that\ the\ silicones\ used\ in\ SBIs\ are\ of\ very\ low\ toxicity\ to\ animals.\ Although\ there\ is\ documented\ evidence\ of\ local\ inflammatory\ reactions\ to\ silicone\ breast\ implant\ materials\ in\ animals,\ there\ is\ no\ convincing\ evidence\ for\ a\ significant\ systemic\ inflammatory\ response.\ The\ local\ reaction\ to\ silicone\ is\ similar\ to\ other\ "foreign\ body\ reactions"\ described\ with\ other\ implanted\ materials.\ ^

^\ There\ is\ some\ evidence\ that\ macrophages\ can\ phagocytose\ small\ droplets\ of\ silicone\ which\ may\ then\ be\ transported\ via\ the\ lymphatics\ to\ other\ tissues\ in\ the\ body.\ This\ process\ appears\ to\ occur\ primarily\ with\ low\ molecular\ weight\ silicone\ fluid\ rather\ than\ the\ high\ molecular\ weight\ gel.\ However,\ even\ with\ phagocytosis,\ there\ is\ currently\ no\ definitive\ evidence\ for\ systemic\ effects\ on\ the\ immune\ system\ or\ for\ processing\ of\ silicone\ as\ an\ antigen\ for\ T\ cell\ activation.\ There\ is\ also\ no\ convincing\ evidence\ from\ animal\ studies\ that\ T\ cells\ can\ be\ activated\ by\ silicone.^

^\ The\ ability\ of\ silicone\ to\ act\ as\ an\ adjuvant\ has\ received\ a\ lot\ of\ attention.\ Even\ though\ some\ silicone\ gels\ and\ fluids\ have\ been\ shown\ to\ possess\ adjuvant\ activity\ when\ antigen\ is\ emulsified\ with\ the\ silicone\ prior\ to\ immunization,\ this\ capability\ has\ little\ bearing\ on\ the\ ^

^issue\ of\ silicone-\ induced\ autoimmune\ disease.\ It\ most\ likely\ reflects\ a\ depot\ effect\ of\ the\ non-degradable\ silicone.\ There\ are\ no\ convincing\ data\ that\ show\ silicone\ acts\ like\ an\ adjuvant\ when\ it\ is\ present\ at\ a\ site\ distant\ from\ the\ antigen\ injection,\ and\ there\ is\ no\ biologically\ plausible\ mechanism\ for\ antigen\ emulsification\ to\ take\ place\ in\ the\ body.\ ^

^\ Immunotoxicity\ testing\ of\ silicones\ revealed\ only\ one\ fairly\ reproducible\ effect\ which\ was\ suppression\ of\ NK\ activity\ in\ both\ rats\ and\ mice.\ The\ degree\ of\ suppression\ was\ variable\ between\ experiments\ and\ not\ of\ sufficient\ magnitude\ to\ affect\ a\ disease\ model\ responsive\ to\ NK\ cell\ activity.\ Although\ these\ results\ are\ of\ interest,\ the\ specific\ role\ that\ NK\ cells\ may\ play\ in\ autoimmune\ disease\ development\ is\ not\ well\ understood.^

^\ \ The\ greatest\ weight\ of\ evidence\ in\ this\ review\ has\ been\ given\ to\ studies\ that\ evaluated\ the\ ability\ of\ silicone\ to\ induce\ or\ promote\ autoimmune\ disease\ in\ whole\ animal\ models.\ Such\ animal\ models\ provide\ the\ most\ holistic\ approach\ to\ identifying\ biologically\ relevant\ effects\ induced\ by\ silicone\ exposure\ that\ might\ lead\ to\ autoimmune\ disease\ induction\ or\ promotion\ in\ humans.\ The\ use\ of\ animals\ that\ are\ genetically\ predisposed\ to\ develop\ autoimmune\ disease\ provide\ the\ advantage\ of\ a\ high\ and\ predictable\ incidence\ of\ spontaneous\ disease.\ If\ an\ alteration\ in\ disease\ induction\ occurs\ with\ silicone\ treatment\ and\ it\ correlates\ with\ changes\ in\ relevant\ clinical\ endpoints,\ the\ evidence\ for\ a\ cause-effect\ relationship\ becomes\ more\ credible.\ If\ clinical\ correlates\ of\ a\ promotional\ effect\ by\ silicone\ can\ be\ identified\ in\ animals,\ it\ would\ provide\ a\ focus\ for\ human\ clinical\ investigations.\ ^

^\ Several\ adequately\ designed\ animal\ studies\ have\ been\ published\ that\ address\ the\ question\ of\ silicone's\ ability\ to\ induce\ or\ exacerbate\ autoimmune\ disease.\ The\ human\ autoimmune\ diseases\ that\ are\ simulated\ in\ these\ animal\ models\ include\ rheumatoid\ arthritis,\ systemic\ lupus\ erythematosus,\ scleroderma,\ and\ autoimmune\ hemolytic\ anemia.\ In\ the\ 17\ experimental\ regimens\ outlined\ in\ Tables\ 1\ and\ 3,\ 15\ indicate\ that\ silicone\ did\ not\ induce\ or\ promote\ the\ development\ of\ autoimmune\ disease\ and/or\ alter\ diagnostic\ clinical\ endpoints.\ The\ other\ two\ experiments\ must\ be\ viewed\ as\ providing\ weak\ but\ suggestive\ preliminary\ evidence\ of\ a\ promotional\ effect\ by\ silicone\ exposure.\ However,\ these\ preliminary\ findings\ must\ be\ confirmed\ in\ independent\ studies.\ Curiously,\ one\ of\ models\ that\ shows\ some\ evidence\ of\ promotion\ by\ silicone\ is\ the\ model\ of\ autoimmune\ hemolytic\ anemia,\ which\ is\ ^

^not\ of\ obvious\ relevance\ to\ the\ SBI\ issue.\ The\ other\ is\ a\ bovine\ collagen-induced\ model\ of\ arthritis\ using\ incomplete\ Freund's\ adjuvant\ to\ lower\ the\ degree\ of\ disease\ in\ the\ controls.\ Limitations\ of\ these\ studies\ relate\ to\ the\ small\ number\ of\ animals\ in\ the\ treatment\ groups\ and\ the\ lack\ of\ clinical\ endpoints\ that\ verify\ the\ exacerbation\ of\ disease.\ ^

^\ On\ the\ other\ hand,\ there\ are\ also\ limitations\ with\ some\ of\ the\ studies\ that\ show\ no\ effect\ of\ silicone\ on\ autoimmune\ disease.\ The\ biggest\ problem\ with\ several\ of\ the\ studies\ is\ that\ disease\ incidence\ is\ so\ high\ in\ the\ controls,\ it\ would\ be\ difficult\ to\ demonstrate\ an\ increase\ in\ disease\ in\ the\ silicone-treated\ mice.\ Although\ a\ promotional\ effect\ in\ such\ cases\ might\ be\ evidenced\ by\ an\ early\ appearance\ of\ the\ disease,\ latency\ was\ not\ an\ endpoint\ that\ was\ documented\ in\ most\ of\ the\ studies.\ The\ second\ limitation\ of\ several\ of\ these\ studies\ is\ the\ lack\ of\ positive\ controls\ that\ demonstrate\ the\ sensitivity\ of\ the\ model\ to\ exogenous\ modulation.\ Without\ a\ positive\ control,\ it\ is\ difficult\ to\ put\ the\ failure\ of\ silicone\ to\ alter\ disease\ in\ a\ relevant\ context.\ Nevertheless,\ the\ data\ from\ these\ studies\ cannot\ be\ ignored\ for\ their\ null\ effects\ on\ the\ disease\ process.\ ^

^\ \ In\ conclusion,\ the\ preponderance\ of\ evidence\ from\ animal\ studies\ indicates\ little\ probability\ that\ silicone\ exposure\ induces\ or\ exacerbates\ systemic\ disease\ in\ humans.\ ^

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# Table 1. Influence of Silicone in Adjuvant Arthritis Disease Models

<table>
<tbody>
<tr class="odd">
<td><p><strong><sup>Model</sup></strong></p></td>
<td><p><strong><sup>Description</sup></strong></p></td>
<td><p><strong><sup>Treatment</sup></strong></p></td>
<td><p><strong><sup>Design</sup></strong></p></td>
<td><p><strong><sup>Significant Results</sup></strong></p></td>
<td><p><strong><sup>Interpretation/conclusions</sup></strong></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>1. Lewis rat model of adjuvant arthritis</sup></p>
<p><sup>Chang (1993)</sup></p></td>
<td><p><sup>Genetically susceptible strain of rat; develops arthritis in
response to CFA injection.</sup></p></td>
<td><p><em><sup>M. tuberculosis</sup></em><sup> was emulsified in
mineral oil or silicone gel that was liquified by
homogenization</sup></p>
<p><sup>Rats were injected subplantar with: </sup></p>
<p><sup>a) saline</sup></p>
<p><sup>b) silicone gel</sup></p>
<p><sup>c) </sup><em><sup>M. tuberculosis</sup></em><sup> in silicone
gel</sup></p>
<p><sup>d)</sup><em><sup> M. tuberculosis</sup></em><sup> in mineral oil
(CFA)</sup></p></td>
<td><p><sup>There were 7 rats/group. </sup></p>
<p><sup>Hind paws were measured on the day of injection and on the 16th
and 23rd day after injection. </sup></p></td>
<td><p><sup>Rats injected with CFA developed arthritis by day
16.</sup></p>
<p><sup>Rats injected with silicone gel with or without mycobacteria did
not develop arthritis.</sup></p></td>
<td><p><sup>This study directly tested the ability of silicone gel to
induce “adjuvant arthritis”. Silicone gel was not
effective.</sup></p></td>
<td><p><sup>2. Lewis rat model of adjuvant arthritis. </sup></p>
<p><sup>Picha and Goldstein (1997)</sup></p></td>
<td><p><sup>Genetically susceptible strain of rat, develops arthritis in
response to CFA injection.</sup></p></td>
<td><p><sup>Rats were injected id into the plantar aspect of the right
foot with:</sup></p>
<p><sup>a) CFA</sup></p>
<p><sup>b) silicone oil + </sup><em><sup>M. tuberculosis</sup></em></p>
<p><sup>c) silica in IFA</sup></p>
<p><sup>d) silicone oil + </sup><em><sup>M. tuberculosis</sup></em><sup>
+ silica + IFA</sup></p></td>
<td><p><sup>There were 15 rats/treatment.</sup></p>
<p><sup>Rats were examined over a 4 month period for development of
arthritis.</sup></p></td>
<td><p><sup>Rats responded to CFA injection with prolonged
arthritis</sup></p>
<p><sup>Rats injected with silicone oil + </sup><em><sup>M.
tuberculosis</sup></em><sup> developed a response at day 8 that declined
by day 15, followed by a second response at 120 days. </sup></p>
<p><sup>Silicone oil injected with silica appeared to decrease the
inflammatory reaction to silica.</sup></p></td>
<td><p><sup>This study directly tested the ability of silicone oil to
induce “adjuvant arthritis”. Silicone oil was ineffective.</sup></p>
<p><sup>The significance of the modified response seen in
silicone-treated rats is difficult to evaluate due to the subjective
description of the data. </sup></p></td>
</tr>
<tr class="odd">
<td><p><sup>3. Dark Agouti (DA) rat adjuvant arthritis model</sup></p>
<p><sup> Naim et al. (1995)</sup></p></td>
<td><p><sup>Genetically susceptible strain of rat, develops arthritis in
response to injection of IFA</sup></p></td>
<td><p><sup>Rats were injected id at base of tail with:</sup></p>
<p><sup> a) silicone oil:gel (1:1)</sup></p>
<p><sup> b) IFA </sup></p></td>
<td><p><sup>There were 10 rats/treatment </sup></p>
<p><sup>The DTH response to collagen was measured on day 18</sup></p>
<p><sup>Rats were killed 89 days after injection</sup></p>
<p><sup>Sera obtained for anti-collagen titers</sup></p></td>
<td><p><sup>IFA induced arthritis in 8/10 rats</sup></p>
<p><sup>Silicone-treated rats did not develop arthritis</sup></p>
<p><sup>No DTH reaction or antibody response to collagen in either
group</sup></p></td>
<td><p><sup>This experiment directly tested the ability of a mixture of
silicone oil and gel to induce “adjuvant arthritis” in a highly
susceptible strain of rat. Silicone oil/gel was
ineffective.</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
</tbody>
</table>

# ^Table\ 2.\ Adjuvant\ Activity\ of\ Silicones^

<table>
<tbody>
<tr class="odd">
<td><p><strong><sup>Model</sup></strong></p></td>
<td><p><strong><sup>Description</sup></strong></p></td>
<td><p><strong><sup>Immunization</sup></strong></p></td>
<td><p><strong><sup>Design</sup></strong></p></td>
<td><p><strong><sup>Significant Results</sup></strong></p></td>
<td><p><strong><sup>Interpretations/Conclusions</sup></strong></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>1. Antibody response to bovine serum albumin (BSA) in
rats</sup></p>
<p><sup>Naim et al. (1993)</sup></p></td>
<td><p><sup>Immune response to a foreign protein (e.g., BSA) is enhanced
when the protein is injected as an emulsified preparation in an
adjuvant</sup></p></td>
<td><p><sup>Sprague-Dawley rats were injected im with 50 ug BSA mixed or
emulsified with:</sup></p>
<p><sup>a) saline</sup></p>
<p><sup>b) silicone oil (20 cs)</sup></p>
<p><sup>c) silicone oil:gel (1:1)</sup></p>
<p><sup>d) CFA</sup></p>
<p><sup>e) IFA</sup></p></td>
<td><p><sup>There were 10 rats/group.</sup></p>
<p><sup>Rats were bled on day 12, 22, 40 and 56 days after immunization.
Sera were tested for anti-BSA antibodies by ELISA.</sup></p></td>
<td><p><sup>High titers of anti-BSA antibodies were found when CFA,
silicone gel, and IFA were used as adjuvant. Antibody titers were low
when silicone oil was used as adjuvant. </sup></p></td>
<td><p><sup>The adjuvanticity of silicone gel was as great as CFA.
Silicone oil possessed only weak adjuvant activity.</sup></p></td>
<td><p><sup>2. Antibody response to BSA in rats</sup></p>
<p><sup>Naim et al. (1995)</sup></p></td>
<td><p><sup>Immune response to a foreign protein (e.g., BSA) is enhanced
when the protein is injected as an emulsified preparation in an
adjuvant</sup></p></td>
<td><p><sup>Sprague-Dawley rats were immunized im with 50 ug BSA mixed
with</sup></p>
<p><sup>a) saline</sup></p>
<p><sup>b) D4</sup></p>
<p><sup>c) silicone oil, 100cs</sup></p>
<p><sup>d) silicone oil, 350 cs</sup></p>
<p><sup>e) silicone oil, 1000 cs</sup></p>
<p><sup>f) silicone oil, 12,500 cs</sup></p>
<p><sup>g) silicone gel in 20 cs oil</sup></p>
<p><sup>h) CFA</sup></p>
<p><sup>A booster injection was given on day 71 in same adjuvant except
CFA group was injected with IFA</sup></p></td>
<td><p><sup>There were 8 rats/group.</sup></p>
<p><sup>Rats were bled at 14, 29, 49, 71, 79 and 98 days after
immunization. Sera were tested for anti-BSA antibodies by
ELISA.</sup></p></td>
<td><p><sup>Antibody titers to BSA were detectable in all rats following
immunization. </sup></p>
<p><sup>All silicones were much poorer adjuvants than CFA.
</sup></p></td>
<td><p><sup>Silicone oils and gel demonstrated weak adjuvant activity
for antibody responses to BSA. Silicone gel was a better adjuvant than
the oils. There was a trend toward increased antibody titers with higher
molecular weight oils. </sup></p></td>
</tr>
<tr class="odd">
<td><p><sup>3. Antibody response to BSA in rats</sup></p>
<p><sup>Naim et al. (1995)</sup></p></td>
<td><p><sup>Immune response to a foreign protein (e.g., BSA) is enhanced
when the protein is injected as an emulsified preparation in an
adjuvant</sup></p></td>
<td><p><sup>BSA was admixed with homogenized silicone gel in 3 separate
preparations that were subjected to varying applied shear
force.</sup></p></td>
<td><p><sup>Rats were bled periodically. Sera were tested for anti-BSA
antibodies by ELISA</sup></p></td>
<td><p><sup>Low titers of anti-BSA antibodies were found in all rats
immunized with BSA in silicone gel. There was no difference in titers
between the treatment groups.</sup></p></td>
<td><p><sup>The homogenization process did not influence the adjuvancy
of silicone gel. </sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>Model</sup></p></td>
<td><p><sup>Description</sup></p></td>
<td><p><sup>Immunization</sup></p></td>
<td><p><sup>Design</sup></p></td>
<td><p><sup>Significant Results</sup></p></td>
<td><p><sup>Interpretations/Conclusions</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><p><sup>4. Antibody response to BSA in rats and mice.</sup></p>
<p><sup>Klykken and White (1996)</sup></p></td>
<td><p><sup>Immune response to a foreign protein (e.g., BSA) is enhanced
when the protein is injected as an emulsified preparation in an
adjuvant</sup></p></td>
<td><p><sup>Sprague Dawley rats or B6C3F1 mice were injected im with BSA
emulsified in </sup></p>
<p><sup>a) saline</sup></p>
<p><sup>b) CFA</sup></p>
<p><sup>c) silicone gel bleed</sup></p>
<p><sup>d) silicone gel</sup></p>
<p><sup>e) D4</sup></p></td>
<td><p><sup>The number of animals in each group was not
indicated</sup></p>
<p><sup>Serum samples were obtained from rats at 2,4,6 and 8 weeks
post-immunization. Serum samples were obtained from mice at 4, 7 and 12
weeks after immunization. </sup></p></td>
<td><p><sup>Administration of BSA in CFA, gel or D4 resulted in an
enhanced antibody response in both rats and mice. Gel bleed tested in
rats did not boost the antibody response above the saline control.
</sup></p></td>
<td><p><sup>Silicone gel and D4 but not gel bleed were effective
adjuvants to increase the antibody response to BSA</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>5. Anti-ovalbumin (OVA) antibody production in
rats.</sup></p>
<p><sup>Naim et al. (1997)</sup></p></td>
<td><p><sup>Immune response to a foreign protein (e.g. OVA) is enhanced
when the protein is injected as an emulsified preparation in an
adjuvant</sup></p></td>
<td><p><sup>OVA was emulsified in </sup></p>
<p><sup>a) CFA</sup></p>
<p><sup>b) Dow Corning gel</sup></p>
<p><sup> lot # HH019581</sup></p>
<p><sup>c) McGhan gel</sup></p>
<p><sup> lot # DP9339</sup></p>
<p><sup>d) McGhan gel </sup></p>
<p><sup> lot # S0400488</sup></p>
<p><sup>Each rat was injected id at base of tail. A booster injection
was given after 48 days in same adjuvant except CFA group was injected
with IFA.</sup></p>
<p><sup>On day 14, rats were challenged in the right ear with OVA and in
left ear with saline.</sup></p></td>
<td><p><sup>There were 4-6 rats/group. </sup></p>
<p><sup>Serum samples were taken at 21, 48, 62 and 84 days after
immunization for measurement of anti-OVA titers. </sup></p>
<p><sup>Delayed-type hypersensitivity (DTH) response to OVA was measured
on day 14 by ear swelling test. </sup></p></td>
<td><p><sup>Antibody and DTH responses to OVA were observed in all rats
except those in group c. </sup></p>
<p><sup>The antibody and DTH responses with silicone as adjuvant were
lower than the response with CFA. </sup></p></td>
<td><p><sup>Different types of silicone gel were compared for their
adjuvant activity. Two of three silicone gels had adjuvant activity in
terms of boosting the antibody response to OVA.</sup></p>
<p><sup>The DTH response did not follow classic kinetics.
</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><p><sup>Model</sup></p></td>
<td><p><sup>Description</sup></p></td>
<td><p><sup>Immunization</sup></p></td>
<td><p><sup>Design</sup></p></td>
<td><p><sup>Significant Results</sup></p></td>
<td><p><sup>Interpretations/Conclusions</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>6. Immune response to keyhole limpet hemocyanin (KLH) in
mice</sup></p>
<p><sup>(Abstract only available)</sup></p>
<p><sup>Gish et al. (1997) </sup></p></td>
<td><p><sup>Immune response to a foreign protein (eg. KLH) is enhanced
when the protein is injected as an emulsified preparation in an
adjuvant</sup></p></td>
<td><p><sup>KLH was emulsified in</sup></p>
<p><sup>a) saline</sup></p>
<p><sup>b) CFA</sup></p>
<p><sup>c) D4</sup></p>
<p><sup>and injected id in the footpad of mice</sup></p></td>
<td><p><sup>There were 3 mice/group.</sup></p>
<p><sup>Footpad swelling was measured at 96 hr</sup></p>
<p><sup>Mice were killed after 1 week and lymph node cells were cultured
with KLH to evaluate proliferation response and cytokine
production</sup></p>
<p><sup>Antibody production was measured after 21 days</sup></p></td>
<td><p><sup>Footpad swelling occurred in mice injected with CFA or D4,
independent of KLH. Swelling with D4 was greater than with
CFA.</sup></p>
<p><sup>D4 was as effective as CFA in inducing immunity to KLH as
measured by lymph node proliferation, IL-2, IL-4 and antibody
production</sup></p></td>
<td><p><sup>The study evaluated the ability of D4, a low molecular
weight cyclosiloxane detected in silicone gel, to act as an adjuvant.
</sup></p>
<p><sup>D4 was as effective as CFA in inducing immunity to
KLH</sup></p></td>
<td><p><sup>7. Antibody response to BSA in rats. </sup></p>
<p><sup>Hill et al. (1996)</sup></p></td>
<td><p><sup>Immune response to a foreign protein (eg. BSA) is enhanced
when the protein is injected as an emulsified preparation in an
adjuvant</sup></p></td>
<td><p><sup>Sprague-Dawley rats were immunized with BSA emulsified
in:</sup></p>
<p><sup>a) IFA</sup></p>
<p><sup>b) silicone oil</sup></p>
<p><sup>c) IFA/silicone oil</sup></p>
<p><sup>d) silicone oil:gel (1:1)</sup></p>
<p><sup>e) silcone oil + 1000 µ elastomer particles</sup></p>
<p><sup>f) silcone oil + 500 µ elastomer particles</sup></p></td>
<td><p><sup>There were 10 rats/group</sup></p>
<p><sup> Blood samples were obtained on days 0, 14, 28, 42, and
55</sup></p>
<p><sup>for measuring anti-BSA IgG levels</sup></p></td>
<td><p><sup>Antibody titers to BSA were enhanced groups a, c, and d
indicating that silicone gel mixed with silicone oil is an effective
adjuvant but silicone oil alone or mixed with elastomer particles is
not. </sup></p></td>
<td><p><sup>This study directly tested the ability of different forms of
silicone to act as an adjuvant in the antibody response to BSA.
</sup></p>
<p><sup>Silicone gel but not silicone oil or silicone elastomer
particles was an effective adjuvant.</sup></p></td>
</tr>
<tr class="odd">
<td><p><sup>8. Immune response to EL</sup><sub><sup>4</sup></sub><sup>
tumor cells in rats </sup></p>
<p><sup>Chang (1993)</sup></p></td>
<td><p><sup>The antibody and cell-mediated immune response of Lewis rats
to mouse EL</sup><sub><sup>4</sup></sub><sup> tumor cells is greatly
enhanced when the cells are mixed with CFA</sup></p></td>
<td><p><sup>Rats were immunized ip with
EL</sup><sub><sup>4</sup></sub><sup> tumor cells in:</sup></p>
<p><sup>a) saline</sup></p>
<p><sup>b) mineral oil</sup></p>
<p><sup>c) CFA</sup></p>
<p><sup>d) silicone + </sup><em><sup>M. tuberculosis</sup></em></p></td>
<td><p><sup>There were 7 rats/treatment group.</sup></p>
<p><sup>Rats were killed 16 days after immunization and spleen cells
were tested for cytotoxic activity to
EL</sup><sub><sup>4</sup></sub><sup> cells.</sup></p>
<p><sup>Rat serum was tested for antibodies to
EL</sup><sub><sup>4</sup></sub><sup> cells. (The paper failed to state
when sera were collected.)</sup></p></td>
<td><p><sup>Rats that were injected with
EL</sup><sub><sup>4</sup></sub><sup> cells in saline developed a low
level of cell-mediated cytotoxicity which was boosted to a high level by
FCA. The response was not boosted by mineral oil alone or by silicone +
</sup><em><sup>M. tuberculosis</sup></em></p>
<p><sup>The antibody response was boosted by CFA but by silicone +
</sup><em><sup>M. tuberculosis</sup></em><sup> (mineral oil alone was
not tested)</sup></p></td>
<td><p><sup>Silicone gel failed to act as an adjuvant in a novel model
of immunity </sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>Model</sup></p></td>
<td><p><sup>Description</sup></p></td>
<td><p><sup>Immunization</sup></p></td>
<td><p><sup>Design</sup></p></td>
<td><p><sup>Significant Results</sup></p></td>
<td><p><sup>Interpretations/Conclusions</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><p><sup>9. Antibody response to BSA in mice.</sup></p>
<p><sup>Nicholson et al. (1996)</sup></p></td>
<td><p><sup>Immune response to a foreign protein (eg., BSA) is enhanced
when the protein is injected as an emulsified preparation in an
adjuvant</sup></p></td>
<td><p><sup>A/J mice were immunized with BSA emulsified in</sup></p>
<p><sup>a) saline</sup></p>
<p><sup>b) IFA</sup></p>
<p><sup>c) silicone oil</sup></p>
<p><sup>d) IFA/silicone oil</sup></p>
<p><sup>e) 1:1 silicone gel:oil</sup></p>
<p><sup>f) D4</sup></p>
<p><sup>g) IFA/D4</sup></p></td>
<td><p><sup>There were 10 mice/group</sup></p>
<p><sup>The mice were bled on days 0, 15, 30, 45, 60, 75 and 90
following immunization.</sup></p>
<p><sup>Serum was tested for IgG antibody to BSA </sup></p></td>
<td><p><sup>Enhanced antibody production compared to the saline group
was observed with all treatments except silicone oil alone.
</sup></p></td>
<td><p><sup>D4 and silicone gel but not silicone oil were effective
adjuvants for the antibody response to BSA in mice.</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>10. Collagen-induced arthritis in DBA/1 mice</sup></p>
<p><sup>Schaefer (1997) </sup></p></td>
<td><p><sup>A genetically susceptible strain of mouse that develops
progressive, inflammatory arthritis in response to immunization with
bovine collagen.</sup></p></td>
<td><p><sup>Mice were immunized with: </sup></p>
<p><sup>a) collagen emulsified in CFA</sup></p>
<p><sup>b) collagen emulsified in silicone oil+</sup><em><sup> M.
tuberculosis</sup></em><sup>)</sup></p>
<p><sup>c) silicone oil + </sup><em><sup>M.</sup></em></p>
<p><em><sup> tuberculosis</sup></em></p></td>
<td><p><sup>There were 18 mice/group for the silicone treatments and 10
mice for the CFA treatment. </sup></p>
<p><sup>Mice were observed for signs of arthritis for 10 weeks after
immunization; joints measured 3x/week.</sup></p>
<p><sup>Sera obtained for measurement of anti-collagen
antibodies</sup></p></td>
<td><p><sup>The incidence of arthritis was 80% in mice immunized with
collagen in CFA. Arthritis did not develop in any mice injected with
silicone oil as adjuvant. </sup></p>
<p><sup>High titers of collagen antibodies developed in mice immunized
with collagen in CFA. Mice immunized with silicone oil as adjuvant did
not develop antibodies to bovine collagen</sup></p></td>
<td><p><sup>This experiment was designed to test the adjuvanticity of
silicone using disease as an endpoint as well as antibody titers. It
does not address the potential of silicone to induce or exacerbate
autoimmune disease.</sup></p>
<p><sup>Silicone oil containing mycobacteria was unable to act as an
adjuvant for collagen in the induction of arthritis. </sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><p><sup>Model</sup></p></td>
<td><p><sup>Description</sup></p></td>
<td><p><sup>Immunization</sup></p></td>
<td><p><sup>Design</sup></p></td>
<td><p><sup>Significant Results</sup></p></td>
<td><p><sup>Interpretations/Conclusions</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>11. Collagen-induced arthritis in Dark Agouti (DA) rat
</sup></p>
<p><sup>Naim et al. (1995) </sup></p></td>
<td><p><sup>DA rats are a genetically susceptible strain that develops
arthritis in response to immunization with bovine
collagen.</sup></p></td>
<td><p><sup>Rats were injected id at the base of the tail with <u>six
</u><u>µ</u><u>g</u> bovine collagen emulsified in </sup></p>
<p><sup>a) saline</sup></p>
<p><sup>b) silicone oil:gel (1:1)</sup></p>
<p><sup>c) IFA</sup></p>
<p><sup>Groups a and b received a booster injection of the emulsified
collagen on day 45</sup></p></td>
<td><p><sup>There were 10 rats/group</sup></p>
<p><sup>Rats were observed periodically for signs of arthritis and
scored for severity of symptoms. </sup></p>
<p><sup>All rats were killed on day 89. </sup></p>
<p><sup>Serum samples were obtained on days 21, 59 and 89 after
immunization for anti-collagen titers </sup></p></td>
<td><p><sup>The incidence of arthritis was:</sup></p>
<p><sup>a) saline 0/10</sup></p>
<p><sup>b) silicone oil:gel 4/10</sup></p>
<p><sup>c) IFA 8/9</sup></p>
<p><sup>Disease severity was lower when silicone was used as adjuvant
compared to IFA</sup></p>
<p><sup>Very low antibody titers were found in both silicone- and IFA-
injected mice. They could not be directly compared because of the
booster injection given to group b but not c. </sup></p></td>
<td><p><sup>This experiment was designed to test the ability of silicone
to act as an adjuvant when emulsified with a foreign protein (bovine
collagen) using disease and antibody titers as endpoints in a highly
susceptible strain of rat. However, it does not directly address the
potential of silicone to induce or exacerbate spontaneous autoimmune
disease.</sup></p>
<p><sup>Silicone gel was an effective adjuvant for collagen-induced
arthritis</sup></p></td>
<td><p><sup>12. Collagen-induced arthritis in DA rat using high dose of
collagen</sup></p>
<p><sup>Naim et al. (1995) </sup></p></td>
<td><p><sup>DA rats are a genetically susceptible strain that develops
arthritis in response to immunization with bovine
collagen.</sup></p></td>
<td><p><sup>Rats were injected id at the base of the tail with <u>125
ug</u> bovine collagen emulsified in: </sup></p>
<p><sup>a) saline</sup></p>
<p><sup>b) silicone gel </sup></p>
<p><sup>c) IFA </sup></p>
<p><sup>d) silicone oil </sup></p>
<p><sup>e) D4 </sup></p>
<p><sup>f) 1% D4 in silicone oil </sup></p></td>
<td><p><sup>There were 10 rats/group</sup></p>
<p><sup>Rats were observed periodically for signs of arthritis and
scored for severity of symptoms.</sup></p>
<p><sup>Serum samples were obtained on days 21, 59 and 89 after
immunization for anti-collagen titers </sup></p>
<p><sup>All rats were killed on day 69 </sup></p></td>
<td><p><sup>The incidence of arthritis was:</sup></p>
<p><sup> a) saline 0/10</sup></p>
<p><sup> b) silicone gel 7/10</sup></p>
<p><sup> c) IFA 10/10</sup></p>
<p><sup> d) silicone oil 3/10</sup></p>
<p><sup> e) D4 0/10</sup></p>
<p><sup> f) D4 in silicone oil 1/10</sup></p>
<p><sup>The severity of arthritis was significantly greater with IFA
compared to any other group.</sup></p>
<p><sup>Only IFA was an effective adjuvant for inducing high titer
anti-collagen antibodies. Antibody production was lower and delayed with
silicone gel as adjuvant. No antibody was produced with D4 as
adjuvant.</sup></p></td>
<td><p><sup>This experiment was designed to test the ability of silicone
to act as an adjuvant when emulsified with a foreign protein (bovine
collagen) using disease and antibody titers as endpoints in a highly
susceptible strain of rat. However, it does not directly address the
potential of silicone to induce or exacerbate spontaneous autoimmune
disease. </sup></p>
<p><sup>Silicone gel and to a lesser extent silicone oil but not D4 was
an effective adjuvant for collagen-induced arthritis and production of
anti-collagen antibodies.</sup></p></td>
</tr>
<tr class="odd">
<td><p><sup>13. Experimental autoimmune thyroiditis (EAT) in Wistar
rats</sup></p>
<p><sup>Naim et al. (1993)</sup></p></td>
<td><p><sup>Thyroiditis is induced in several species of animals by the
injection of thyroglobulin (Tg) emulsified in CFA. The induction of
disease in the rat is rapid and predictable. </sup></p></td>
<td><p><sup>Rats were injected once id at base of tail with 2 mgTg
emulsified in </sup></p>
<p><sup>a) saline</sup></p>
<p><sup>b) CFA</sup></p>
<p><sup>c) silicone oil:gel (1:1)</sup></p></td>
<td><p><sup>There were 6-7 rats/group.</sup></p>
<p><sup>Rats were bled on day 15 and 28 after immunization for
measurement of anti-Tg antibodies</sup></p>
<p><sup>Rats were killed on day 28 and thyroids were processed for
histological exam. </sup></p></td>
<td><p><sup>The incidence of thyroiditis was:</sup></p>
<p><sup> a) saline 0/6</sup></p>
<p><sup> b) CFA 7/7</sup></p>
<p><sup> c) silicone oil:gel 0/7</sup></p>
<p><sup>All rats immunized with Tg in CFA developed high antibody titers
to Tg. Three of 7 rats injected with Tg in silicone produced low titers
of anti-Tg antibodies</sup></p></td>
<td><p><sup>This experiment was designed to test the adjuvanticity of
silicone using disease and antibody production as endpoints. It does not
address the potential of silicone to induce or exacerbate spontaneous
autoimmune disease.</sup></p>
<p><sup>Silicone gel was a weak adjuvant for induction of anti-Tg
antibodies but not for induction of disease.</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
</tbody>
</table>

**^Table\ 3.\ Influence\ of\ silicone\ injections/implants\ on\ spontaneous\ or\ induced\ autoimmune\ diseases^**

<table>
<tbody>
<tr class="odd">
<td><p><strong><sup>Model</sup></strong></p></td>
<td><p><strong><sup>Description</sup></strong></p></td>
<td><p><strong><sup>Treatment</sup></strong></p></td>
<td><p><strong><sup>Design</sup></strong></p></td>
<td><p><strong><sup>Significant Results</sup></strong></p></td>
<td><p><strong><sup>Interpretation/Conclusions</sup></strong></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>1. DBA/1 mouse model of arthritis</sup></p>
<p><sup>Schaefer (1997) Schaefer et al. (1997)</sup></p></td>
<td><p><sup>An arthritis-prone strain; spontaneously develops arthritis
at low incidence. </sup></p>
<p><sup>Increased incidence of arthritis with mineral oil (pristane)
injection.</sup></p></td>
<td><p><sup>Mice were injected ip with:</sup></p>
<p><sup>silicone elastomer (0.1 mg segment)</sup></p>
<p><sup>silicone gel (0.1 ml)</sup></p>
<p><sup>sham injection</sup></p>
<p><sup>Some of the mice were injected with CFA at the base of tail
</sup><em><sup>3 days</sup></em><sup> after implant surgery because
these mice served as controls for collagen-immunized mice reported in
entry 12, this table)</sup></p></td>
<td><p><sup>There were 10 mice/treatment </sup></p>
<p><sup>Mice were observed for signs of arthritis for 12 weeks; joints
were measured.</sup></p>
<p><sup>Sera were obtained:</sup></p>
<p><sup>a) after 28 days,</sup></p>
<p><sup>b) at onset of arthritis (when applicable)</sup></p>
<p><sup>c) at termination, for measurement of anti-collagen antibodies,
cytokines, and “silicone- binding antibodies”</sup></p></td>
<td><p><sup>No arthritis observed in any treatment group</sup></p>
<p><sup>No anti-collagen antibodies observed in any group </sup></p>
<p><sup>No differences in cytokine levels in any group</sup></p></td>
<td><p><sup>This experiment directly tested the ability of silicone to
induce arthritic disease.</sup></p>
<p><sup>The absence of a positive control group (e.g., ip pristane)
limits interpretation of the negative data. CFA injected id did not
induce arthritis in this mouse model (in contrast to the DA rat model)
</sup></p>
<p><sup>The data on “silicone binding antibodies” and “silicone-bound
proteins” were not convincing of anything more than nonspecific protein
binding.</sup></p></td>
<td><p><sup>2. DBA/1 mouse model of arthritis</sup></p>
<p><sup>Schaefer (1997)</sup></p></td>
<td><p><sup>An arthritis-prone strain; spontaneously develops arthritis
at low incidence. </sup></p>
<p><sup>Increased incidence of arthritis with mineral oil (pristane)
injection.</sup></p></td>
<td><p><sup>Mice were injected ip with:</sup></p>
<p><sup> silicone oil</sup></p>
<p><sup> silicone gel</sup></p>
<p><sup> silicone elastomer</sup></p>
<p><sup> sham injection</sup></p>
<p><sup>(Some of the mice in each treatment group were injected with CFA
at base of tail</sup><em><sup> 9 months</sup></em><sup> after implant
surgery because these mice served as controls for collagen-immunized
mice reported in entry 13, this table)</sup></p></td>
<td><p><sup>There were 18-20 mice/ treatment Implants were in place for
12 months prior to termination. </sup></p>
<p><sup>(Many of the specific details of this study were not included in
the chapter of the thesis from which these data were obtained. It was
assumed that the same experi-mental methods as described in entry 1,
this table, were used.) </sup></p></td>
<td><p><sup>No arthritis observed in any treatment group (data not
shown)</sup></p>
<p><sup>No anti-collagen antibodies were observed in any group.
</sup></p>
<p><sup>Several changes in cytokine levels were noted in different
groups of silicone-treated mice but effects were not consistent between
mice that were injected with CFA and those that were not.</sup></p></td>
<td><p><sup>This experiment directly tested the ability of silicone to
induce arthritic disease. The absence of a positive control group (e.g.,
ip pristane) limits interpretation of the negative data. </sup></p>
<p><sup>The significance of altered cytokines is not known. </sup></p>
<p><sup>Silicone implantation for as long as 12 months did not induce
arthritis in this susceptible strain of mouse.</sup></p></td>
</tr>
<tr class="odd">
<td><p><sup>Model</sup></p></td>
<td><p><sup>Description</sup></p></td>
<td><p><sup>Treatment</sup></p></td>
<td><p><sup>Design</sup></p></td>
<td><p><sup>Significant results</sup></p></td>
<td><p><sup>Interpretation/conclusions</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p>3. Balb/cAnPt mouse model of arthritis</p>
<p>MacDonald et al. (1998)</p></td>
<td><p>This strain of mouse develops plasmacytomas and arthritis from ip
pristane; otherwise not considered autoimmune-prone</p></td>
<td><p>Mice were injected sc with:</p>
<p>0.2 ml saline</p>
<p>0.2 ml silicone gel</p>
<p>0.5 ml pristane</p>
<p>0.2 ml silicone gel + capsulotomy</p>
<p><em>Staph. epidermidis</em></p>
<p>0.2 ml gel +<em> Staph</em></p>
<p>0.2 ml saline 3X</p>
<p>0.2 ml silicone gel 3X</p>
<p>0.5 ml pristane 3X</p></td>
<td><p>There were 10 mice/group.</p>
<p>Survival was recorded daily for 10 mos.</p>
<p>Urinary protein was measured biweekly.</p>
<p>At termination:</p>
<p>hematocrit, anti-RBC titers, anti-nuclear antibody titers; anti-Type
I collagen titers</p></td>
<td><p>None of the treatment groups developed signs of arthritis.</p>
<p>Survival was 100% in all groups except for group injected 3x with
silicone in which survival was 80%. The cause of death was not
stated.</p>
<p>There were no effects of any treatment on hematocrit values or
urinary protein levels, and no antibodies to RBC were found.</p>
<p>Pristane 3x induced ANA in 5/5 mice at a titer of 96. No other group
had more than 1out of 5 animals with a low ANA titer.</p>
<p>Anti- collagen titers were increased in mice that were injected 3x
with pristane or 3x with silicone.</p></td>
<td><p>This experiment directly addressed the ability of silicone to
induce autoimmune disease in a mouse model that is not considered
autoimmune prone.</p>
<p>No evidence of autoimmune disease was found with any treatment.</p>
<p>The presence of a bacterial infection or multiple injections of
silicone did not induce disease.</p>
<p>The lack of response in the positive control group (pristane) limits
interpretation of the negative data.</p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><p><sup>4. BALB/cAnPt-A mouse model of arthritis</sup></p>
<p><sup>Potter et al. (1994) </sup></p></td>
<td><p><sup>BALB/cAnPt mice injected ip with pristane frequently develop
arthritis (Potter and Wax, 1981) </sup></p></td>
<td><p><sup>Mice received multiple ip injections of different silicone
gels, silicone oil, corn oil or pristane for the primary purpose of
assessing plasmacytoma induction. </sup></p></td>
<td><p><sup>The mice were examined for plasmacytoma development over a
period of 125-400 days. </sup></p></td>
<td><p><sup>The authors noted that “ silicone- treated mice did not
develop arthritis frequently found in pristane-treated mice”. The
frequency of arthritis in the pristane-treated mice was not reported.
</sup></p></td>
<td><p><sup>These studies indirectly provided evidence regarding the
inability of various silicone gels to induce arthritis in this
genetically susceptible mouse strain.</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>Model</sup></p></td>
<td><p><sup>Description</sup></p></td>
<td><p><sup>Treatment</sup></p></td>
<td><p><sup>Design</sup></p></td>
<td><p><sup>Significant results</sup></p></td>
<td><p><sup>Interpretation/conclusions</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><p>5. MRL <sup>lpr/lpr</sup> mouse model of SLE</p>
<p>Schaefer (1997)</p></td>
<td><p>By 8 weeks of age,</p>
<p>MRL<sup>lpr/lpr</sup> mice spontaneously develop lymphadenopathy,
arthritis, proteinuria and glomerulonephritis.</p>
<p>Death is at 16-24 weeks due to renal failure.</p></td>
<td><p>At 5 weeks of age, mice received sc implants of</p>
<p>a) sham</p>
<p>b) silicone gel</p>
<p>c) silicone oil</p></td>
<td><p>There were 6 mice/treatment.</p>
<p>Mice were killed 12 weeks after implant surgery.</p>
<p>Disease was assessed by palpation of cervical lymph nodes, urinary
protein levels, and immune complex deposition in kidney.</p>
<p>At termination, serum was tested for antibodies to collagen and DNA,
rheumatoid factor, and total Ig. IL-1, IL-2, IL-4, TNF-" levels in serum
were measured at 3 time points.</p></td>
<td><p>After 15 weeks of age, disease severity was similar in all three
groups.</p>
<p>Anti-collagen antibodies were similar in all groups.</p>
<p>Anti-DNA antibodies were higher in silicone gel implanted mice.</p>
<p>IL-2 levels in serum were elevated in mice with silicone oil
implants.</p></td>
<td><p>This model directly tested the ability of silicone gel and oil to
modify genetically-determined autoimmune disease. However, because of
the severity of the disease in control mice, it may be difficult to
detect exacerbation in this model. The authors did not report
time-to-onset data.</p>
<p>The significance of anti-DNA antibodies and IL-2 to disease in this
model are not known.</p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>6. MRL+/+ mouse model of SLE</sup></p>
<p><sup>Schaefer (1997)</sup></p></td>
<td><p><sup>MRL+/+ mice spontaneously develop mild autoimmune
glomerulonephritis late in life. </sup></p></td>
<td><p><sup>At 5 weeks of age, mice received sc implants of </sup></p>
<p><sup> a) sham</sup></p>
<p><sup> b) silicone gel</sup></p>
<p><sup> c) silicone oil</sup></p></td>
<td><p><sup>There were 6 mice/treatment. </sup></p>
<p><sup>Mice were killed 12 weeks after implantation. </sup></p>
<p><sup>Disease was assessed by palpation of cervical lymph nodes,
urinary protein levels, immune complex deposition in kidney.</sup></p>
<p><sup>At termination, serum was tested for antibodies to collagen and
DNA, rheumatoid factor, and total Ig. IL-1, IL-2, IL-4, TNF-" levels in
serum were measured at 3 time points.</sup></p></td>
<td><p><sup>Lymphadenopathy was not detected in any group.</sup></p>
<p><sup>Immune complex deposition in glomeruli was minimal in all
groups.</sup></p>
<p><sup>Antibodies to DNA were slightly higher in silicone gel and oil
implanted mice.</sup></p></td>
<td><p><sup>Because of the minimal disease that develops in control
MRL+/+ mice, this model directly tested the ability of silicone to
exacerbate genetically-determined autoimmune disease. Silicone failed to
induce disease. </sup></p>
<p><sup>The significance of the anti-DNA antibodies to disease in this
model are not known.</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><p><sup>Model</sup></p></td>
<td><p><sup>Description</sup></p></td>
<td><p><sup>Treatment</sup></p></td>
<td><p><sup>Design</sup></p></td>
<td><p><sup>Significant results</sup></p></td>
<td><p><sup>Interpretation/conclusions</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>7. C57Bl/6lpr/lpr</sup></p>
<p><sup>model of SLE</sup></p>
<p><sup>Osborn et al. (1995) (abstract)</sup></p></td>
<td><p><sup>C57Bl/6 lpr/lpr mice spontaneously develop autoimmune
disease characterized by lymphadenopathy, antinuclear antibodies and
early mortality</sup></p></td>
<td><p><sup>At 6 weeks of age, mice were injected sc with:</sup></p>
<p><sup>a) silicone oil containing 5% D4</sup></p>
<p><sup>b) saline</sup></p></td>
<td><p><sup>There were 20 mice/group. </sup></p>
<p><sup>Animals were monitored at 0, 1, 3, 6, and 12 mos. for
antinuclear antibodies, rheumatoid factor, lymph node enlargement and
death.</sup></p></td>
<td><p><sup>At 48 weeks, mortality was: silicone 10/20 </sup></p>
<p><sup>saline 11/20 </sup></p>
<p><sup>The frequency and latency of other disease symptoms did not
differ between the groups. </sup></p></td>
<td><p><sup>This model directly tested the ability of silicone to
exacerbate genetically-determined autoimmune disease. Silicone failed to
alter disease.</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><p><sup>8. TSK/+ mouse model of scleroderma</sup></p>
<p><sup>Frondoza et al. (1996)</sup></p></td>
<td><p><sup>Tight skin (TSK/+) mice spontaneously develop skin fibrosis
and characteristic auto-antibodies which resemble human
scleroderma</sup></p></td>
<td><p><sup>1 month old TSK/+ mice or their normal litter-mates were
injected sc with:</sup></p>
<p><sup>a) silicone oil</sup></p>
<p><sup>b) silicone gel</sup></p>
<p><sup>c) IFA</sup></p>
<p><sup>d) saline</sup></p></td>
<td><p><sup>There were 5-6 mice/group</sup></p>
<p><sup>Mice were bled on day 0 and day 30 after implant surgery for
measurement of antibodies to BSA, RNA polymerase protein, and
topoisomerase.</sup></p>
<p><sup>Samples of skin, kidney and liver were examined
histologically.</sup></p></td>
<td><p><sup>There were no significant differences in skin histology or
antibody titers in the silicone injected mice compared to those that
received saline or IFA.</sup></p></td>
<td><p><sup>This study directly assessed the ability of silicone to
modify the development of a spontaneous autoimmune disease. </sup></p>
<p><sup>Silicone oil or gel injected sc did not alter the progression of
disease in the TSK mouse model of scleroderma. The positive control
group (IFA-treated mice) also failed to alter disease progression.
</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>9. NZB/W F1 model of SLE </sup></p>
<p><sup>White et al. (1997)</sup></p></td>
<td><p><sup>The NZB/W &amp; F1 mouse develops a gradual systemic
autoimmune disease with characteristics including elevated titers of
antinuclear antibodies and serum IgG, polyclonal B cell activation, and
ultimately a fatal immune-complex mediated glomerulo-nephritis.
</sup></p></td>
<td><p><sup>Mice, 7-8 weeks of age, were implanted sc with 1, 2 or 3 ml
of silicone gel for 78 days. Saline (3 ml) was injected in control mice.
</sup></p>
<p><sup>Two positive control groups of mice were also evaluated in
separate studies: 1 mg/kg HgCl</sup><sub><sup>2</sup></sub><sup>
injected sc 3x/week for 2 wks or 450 mg/kg d-penicillamine orally for 28
days </sup></p></td>
<td><p><sup>All mice were bled on day 79 following implantation of
silicone gel. </sup></p>
<p><sup> It was not stated when the positive control mice were sampled.
</sup></p></td>
<td><p><sup>Exposure to silicone gel did not alter serum levels of IgG,
or levels of antibodies to dsDNA, laminin , DNP-HSA , or SRBC. Spleen
weight was not increased. </sup></p>
<p><sup>All of these endpoints were increased in mice treated with
HgCl</sup><sub><sup>2</sup></sub><sup> or d-penicillamine.</sup></p>
<p><sup>Proteinuria was noted in HgCl</sup><sub><sup>2</sup></sub><sup>
and d-penicillamine treated mice but not in silicone-treated mice(data
was not shown)</sup></p></td>
<td><p><sup>This study did not measure autoimmune disease; only clinical
endpoints that correlate with disease progression. </sup></p>
<p><sup>The data from the positive controls demonstrate that clinical
measurements of disease can be influenced by exposure to chemicals that
are known to induce autoimmune disease. How-ever, these data were
obtained in independent studies and are thus not directly comparableto
the silicone-treated mice. The responses of the sham controls differed
between the studies. </sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><p><sup>10. TSK/+ mouse model of scleroderma</sup></p>
<p><sup>Osborn et al. (1995)</sup></p></td>
<td><p><sup>Tight skin (TSK/+) mice spontaneously develop skin fibrosis
and characteristic auto-antibodies which resemble human
scleroderma</sup></p></td>
<td><p><sup>3 week-old TSK mice were injected sc with:</sup></p>
<p><sup>a) saline</sup></p>
<p><sup>b) silicone + 5% D4 </sup></p></td>
<td><p><sup>There were 12 mice/group </sup></p>
<p><sup>Mice were sacrificed at 1, 6, and 12 months. Skin thickness of 6
mm biopsies and serum levels of antinuclear antibodies were measured.
</sup></p></td>
<td><p><sup>There were no significant differences in skin thickness
measurements or antinuclear antibody titers between saline-treated and
silicone-injected mice. </sup></p></td>
<td><p><sup>This study directly assessed the ability of silicone to
modify the development of a spontaneous autoimmune disease. </sup></p>
<p><sup>Silicone gel containing 5% D4 when injected sc did not alter the
progression of disease in the TSK mouse model of scleroderma. There was
no positive control group.</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>11. NZB mouse model of autoimmune hemolytic anemia</sup></p>
<p><sup> MacDonald et al. (1998) </sup></p></td>
<td><p><sup>NZB mice</sup></p>
<p><sup>spontaneously develop autoantibodies and autoimmune hemolytic
anemia. Death is due to anemia. </sup></p></td>
<td><p><sup>Mice were given the following sc treatments: </sup></p>
<p><sup>sham </sup></p>
<p><sup>0.2 ml saline</sup></p>
<p><sup>0.2 ml silicone gel</sup></p>
<p><sup>0.5 ml pristane</sup></p>
<p><sup>0.2 ml silicone gel + </sup></p>
<p><sup> capsulotomy at 3 mos</sup></p>
<p><em><sup>Staph. epidermidis</sup></em></p>
<p><sup>0.2 ml silicone gel + </sup><em><sup>
Staph</sup></em><sup>.</sup></p>
<p><sup>0.2 ml saline 3X</sup></p>
<p><sup>0.2 ml silicone gel 3X</sup></p>
<p><sup>0.5 ml pristane 3X</sup></p></td>
<td><p><sup>There were 10 mice/group</sup></p>
<p><sup> Survival was recorded daily for 10 months and proteinuria
biweekly.</sup></p>
<p><sup>At termination, hematocrits and hemagglutination titers were
measured.</sup></p>
<p><sup>Other endpoints included:</sup></p>
<p><sup>anti-nuclear antibody titers and anti-Type I collagen
titers</sup></p></td>
<td><p><sup>At 12 months of age, the percentage mortality was:</sup></p>
<p><sup>untreated 20</sup></p>
<p><sup>saline 1x 30</sup></p>
<p><sup>pristane 1x 20</sup></p>
<p><sup>silicone gel 1x 50 </sup></p>
<p><em><sup>Staph. epidermidis</sup></em><sup> 20</sup></p>
<p><sup>gel +</sup><em><sup> Staph</sup></em><sup>. 40</sup></p>
<p><sup>gel + capsulotomy 20</sup></p>
<p><sup>saline 3X 30</sup></p>
<p><sup>pristane 3X 80</sup></p>
<p><sup>silicone gel 3X 60 </sup></p>
<p><sup>Mortality was significantly increased compared to controls only
in mice given 3 injections of pristane.</sup></p>
<p><sup>Hematocrits in survivors at 12 mos of age were lower compared to
controls in all silicone-treated mice and in mice injected 1x with
pristane.</sup></p>
<p><sup>Multiple injections of silicone or pristane increased urinary
protein levels</sup></p></td>
<td><p><sup>This model directly tested the ability of silicone to
exacerbate an autoimmune disease other than arthritis.</sup></p>
<p><sup>The data on clinical endpoints reflect only survivors which
likely changes the significance of effects. </sup></p>
<p><sup>It is not clear what statistical comparators were used to
determine the significance of the clinical data (eg., 3x saline as
comparator for 3x pristane and 3x silicone)</sup></p>
<p><sup>The relevance of certain endpoints measured (eg., urinary
protein) in this model are not known.</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><p><sup>Model</sup></p></td>
<td><p><sup>Description</sup></p></td>
<td><p><sup>Treatment</sup></p></td>
<td><p><sup>Design</sup></p></td>
<td><p><sup>Significant results</sup></p></td>
<td><p><sup>Interpretation/conclusions</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>12. Collagen-induced arthritis in DBA/1 mice</sup></p>
<p><sup>(short-term implants)</sup></p>
<p><sup>Schaefer (1997) Schaefer et al. (1997) </sup></p></td>
<td><p><sup>A genetically susceptible strain that develops progressive
inflammatory arthritis in response to immunization with bovine
collagen.</sup></p></td>
<td><p><sup>Mice were implanted ip with:</sup></p>
<p><sup> Sham</sup></p>
<p><sup> elastomer (0.1 mg segment)</sup></p>
<p><sup> silicone gel (0.1 ml)</sup></p>
<p><sup>3 days after implant surgery, all mice were injected at base of
tail with 100 µg bovine collagen type II emulsified in
CFA.</sup></p></td>
<td><p><sup>There were 10 mice/group.</sup></p>
<p><sup>Mice were observed for signs of arthritis for 10 weeks after
immunization; joints measured 3x/week.</sup></p>
<p><sup>Sera obtained:</sup></p>
<p><sup> a) after 28 days</sup></p>
<p><sup> b) at onset of arthritis (when applicable)</sup></p>
<p><sup> c) at termination</sup></p>
<p><sup>for measurement of anti-collagen antibody, cytokines (IL-1, TNF,
IFN(, IL-2), and silicone-binding antibodies</sup></p></td>
<td><p><sup>The incidence of arthritis was high in all groups.
</sup></p>
<p><sup>The severity of disease was not different between
groups.</sup></p>
<p><sup>Titers of anti-collagen antibodies were similar between
groups.</sup></p>
<p><sup>TNF levels were lower in silicone gel-implanted mice.</sup></p>
<p><sup>IL-2 levels were elevated in elastomer-implanted mice.
</sup></p></td>
<td><p><sup>This model directly tested the ability of silicone to modify
collagen-induced arthritis. However, because of the high incidence of
disease in control mice, the model was not sensitive to detect increased
frequency of disease. The authors did not report time-to-onset
data.</sup></p>
<p><sup>The cytokine data are contrary to a hypothesized proinflammatory
effect of silicone</sup></p>
<p><sup>The data on “silicone binding antibodies” and “silicone-bound
proteins” were not convincing of anything more than nonspecific protein
binding.</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><p><sup>13. Collagen-induced arthritis in &amp; DBA/1 mice</sup></p>
<p><sup>(long-term implants)</sup></p>
<p><sup>Schaefer (1997)</sup></p></td>
<td><p><sup>A genetically susceptible strain that develops progressive,
inflammatory arthritis in response to immunization with bovine
collagen.</sup></p></td>
<td><p><sup>Mice were implanted ip with:</sup></p>
<p><sup> sham</sup></p>
<p><sup> silicone elastomer (0.1 mg segment)</sup></p>
<p><sup> silicone gel (0.1 ml)</sup></p>
<p><sup> silicone oil</sup></p>
<p><sup>9 months after implantation, all mice were injected with bovine
collagen in CFA at base of tail.</sup></p></td>
<td><p><sup>There were 10 mice/treatment</sup></p>
<p><sup>Mice were observed for signs of arthritis for 10 weeks after
immunization; joints measured 3x/week.</sup></p>
<p><sup>Sera obtained for measurement of</sup></p>
<p><sup>anti-collagen Abs, cytokines, silicone-binding
antibodies</sup></p></td>
<td><p><sup>The incidence of arthritis was high in all groups.
</sup></p>
<p><sup>The severity score was higher in silicone oil implanted mice
compared to all other groups.</sup></p>
<p><sup>Titers of anti-collagen antibodies were similar between
groups</sup></p>
<p><sup>IL-1 was higher in oil-implanted mice. IL-2 levels were lower in
gel-implanted mice. IL-4 levels were lower in oil- and elastomer-
implanted mice. IL-5 levels were higher in gel- and elastomer-implanted
mice. IFN levels were lower in oil- and higher in elastomer-implanted
mice.</sup></p></td>
<td><p><sup>This experiment directly tested the ability of silicone to
exacerbate collagen-induced autoimmune disease. However, the high
incidence and severity of disease in the control mice limits the
sensitivity of the model to detect exacerbation.</sup></p>
<p><sup>The significance of altered cytokine levels is not known but did
not correlate with disease severity in any recognizable pattern.
</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><p><sup>Model</sup></p></td>
<td><p><sup>Description</sup></p></td>
<td><p><sup>Treatment</sup></p></td>
<td><p><sup>Design</sup></p></td>
<td><p><sup>Significant results</sup></p></td>
<td><p><sup>Interpretation/conclusions</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><p><sup>14. Collagen-induced arthritis in DBA/1 mice</sup></p>
<p><sup>(long-term implants) (collagen emulsified in IFA).</sup></p>
<p><sup>Schaefer (1997)</sup></p></td>
<td><p><sup>A genetically susceptible strain of mouse that develops
progressive, inflammatory arthritis in response to immunization with
bovine collagen.</sup></p></td>
<td><p><sup>Mice were implanted ip with:</sup></p>
<p><sup> sham</sup></p>
<p><sup> silicone elastomer (0.1 mg segment)</sup></p>
<p><sup> silicone gel (0.1 ml)</sup></p>
<p><sup> silicone oil</sup></p>
<p><sup>9 months after immunization, all mice were injected with bovine
collagen emulsified in </sup><em><sup>IFA</sup></em><sup> at base of
tail</sup></p></td>
<td><p><sup>There were 9-10 mice/treatment. </sup></p>
<p><sup>Mice were observed for signs of arthritis for 3 months following
immunization; joints were measured.</sup></p>
<p><sup>Sera obtained for measurement of</sup></p>
<p><sup>anti-collagen antibodies, cytokines, and silicone-binding
antibodies</sup></p></td>
<td><p><sup>The incidence of arthritis was:</sup></p>
<p><sup> sham 3/10</sup></p>
<p><sup> elastomer 8/9</sup></p>
<p><sup> silicone gel 6/9</sup></p>
<p><sup> silicone oil 6/9</sup></p>
<p><sup>Average severity score was higher in silicone elastomer-
implanted mice </sup></p>
<p><sup>Titers of anti-collagen antibodies were similar between
groups.</sup></p>
<p><sup>IL-2 levels were lower in gel-implant mice. IL-4 and IL- 5
levels were lower in oil-injected mice. IL-10 levels were higher in
elastomer implanted mice.</sup></p></td>
<td><p><sup>This experiment directly tested the ability of silicone to
exacerbate collagen-induced autoimmune disease. The sensitivity to
detect exacerbation was improved by using IFA as adjuvant, which reduced
the incidence and lowered the severity of arthritis in the control mice.
</sup></p>
<p><sup>The data indicate enhanced disease in silicone elastomer
implanted mice.</sup></p>
<p><sup>The significance of altered cytokines is not known. However,
overall levels of cytokines were similar to levels seen in mice
immunized with CFA indicating lack of correlation with disease
severity.</sup></p></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
</tbody>
</table>

[^1]: It is not clear why this endpoint was included in this study. This
    NZB model should not to be confused with the NZB/NZW F1 mice that
    spontaneously develop an SLE-like disease, as described in part 2 of
    this section.

[^2]: According to Wilson and Munson, (1996): Assuming a specific
    density of 1 g/ml for the gel, a dose of 1-3 ml of gel in the mouse
    constitutes approximately 5-15% of the body weight. In the rat, a
    gel implant ranging from 1 to 20 ml represents approximately 0.6 to
    13% of body weight. In women, the implantation of two 300 ml mammary
    implants is not unusual. In a 50-kg woman, this volume of implanted
    material would correspond to 1.2% of her body weight.

[^3]: If two 300-ml implants contain gel with D4 at a concentration of
    500 ppm , this equals a total of 0.3 g D4. If a 50 kg woman were
    exposed to all of the D4, it would represent a total dose of 6
    mg/kg. Any exposure to D4 from a leaking implant would be acquired
    over a long exposure period, such that on a daily basis the dose is
    much lower.