Dillingham1975.biological evaluation of polymers

J. BIOMED. MATER. RES. VOL. 9, PP. 569-596 (1975)
Biological Evaluation of Polymers
I. Poly(methy1 Methacylate)
E. 0. DILLINGHAM, N. WEBB, W. H. LAWRENCE, and J.
AUTIAN, Materials Science Toxicology Laboratories, The University
of Tennessee,Centerfor the Health Sciences, Memphis, Tennessee 38163
Summary
A series of poly(methy1 methacrylate) formulations differing widely in chemical
and physical properties was employed for the evaluation of primary screening
methods for the assessment of acute toxicity. Materials and USP extracts of
materials were tested in parallel. Tissue culture, hemolysis, intradermal irrita-
tion, systemic toxicity, muscle implant and histopathologic responses were
determined for each of 27 formulations. A determination of the nonvolatile
methanol extractable components was carried out on each formulation. The
formulations varied with respect to percent, w/w, methyl methacrylate, N,N-
dimethyl-p-toluidine, stannous octoate, 3,4-diamino-toluene and, also, with
respect to curing conditions. Volatile components, primarily methyl methacry-
late, of three selected formulations were determined quantitatively by vacuum
distillation and mass spectrographic analysis.
Statistical analysis of the primary data indicated a significant correlation of
residue weight (methanol extractable) with hemolytic activity (r = 0.93) and
with the cumulative biological response (r = 0.9). Multiple linear regression
analysis of residue weights with hemolysis and intradermal irriation responses
gave the highest overall correlation (r = 0.96). Hemolytic activity and tissue
culture responses were significantly correlated (r = 0.87). It was concluded
that the observed variation of biological test results reflected significant differ-
ences in the toxicity of the test materials. The poly(methy1 methacrylate)
series examined was relatively low in toxicity and the biological tests examined,
particularly the in vitro tests, were found to be responsive to formulation and
curing conditions which indicated their suitability for primary toxicity screening.
INTRODUCTION
Synthetic polymeric materials have found wide use in medical
practice as restorative structural substances and as components of
implanted prosthetic devices. At the present time, the selection
569
@ 1975by John Wiley & Sons, Inc.

570 DILLINGHAM ET AL.
and use of a specific polymer material rests primarily on the judg-
ment of the investigator or practitioner, since there are no generally
accepted criteria or standards for preclinical evaluation. The
variable clinical success with a given polymer material, and the fact
that many different sources and formulations are available, points
to the need for evaluation prior to introduction into medical or dental
practice. Thisstudy was directed toward an evaluation of procedures
for preclinical evaluation.
Poly(methy1 methacrylate), PMMA, a polymer which has been
widely used as a restorative material and as a componcnt of pros-
thetic devices, was employed in an investigation of physical, chemical,
and toxicological properties with respect to formulation variables.
The principal objective of the work was the discrimination of test
methods providing the highest correlation between formulation and
toxicological parameters.
Biological Compatibilityof Poly(methy1Methacrylate)
Poly(methy1 methacrylate) has been used extensively as a pros-
thetic material in dental and mandibular corrections, and as a bone
cement in hip joint restructure. When used as a dental restorative
material, there are reports of little or no inflammatory response,1-4
while others have reported inflammation, formation of connective
tissue, and loss of bon~.~-'O Studies of orthopedic restorations
(1960-1970) indicated no lasting adverse effects of PMMA.11-13
Two long-term studies reported carcinomas associated with PMMA
restorati~ns.'~-'~Adverse response was noted in some cases of
"cold cured" restorations which suggested toxicity from the residual,
unpolymcrized, methyl methacrylate monomer. The clinical use of
PMMA in orthopedic surgery was approved by the Food and Drug
Administration in October, 1971. Since that time, many adverse
effects of PMMA bone cement have been Cardiac
arrests and pulmonary complications during hip joint repair with
PMMA have been noted; the adverse responses, e.g., hypotension16.
18,23,24928*30,35 or pulmonary embolism, 21,22e25-27 were in some cases
attributed to free monomer. Other reports have cited contact
dermatiti~,~Zchanges in urinary level of amino nitrogen,33 and
histological response of tissues with respect to blood levels.34 Homsy
et a1.35determined blood levels and clearance of methyl methacrylate
monomer in humans and canines exposed to subcutaneous PMh4A
implants and intravenous administration of the monomer.

BIOLOGICAL EVALUATION OF PMMA 571
Adverse biological response to plastic materials is primarily re-
lated to the kind and quantity of low molecular weight residual
components having adequate solubility in tissue fluids to leach from
the material into adjacent tissues and the systemic circulation.
The physical properties and chemical composition of the surface of
the material also influmce compatibility with tissues, hemolytic
activity and thrombogenic activity.
A polymeric material such as PMMA can possess a wide range of
toxic potentials, depending upon the kind and quantity of starting
materials usrd in its formulation and the physical history of the
polymer. Each polymer formulation must, therefore, be considered
to have a unique potential toxic liability.
MATERIALS AND METHODS
Formulationof Poly(methy1Methacrylate)
The starting materials for PMMA formulations are given in
Table I. The formulations were varied systematically with respect
to methyl methacrylate (MMA) and N,N-dimethyl-p-toluidinr
(DPT) concentrations. Stannous octoate (SO) and 3,4-diaminoto-
luene (DAT) were each used in two formulations. Details of for-
mulations were designed to provide diversity of final polymer proper-
TABLE I
Starting Materials: Poly(methy1Methacrylate) Formulations
Component Source
Hue-Lon Clear: poly(methy1methacrylate);
polymer beads with initiator, benzoyl
peroxide ( <2%)
Hue-Lon Liquid: methyl methacrylate (MMA)
with stabilizer, hydroquinone (<0.006%),
and crosslinkingagent, ethylene di-methacry-
late (l-Z%)
N,N-dimethyl-p-toluidine (DPT)
3,4-diaminotoluene(DAT)
Stannous octoate (SO)
L. D. Caulk Company
P. 0.Box 359
Milford, Delaware
J. T. Baker Chemical Co.
Phillipsburg, New Jersey
Eastman Chemical Co.
343 State Street
Rochester, New York
Dow-Corning
P. 0. Box 5074
Atlanta, Georgia

ties and have no direct relationship to specific end uses, although
highly cured formulations without additives approximated those
employed in the fabrication of prosthetic devices such as dentures.
The basic formulation procedurc consisted of mixing monomer
(Hue-Lon Liquid, MMA) and polymer beads (Hue-Lon Powder),
1:2, w/w, until the product reached a nontacky dough stage. Thr
dough was transferrrd to standard dental stone molds lined with
aluminum foil and pressure (20psi) was applied. The press asscmbly
was heatrd in a 65°C water bath 45 rnin and quenched in an ice bath
for 1hr (Table11, Code 1). The product, a hard, transparent plastic
sheet 5 mm thick, was stored at 4°Cuntil usrd. Table I1 describes
the variations in curing conditions and additives employed in the
formulation of PMRIA test materials. Additives, including excess
TABLE I1
Formulation and Curing of Poly(methy1 Methacry1ate)a
Formulation
Code Additives, O/~(W/W)~
______ _ ~
No. I I1 Method
1
2
3
4
5
6
7
8 IMMAc
6.0 DPT
4.0 DAT
4.0 SO
.6.0SO
Monomer pipetted into polymer powder (1 :2,
w/w) with stirring (20 min) ;'dough placed in
dental press (20psi, 65"C, 45 min) followed by
O'C, 1hr.
# I procedure except cured at 65"C, 18 hr,
followed by lOO"C, 1 hr, and cooled 15 min
with tap water.
#1 Procedure
# 1 procedure except DPT added 5 min after
initiating stirring.
DAT added to monomer and suspension
added to polymer powder (monomer: polymer,
l:l, w/w); stirred until dough-like (about 20
rnin); cured 65"C, 18 hr, cooled (tap water).
# 1procedure except SO added at 5 min.
#6 Procedure
Monomer added to polymer powder (l:l,
w/w), stirred 65 min; cured (room temp.,
1hr) followed by O"C, 1 hr.
~~
(continued)

TABLE 11-(Continued)
Formulation
Code Additives,
No. I
9 SMMA
10 SMMA
11 PMMA
12 2MMA
13 2MMA
14 SMMA
15 2MMA
16 2MMA
17 4 MMA
18 4MMA
19 4MMA
20 GMMA
21 GMMA
22 GMMA
23 GMMA
24 GMMA
25 GMMA
26 6MMA
27 GMMA
28 2MMA
%(W/WP
I1
0.25 DPT
0.75 DPT
1.25 DPT
0.25 DPT
0.75 DPT
1.25 DPT
0.25 DPT
0.75 DPT
1.25 DPT
6.0 DPT
0.25 DAT
0.5 DAT
Method
# 1procedure except cured at room tempera-
ture, 1hr, followed by O'C, 1 hr.
#2 Procedure
# 1 procedure except cured at 65"C, 90 min;
100°C, 1 hr, and cooled (tap water) 15 min.
# 1 procedure execpt cured at 65"C, 90 min;
lOO"C, 1 hr; cooled, room temperature.
# I Procedure
#4 Procedure
#4 Procedure
#4 Procedure
#4 Procedure
#4 Procedure
#4 Procedure
# 1Procedure
#1 Procedure
#4 Procedure
#4 Procedure
#4 Procedure
#4 Procdeure
# 1 procedure except DAT mixed with poly-
mer powder before adding monomer.
# 26 Procedure
#9 Procedure
*Abbreviations: MMA = methyl methacrylate; DPT = N,N-dimethyl-p-
bAmount added &s a percent of the total weight of the stated base formulation.
OExcess MMA was added to the MMA of the base formulation before mixing.
toluidine; DAT = 3,4-diaminotoluene;SO = stannous octoate.
monomer, were added as a wt '% of the total weight of the basic
formulation.
Biological Tests
Agar Overlay Tissue Culture Test.-Tissue culture toxicity was
evaluated using nonreplicating mouse fibroblast cells.36 Monolayers
of cells in Petri dishes were covered with a thin layer of agar contain-
ing minimum nutrients to maintain cell viability. The cells were

vitally stained with neutral red and the test samples were applied
to the surface of the agar, 2 samples per Petri dish, along with one
positive (toxic) control material and one negative (nontoxic) control
material.* The standard assay consisted of duplicate test plates.
Solid samples were applied as squares of approximately 1 cm.
Liquid samples (USP extracts) were applied to 1cm filter paper assay
disks, 0.2 ml per disk. The test plates were incubated for 24 hr
and the monolayer responses reported in the form of a Response
Index (RI), as shown in Table 111. A toxic response was detected
by loss of the vital dye followed by lysis if the concentration of leach-
able toxic components was high enough. The zone size and percent
of cells lysed within the zone were determined microscopically,
although zones were apparent macroscopically.
Extraction of Materials for Biological Tests.-The USP procedure37
for extraction of materials (4g materials/20 ml extraction medium,
121"C, 1 hr) was employed to obtain extracts for both in vitro and
in vivo tests. The PRIMA material was prepared for extraction by
breaking the 5 mm thick sheet into small pieces. Four or 5 pieces
of approximately equal dimensions totaling 4 g were selected for
extraction. Cottonseed oil (C), polyethylene glycol-400 (P), and
saline (S) extracts were prepared for tissue culture (TC), intradermal
irritation (ID) and systemic toxicity tests (IV or IP), Table IV.
All tests were carried out on each extract within 6 hr of extraction.
Hemolysis Test.-Five g of material (prepared as for USP extraction
was added to 10 ml of saline (0.9% sodium chloride, aqueous) in
.16 X 150 mm assay tubes and equilibrating at 37°C for 30 min.
Freshly collected rabbit blood (1ml of 2y0 sodium oxalate per 20 ml
of blood) was diluted with saline to a positive control optical density
of 0.9-1.0. Two-tenths ml was added to each assay tube and mixed
by gentle inversion, followedby incubation, 37°C for 1hr. The tubes
were centrifuged, 500 X 9, 5 min, and the supernatant removed for
determination of optical density (O.D.) at 545 nm (saline reference).
The negative control was saline and blood only. The positive control
(loo'%hemolysis) was obtained by addition of 0.2 ml blood to 10 ml
of 0.1% sodium carbonate. Treated and control tubes were handled
* Positive control: Poly(viny1 chloride) tubing demonstrated to be consistently
Negative control: Polyethylene film demonstrated to be consistentlycytotoxic.
noncytotoxic.

BIOLOGICAL EVALUATION OF PMMA 57.5
TABLE 111
Rating Scales For Biological Response
Agar Overlay Tissue Culture Test:
Zone Index (ZI)
0 No detectable zone around or under sample
1 Zone limited to area under sample
2 Zone not greater than 0.5 cm in extension from sample
3 Zone not greater than 1.0 cm in extension from sample
4 Zone greater than 1 cm in extension from sample but not
5 Zone involving the entire plate
0 No observable lysis
1 Less than 20% of zone lysed
5 Greater than 80% lysis within zone
involving the entire plate
Lysis Index (LI)
Response Index (RI) = ZI/LI
Percent Hemolysis (HY,): 0 to l0Oy'
Percent Inhibition fo Cell Growth (ICGY,): 0 to 100%
Rating Scalea
Hemolysis Test:
Inhibition of Cell Growth:
Intradermal Irritation (I. D.):
0 No detectable irritation
1
2, 3, 4
Systemic Toxicity (I. V. or I. P.):
Intramuscular Implant Test (IM):
.5 Minimal irritation detected
Very slight but distinct irritation
Increasing intensity of inflammation and wheal
Rating Scale = Number of deaths after 7 days: 0 to 5
Rating scale was analagous to that for I. D.: intensity of gross visible
inflammation and necrosis around implant primary factors.
Histopathologic Rating (HR) :
Rating Scale:
0
1 Minimal response detected
2, 3, 4
Not different from negative control
Increasing tissue reaction around implant; the degree
of necrosis primary factor
UOriginal data converted as follows for computer analysis:
0 = 0 ; % = .5, f = 1 , 1 = 2 , 2 = 3 , 3 = 4 , > 3 = 5

TABLEIV
Poly(methy1Methacrylate):PrimaryTestResults"
~~~
FormulationbU
CodeAdditives,'%TissueCultureIDIPIVP
No.II1MCPSHMICPSsHK.CIICGHE^,%^RW50
HighSet:
27GMMA
92MMA
8lMMA
3
20GMMA
26GMMA
24GMMA
LowSet:
5
132MMA
21GMMA
1
7
122MMA
0.5DAT4/21/21/21/2830222005"1408
4/31/21/21/28402200002394
4/41/31/31/2650.5200000318
5/51/31/21/25400000001279
5/51/21/11/06400000001249
0.25DAT3/21/12/22/03310000002238
1.25DPT1/11/21/11/1001100001165
4.0DAT5/53/31/32/39914000000474
2/21/21/21/26100000000191
2/21/21/11/18300100000218
2/21/21/21/26500000001220
6.0SO2/21/01/01/0800000002128
0/01/01/01/000000000030
P182.96.07g
6.60M
2.54.864
191.626.23.38
192.38.43.09
251.02.57
222.70.53
65.012.22
225.35.29
194.410.25.85
4.85.11
130.72.72
0.00.51

" r -o o
? ?
0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 3 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 3 3 3 3 4 0 0 3 3 3 3 0 3
3 0 0 0 3 3 3 3 3 0 3 1 3 0
2 2 2 2 2 2 2 2 2 2 2 2 2 2
3 O N O 3 3 3 3 3 0 3 3 3 0

3 0 0 0 N 3 3 3 3 0 3 3 3 0
e.33 3 3 3 3 H 3 F i 3 3 3 3
22222222222222
4:

TABLE IV-A
Explanation of Headings: Table IVa
M = Material sample
C = USP cottonseed oil extract
P = USP polyethylene glycol-400 extract
S = USP saline extract
H = Hemolysis, % (polymer test sample)
MI = Intramuscular implant response
I D = Intradermal irritation response
IP = Systemic toxicity, I. P. administration
IV = Systemic toxicity, I. V. administration
HR = Histopathologic rating of MI
CI = Cumulative biological index (see under “Statistical Analysis”)
ICG = Inhibition of cell growth, %; tissue culture
HE^ = Hemolysis, yo(USP saline extract of polymer)
IRW = Weight of methanol extractable substances,%
93ee Table I11for rating scales.
alike. The assay was carried out with 5-fold replication, and the
percent hemolysis was estimated using the average O.D. values:
Hemolysis yo =
O.D. (treated) - O.D. (negative control)
x 100
O.D. (positive control) - O.D. (negative control)
The hemolytic activity of USP saline extracts was carried out in
the above manner except that the blood was added directly to the
saline extract.
The influence of surface properties of materials on hemolytic
activity was determined by carrying a set of assay tubes in parallel
with a standard assay of a material, but without the addition of blood.
At the end of the 1 hr incubation period, the standard set with blood
was centrifuged and assayed normally. The set without blood was
decanted into new assay tubes, taking care not to transfer any of the
material test sample, 0.2 ml blood was added to the decantate,
incubated for 1 hr at 37”C, and assayed normally.
Inhibition of Cell Growth.-Distilled water extracts of materials,
USP, were incorporated into Eagle’s medium* 38 at a final extract
* Modified Basal Medium Eagle supplemented with 5% newborn calf serum,
1% tglutamine, and 50 mcg/ml of streptomycin sulfate, magnesium being
added as magnesium chloride, 177 mg/l.

concentration of 50% with respect to the original extract by mixing
equal volumes of double strength Eagle’s medium and distilled
water extract. Growth in the extract-treated medium was compared
to growth in the untreated Eagle’s medium. Two X 105 mouse
fibroblast cells (strain L-929) in Eagle’s medium were added to each
of 20 assay tubes. The tubes were centrifuged, 500 X g, 5 min,
and the medium decanted, taking care to maintain asepsis and not to
lose cells. Ten received 2 ml Eagle’s medium and ten received 2 ml
treated medium. Five of the treated set and 5 of the control set
were centrifuged, decanted, washed twice with saline (centrifugation),
and stored at 4°C until assayed for cell protein by the Lowry modifi-
cation of the Oyama and Eagle 40,41 protein assay procedure (zero-
time controls). The remaining 10 tubes were incubated for 72 hr
(37OC, 5% COn-air atmosphere), centrifuged, washed, and assayed
for total protein along with the zero-time controls. The inhibition
of cell growth was calculated as the decrease in net (72 hr minus zero-
time) cell protein in the treated set as a percent of the net cell protein
of the control set.
Intradermal Irritation Test.-Irritant response42was determined
by injection of 0.2 ml of a USP extract (C, P, or S) of a test material
intracutaneously into 10 sites of each of 2 rabbits, previously clipped
of hair on the dorsal surface. Two-tenths ml of a negative control
solution (saline) and a positive control solution (20% ethyl alcohol)
were injected intracutaneously in each animal. After 3 days the
irritant response was rated on a 6-point scale (Table 111) with respect
to intensity. The protocol was essentially that described in the
United States Pharmacopeia, XVIII.37
Systemic Toxicity Test.-Extracts were administered to 5 mice with
5 control mice receiving the untreated extraction solution. Saline
extracts were administered intravenously (IV), 50 ml/kg. Poly-
ethylene glycol-400 and cottonseed oil extracts were administered
intraperitoneally (IP), 50 ml/kg and 10 ml/kg, respectively. The
animals were observed for signs of toxicity and mortality at 1 week
(0-5 scale, Table 111).
Intramuscular Implantation Tests.-The protocol was essentially
that described in the United States Pharmacopeia, XVIII.37 A
trocar needle was employed to implant thin sections of the test mate-
rial into the paravertebral muscle of rabbits, lightly anesthetized
with sodium pentothal and shaved on the dorsal surface. Each

$80 DILLINGHAM ET AL.
animal received 2 negative (nontoxic), 2 positive (toxic) control
implants, same control materials as used in tissue culture, and 6
implants of the test material. After 1week the rabbits were sacri-
ficed; the implant sites were surgically removed and examined for
grosstoxicity. The intensity of response was recorded using a 6-point
scale (Table 111). The tissue including the implantation site was
preserved in formalin, examined histologically, and a Histopatho-
logic Rating, HR,41was recorded on a &point scale (0-4, Table 111).
Twelve-Week Intramuscular Implantation Test.-This test followed
the identical protocol for the 1week implantation test, except that
14rabbits were implanted with material tested and 1rabbit sacrificed
and evaluated each week.
Physical and Chemical Analytical Procedures
VolatileComponentsof PMMA Formulations.-Volatile components
of 3 selected PMMA Samples (4,7, and 27, Table 11)were obtained
by heating 1.5-2.5 g of polymer under vacuum (85°C f 3"C, 10-3
torr) in an all glass system for 90 min. The collection vessel, a
5 mm i. d. glass tube (- 180°C)was heat-sealed, removed from the
system, the weight of volatile material determined and the contents
analyzed by gas chromatography and mass spectrometry, Table V.
Extraction of Polymer Formulations.-Methanol was used for the
extraction of all PMMA formulations (Table 11). The test sample
was broken into small pieces and 20-25 pieces of approximately
equal size totaling 10 g (=I=0.1mg) were extractedin 150ml of meth-
anol with continuous agitation, 23"C, for 24 hr. After extraction,
the solvent was decanted and held at 4°C until used.
Three samples (4, 7, and 27, Table 11)employed for the determina-
tion of volatile components were subsequently extracted with 100ml
of methanol for 15 hr by refluxing under ambient conditions (bp
64.5 =t0.3"C). The solvent was decanted and evaporated at 50°C.
The residue was weighed and analyzed by infrared and mass spec-
trometry.
Quantitation of Methanol Extractable Residue.-Eighty to 90 ml of
the 24 hr extract used in the biological tests was reduced to approx-
imately 10ml under reduced pressure in a rotary evaporator (40°C).
The concentrate was quantitatively transferred to a tared crucible,
reduced to dryness (25"C, 4 days, 15-20 mm Hg), and weighed. The

TABLEV
GasChromatographyandMassSpectrometryAnalysesofPMMAFormulations
Percent(w/w)Identityof
MethanolMethanol
Formulation
Volatile
CodeAdditives'%(w/w)Components,"IdentityofExtractableExtractable
VolatilecomponentsComponentsComponentsNo.II1Percent(w/w)
46DPT00.9DPT+Unknown
76SO1.1MethylMethacrylatea1.3Organotin
276MMA.5DAT11.0MethylMethacrylate2.0DAT
*SeeTable11.
bVacuumdistillation.
CPresumptive.

amount of residue (RW, % w/w) was calculated as a percent of the
original sample weight.
Gas Chromatographic Analysis of Methanol Extract.-Fifty ml of
methanol extract was concentrated to 10 ml on a rotary evaporator,
and further reduced to approximately 2 ml under a stream of dry
nitrogen in a graduated cylinder. The final volume was recorded
(=t0.05ml). The sample was filtered on Whatman No. 1 filter paper
and chromatographed on a Varian Model 2100 Gas Chromatograph
with flame ionization detection.
StatisticalAnalysis
A stepwise multiple linear regression45was performed on residue
weight %, versus the 9 biological test results showing significant
variation. A similar analysis of hemolysis % versus other biological
tests was carried out. A test was also made of the correlation betwecn
the Cumulative Biological Index (CI) and residue weight %. CI
was defined as the sum of all biological test results, except inhibition
of cell growth %, normalized to a value of 100 for the maximum or
positive control response. In the case of tissue culture data, the
sum of Zone Index (ZI, maximum of 5) plus Lysis Index (LI, maxi-
mum of 5 ) was normalized to 100 (e.g., the sum was multiplied by
10).
RESULTSAND DISCUSSION
Twenty-seven formulations of PMMA were tested for toxicity.
Table I1 presents the formulation data, and Table IV, the primary
biological test results, along with residue weight yoof the methanol
extractable components. The PMMA formdations gave a wide
range of toxic responses in vitro, but relatively little response in vivo.
The residue weight yo showed sufficient systematic quantitative
variation for statistical analysis. Gas chromatography and infrared
and ultraviolet absorption data obtained on extracts or residues
were qualitatively related to formulation and curing protocols, but
were not sufficiently quantitative for statistical analysis.
CumulativeBiological Index
The correlation of the Cumulative Biological Index (CI)” with
The apparentresidue weight % was significant (r = 0.9), Figure 1.
*Defined under “statistical analysis.”

CUMULATIVE BIOLOGICAL INDEX
Fig. 1. Poly(methy1methacrylate):regression of residue weight on cumulative
The curves indicate the approximate upper andbiological response (T = 0.90).
lower limits of biological response.
upper and lower limits of CI, indicated by the 2 linear curves,
differed by approximately 150 CI units over the observed range of
residue weights. The formulations that included N,N-dimethyl-p-
toluidine (DPT), an activator of the polymerization reaction, showed
uniformly low biological response and residue weights of 1% or less,
which was consistent with the assumption that free monomer contrib-
utes significantly to biological response. The addition of 6% DPT
(formulation 4) resulted in one of the lowest values of CI. DPT
was detected by gas chromatography in methanol extracts of all
samples formulated with 20.75% DPT. Biological response was
significantly affected by the presence of DAT (formulations 5, 26,
and 27).
Benzoyl peroxide and its degradation products may contribute
to biological response and residual benzoyl peroxide would be expected
to be greatest in the less polymerized formulations which showed the
highest biological response. Benzoic acid, a primary degradation
product of benzoyl peroxide, should be greatest in the more highly
polymerized formulations. Hydroquinone, and unstable compound,

was also present. The presence of these compounds and their
relative leachability may contribute to the 150 CI variation of CI
observed.
The influence of curing conditions was evident in formulations
9-13 (Table VI), which were identical in composition, but,were cured
TABLE VI
Influence of Curing Conditions on Biological Response
Poly(methy1Methacrylate)*
Degree of Tissue
Sampleb Curing Culture Hemolysis CI
9 Lowest 4/3 84 394
13 2/2 61 191
11 1/1 1 116
12 o/o 0 30
10 Highest 0/0 1 81
a2y0 excess monomer.
bSee Table 11.
under different conditions. The degree of curing increased in the
order 9, a highly quenched formulation, 13, 11, 12 and 10. The
latter two were raised to 100°C for the last hr of curing and were
considered highly cured. The CI values for those formulations were
394, 191, 116, 30 and 81, respectively, which further supported the
assumption that free monomer, a function of curing conditions, was
a significant factor in the biological response of PMMA formulations.
Curing conditions were also found to be correlated with the hemolytic
activity of both the materials and USP saline extractsof the materials;
see Tables I1 and IV.
The regression of RW on CI and the empirical correlation of CI
with curing conditions and type of additives in the formulations in-
dicated that the biological tests employed provided a sensitive index
of leachable toxic components. The fact that the lower limits of CI
showed a linear regression through the origin (Fig. 1) supported the
conclusion that little or no biologically inert substances (e.g., polymer)
contributed to RW. It is also consistent with the conclusion that the
variability of CI at a given RW reflects differences in biological
activity resulting from differential leachability of low molecular
weight components of diffcrent formulations.

The results of a 5 week study of reproducibility of biological test
results are given in Table VII. The major part of the variation
could be accounted for on the basis of decreasing biological activity
with respect to time, particularly apparentin the in vivo andhemolysis
results. It was concluded that the principal component of variation
was differences in biological activity of samples rather than the
variability of test results.
The Influence of SampleAge on Biological Test Results
Table VIII summarizes tissue culture and hemolysis test results
obtained on samples of different ages. The tissue culture test
results varied little for tests on materials and on extracts of materials.
However, the hemolysis test results declined significantly with time,
with the exception of formulation 5 which contained a high (4%)
level of DAT. There was an overall correlation of 0.87 between
tissue culture and hemolysis test results for samples assayed within
the first 50 days after formulation. Residual hemolytic activity of
1.9% or higher (128 to 206 days after formulation ) was consistently
associated with a distinct tissue culture response (RI = 2/2 or
higher). The relative insensitivity of tissue culture test results with
regard to sample age supports the selection of tissue culture tests for
comparative studies, although under fixed conditions the hemolysis
test provides essentially the same relative information and is con-
siderably easier to perform.
Chronic Toxicity of PMMA Implants
The results of a 12 week study of muscle implants of formulations
3, 5, 6, and 24 are shown in Table IX. The highly cured sample
(formulation 5) showed the lowest overall response in spite of the
presence of DAT, which in other tests gave a very high overall re-
sponse (CI = 474). There was no significant response after 1week
for any of the formulations, and only formulation 6 with stannous
octoate gave a distinct response (HR = 2) at 1week. H R ratings
of 2 for all samples at 3 days indicated a significant degree of initial
tissue reaction (primarily due to trauma);however, chronic toxicity
was not great for the 4 samples tested.
CorrelationsBetween Tests
The multivariate correlation of residue weight % (RW) with
biological tests (Fig. 2) indicated that residue weight was most

TABLEVII
Five-WeekStudyofPMMAFormulationNo.28"
IPIV~-SampleAge,TissueCultureID
WeeksMCPSHMICPSCPSHRCIICGRW
15/51/22/21/26302200000363118.06.41
25/52/21/21/2590110000030956.05.95
35/52/21/21/2590100000028494.45.47z
M
45/51.5/21/21/2470010000026783.06.25cl
55/51/21/21/2530120000031837.66.08b
r(
%SeeTableIV-Aforexplanationofheadings;TableI1forformulationdata.

BIOLOGICAL EVAL,UATION OF PMMA 587
D
W
>
W
m
0
a
m
c
2
RW=0.65(H,o/o)t .93 (lD,C) + .29
r = 0.96
I I I I I I I 1 I
0 I 2 3 4 ' 5 6 7 8 9 10 I1 12
RW, PREDICTED
Fig. 2. Prediction of residue weight using hemolytic activity of material
The
Formulation numbers are indicated for
samples and the cottonseed oil extract intradermal irritation responses.
curve is the line of perfect correlation.
selected data points.
highly correlated with hemolysis and intradermal irritation of cotton-
seed oil extracts.
RW % = 0.65(H %) +0.93(ID,C) +0.29 T = 0.96
Intradermal irritation (ID,C) contributed to the correlation only
slightly. The univariate correlation coefficient of RW % with H %
was 0.93.
The correlation of H '% with all other biological tests (Fig. 3)
indicated it to be most highly correlated with tissue culture param-
eters.
H % = 14.9 (ZI) + 11.4 (LI) - 12.2 T = 0.87

TABLEVIII
InfluenceofSampleAge:TissueCultureandHemolysisAssaysPoly(methy1Methacrylate)"
n
FormulationTissueCulture
MaterialsExtractsHemolysis
CodeAdditives,yo
No.II1DaysbRIcDaysRIDaysCPSDaysCPSDays'%Days%
1OMMA02/21923/310651953
2OMMA221/11831/1211/11/11/1181/21/11/1711860
3OMMA285/51474/445415011
40MMA6DPT391/01631/0311760
50MMA4DAT115/52035/52110020698
60MMA4SO440/01680/0811610
7OMMA6SO281.5/1.51524/4481750
8lMMA74/41995/417652023
92MMA64/31984/4
10SMMA22o/o198o/o
13SMMA12/21982/2
11SMMA41/12011/12011930
122MMA30/01950/01301981
17611912

142MMA0.25
152MMA0.75
162MMA1.25
174MMA0.25
184MMA0.75
194MMA1.25
206MMA
216MMA
226MMA0.25
236MMA0.75
246MMA1.25
256MMA6.0
266MMA6.25
276MMA0.5
DPT
DPT
DPT
DPT
DPT
DPT
DPT
DPT
DPT
DPT
DAT
DAT
161/1182
161/1182
171/1178
171/1178
180/0185
211/1182
455/5164
282.5/2147
251/1186
241/1185
231/1190
431/2161
733/2197
114/2.5135
1/0151/11/11/1
1/0151/11/11/1
1/1161/11/11/1
1/1161/11/11/1
1/2171/01/01/0
1/1201/11/11/1
4/4
3/25
1/0241/11/11/1
1/0.5231/11/11/1
1/15
1/1221/21/11/1
3/3
4/3
67
67
68
73
74
77
81
80
79
1/21/11/171175
1/21/21/170175
1/21/11/181181
1/11/01/031178
1/21/11/161185
965167
483150
1/21/11/190188
1/11/11/180183
71164
5033190
783128
1/11/21/180181
1/11/11/1101189
aSeeTableIV-Aforexplanationofheadings;TableI1forformulationcode.
bAgeofformulationattimeoftesting.
cRI=ResponseIndex(seeTable111).
138td
0s

cnW
0
TABLEIX
Twelve-WeekIntramuscularImplantationofPMMAFormulations
U
FormulationcWeeks
CodeAdditives,yo3Days12345-12i:
F
r,
z
No.II1MI*HRbMIHRMIHRMIHRMINRMIHR
D
54DATf2f0-0-0-0-0
E246MMA1.25DPTf2-1-1-0-0-0
64SO+12-2-0-0-0-0M
3(Control)+12-1-0-0-1-0cj
+?8MI=Grossmuscleresponse.
bHR=Histopathologicrating.
cSeeTableI1forformulationcode.

BIOLOGICAL EVALUATION OF PMMA
100
90
80
70
60
50
40
30
20
10
591
-
-
-
-
-
-
-
-
-
-
(3)
-@-
-10 0 lo I 2
0
W
>
[L
W
v)
0
m
8
I
0
0
H ,X PREDICTED
Fig. 3. Prediction of hemolytic activity using tissue culture response: ZI(M) =
Zone index of material samples; LI(S-1) = Lysis index of USP saline extracts.
The curve is the line of perfect correlation. Numbers in parentheses indicate
the number of values at the indicated point.
The PMMA formulations employed in this study were relatively
low in toxicity, showing few significant in vivo responses, which
accounts for their lack of significant contribution to the above
correlations. The correlations obtained with polymer formulations
of higher toxicity will be presented in a second publication.
Inhibition of Cell Growth by Extracts.-A striking difference was
observed between the percent inhibition of cell growth (% ICG) by
extracts and the other biological data. All 12 extracts assayed com-
pletely inhibited growth (131-253%) and showed no signficant
trend regarding formulation variables (least significant difference,
95% confidence = 12%). The ID5, (concentration producing 50%
inhibition of growth) of MMA was determined to be 2.0 X M

(moles/liter). The intrinsic toxicity (slope of the dose-response
curve) was 3.7 X lo2% inhibition/mole. On the assumption that
methyl methacrylate was the active component in the ICG extracts,
concentrations in excess of 10" M were indicated for the extracts.
Mir et a1.4+48 reported 17% inhibition of contraction of guinea pig
illeum at 4.6 x 10-3 M ; 42% depression of blood pressure, 9.6%
depression of heart rate, and 65% increase in respiration rate, in
male dogs at 1.4 X They also reported 50% depression
of heart rate (8 x M ) ,
and coronary flow (1 X M ) in isolated rabbit heart assays.
Approximately 2% ethylene dimethacrylate was present in the
monomer employed in the present study and those reported by Mir
et al. It was detected in some of the methanol extracts of less
well-polymerized formulations of PMMA, but its contribution to
biological response was not determined. Since the limit of gas
chromatographic detection was f 1 ng, it was unlikely that ethylene
dimethacrylate was a significant factor for the more highly cured
formulations where it was not found in the methanol extract.
M .
M ) ,force of heart contraction (1 X
The Influence of Additives on Hemolytic Activity
Fifteen selected formulations were assayed in parallel for hemo-
lytic activity of the material and USP saline extracts of the material
(Fig. 4). There is no significant difference in the rank order of
response in the 2 assay systems, which indicated that the hemolytic
activity was due to readily soluble components of the formulations,
and that the physical presence of the solid material in the assay system
had no apparent effect. In view of the very steep dose-response
curves typically obtained in the hemolysis assay, consistency of the
2 rank orders was excellent.
Physical and Chemical Analyses of PMMA
Volatile Components.-Since the routine extraction and concen-
tration procedure used for gas chromatographic analysis of the
methanol extracts did not recover the highly volatile components of
the extracts, a vacuum distillation procedure which permitted quan-
titative recovery of those components was used with 3 typical
formulations, 4, 7 and 27 (Table V). Formulation 4, which con-
tained 6% DPT and was very low in biologica1,responses (CI = 91),
gave 0% volatile material. Similar low biological responses were

100
40
20
W
OAT
Fig. 4. Poly(methy1 methacrylate) : Rank order of hemolytic activities of
M = methylUSP extracts and material samples with respect to additives.
methacrylate (see Table IV).
obtained with all formulations containing DPT or highly cured sam-
ples without additives other than excess monomer. Formulation
7, with 6% stannous octoate, contained 1.1% volatile components
and had moderate biological activity (CI = 130.7). Formulation
27, with 6% excess monomer and 0.5% DAT, contained 11.0%
volatile material and was very toxic (CI = 408). Comparison of the
biological response of the three formulations indicated the volatile
component, identified by infrared spectrophotometry and mass
spectrometry to be methyl methacrylate, contributed substantially
to the biological response and both parameters were correlated with
the level of methanol extractable components. The relatively com-
plete curing obtained with DPT under the conditions of curing
used in this study cannot be extrapolated to conditions of "cold
curing" employed in situ, since all formulations with DPT were
cured in a dental press at elevated temperatures. Formulations 8
and 9 (Table 11),which contained 1 and 2% excess monomer re-
spectively, and cured at ambient temperature, gave two of the

highest biological responses (CI = 318 and 394, respectively), a
further indication of the importance of free monomer in influencing
biological response.
Gas Chromatographic Analysis of Extracts.-Methanol extracts
of formulations having 2 0.75% DPT contained quantities of DPT
correlated with concentrations in the initial formulation, The
quantity of ethylene dimethacrylate extracted by methanol was
inversely proportional to the degree of curing, none being found (less
than 1ng) in the extracts of highly cured samples. These two com-
pounds and MMA were the principal components found in methanol
extracts; other very minor unidentified components were sometimes
present.
CONCLUSIONS
The results of this investigation support the following conclusions.
1. The biological tests examined, particularly the in vitro tests,
are responsive to formulation and curing conditions of PMMA and
are suitable for primary toxicity screening.
The results of this study suggest that methanol-extractable
residues of PMMA formulations in excess of 0.7% are significantly
related to adverse (toxic) biological response, and that residue weights
might serve as a useful index of formulation viability in the quality
control of PiLIRIIA formulations when used in conjunction with
biological tests.
PMMA can be expected to vary widely in toxicity, depending
on formulation, curing conditions, and physical history of the
material, a higher degree of variability being expected for “cold
cured” formulations. A need for rigorous control of formulation
procedures and protocols, particularly for “cold curing,” is indicated.
In vitro tests, particularly tissue culture and hemolysis tests
on materials, are the most sensitive to formulation variables and yield
the greatest information on acute toxicity. Erst-round testing of
new formulations, especially those containing additives, should in-
clude the intramuscular implantation and the intracutancous irri-
tation test with a cottonseed oil extract. On the basis of this study,
it is unlikely that a formulation having significant acute toxicity
would not be detected using the above series of tests.
2.
3.
4.

5. The results of this investigation are pertinent primarily to the
evaluation of test procedures, and do not relate to specific end use
conditions of PMMA beyond the conclusions drawn above.
This study was supported through a research contract, no. 223-73-5231,of the
Food and Drug Administration, Washington, D. C.
The technical assistance of the staff of the Materials Science Toxicology
Laboratories, particularly Mrs. Alice Spralling, Mr. Michael Malik, Mrs. Eula
Baldwin, Mrs. Ramone Marquardt, and Dr. George Hung is gratefully acknowl-
edged. Computational serviceswere provided through USPHS Grant HL-09495.
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Revised February 4, 1975

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