JOURNAL OF ENDODONTICS I VOL 2, NO 4, APRIL 1976

E v a l u a t i o n of t h e c y t o t o x i c i t y of root c a n a l
s e a l i n g a g e n t s on t i s s u e c u l t u r e c e l l s in vitro:
Grossman's sealer, N2 (permanent),
R i c k e r t ' s s e a l e r , a n d Cavit

Donald D. Antrim, DDS, MS, Loma Linda, Calif

A n in vitro s t u d y with r a d i o a c t i v e l y l a b e l e d tissue culture cells tissue to preclude any possible harm-
w a s c o n d u c t e d o v e r a s e v e n m o n t h period on G r o s s m a n ' s sealer, ful responses. One of the requirements
N2 (permanent), Rickert's sealer, a n d Cavit to d e t e r m i n e the of an ideal root canal sealer or filling
l a s t i n g a n d relative tissue toxicity of these materials. In this material is that it be nonirritating to
s t u d y it w a s o b s e r v e d that all the m a t e r i a l s s t u d i e d p o s s e s s lasting the periapical tissues. Studies of the
biologic response to endodontic ma-
tissue toxicity. W h e n relative toxicity is c o n s i d e r e d , Grossman.'s
terials have been conducted with
is the most toxic, f o l l o w e d b y N2, Rickert's, a n d Cavit. Both various species and methods. These
G r o s s m a n ' s a n d N2 s h o u l d b e c o n s i d e r e d h i g h l y toxic; Rickert's pre,cious studies have involved pri-
d i s p l a y s m o d e r a t e toxicity. Cavit s h o u l d b e c o n s i d e r e d mild to marily tissue culture experiments,
m o d e r a t e in toxicity. The u s e of r a d i o a c t i v e - l a b e l e d tissue cells implant studies, or usage tests in
a s a m e t h o d of testing root c a n a l filling m a t e r i a l s is a rapid, animal or human teeth. The biologic
h i g h l y sensitive m e t h o d that a l l o w s a d e q u a t e cell-material c o n t a c t effect of various root filling materials
a n d permits objective q u a n t i t a t i o n of cell d a m a g e with a c c u r a c y . can be tested by the examination of
animal and human biopsy prepara-
tions at different periods of time after
implantation or usage. Such examina-
The objective of endodontic therapy concern to all dentists who perform tions are subject to great biologic
is restoration of the treated tooth to endodontic therapy. variation. For successive examinations
its proper form and function in the The culmination of careful endo- it is scarcely possible for sufficient
masticatory apparatus. In doing so, dontic treatment involves the ~illing human biopsy preparations to be ob-
the least amount of irritation pro- or obliteration of the canal to obtain tained.
duced by filling substances is of an adequate seal as close as possible Preliminary examination of the
primary importance. Filling materials to the cementodentinal junction, with biologic effect of alien substances can
inadvertently extruded beyond the an inert material. To accomplish this be carried out conveniently in tissue
confines of the root canal into the the dentist has at his disposal many or cell culture studies. The toxicity of
periapical tissue can cause inflamma- cements, pastes, plastics, and solids. root canal filling materials has been
tion and resultant pain. The degree of Sealers and filling materials used studied extensively by in vitro test-
discomfort experienced by the patient in endodontic treatment should be ing. 1-z The results of such experiments,
after a root canal filling should be of compatible with living connective however, have varied not only with

111

the root canal filling material in
question, but also with the testing
method. Therefore, it became impor-
tant to develop a more objective
method for such evaluation. Such a
method was developed by Spang-
berg. 4 It involved (1) complete ma-
terial-cell contact; (2) the possibility
of varying exposure time related to
the stage of setting of the material;
and (3) quantitation of the results.
The advantages of this in vitro
method lie in its simplicity, in the
possibility of control, and in the
standardizing of experimental condi-
tions.
The objective of this study was to
compare, by using Spangberg's in
vitro method, 4 the lasting and relative
tissue toxicity of four root canal
sealing materials: Grossman's sealer*;
Rickert's sealert; N2 (permanent)z~;
and Cavit.w
MATERIALS AND METHODS
The root canal filling materials
had the following compositions:
Grossman's root canal sealer: zinc
oxide, 42%; Staybelite Resin, 27%,
bismuth subcarbonate, 15%; barium
sulfate, 15% ; borax, t % ; and
eugenol as the liquid component.
Rickert's root canal sealer: molecular
silver, 25%; zinc oxide, 34%;
dithymol diiodide, 11% ; oleo resins,
30%; and clove oil and Canada
balsam as the liquid component. N2
(permanent) (RC-2B formula) : hy-
drocortisone, 1.5%; titanium dioxide,
2.0%; trioxymethylene, 7.0%; lead
oxide, 16.5%; zinc oxide, 73.0%; and
eugenol ~as the liquid component.
Cavit: zinc oxide, calcium sulfate,
zinc sulfate, glycol acetate, polyvinyl
acetate, polyvinyl chloride-acetate,
triethanalamine, and red pigment.
All materials were mixed according
to the manufacturers' instructions.
Cells
Three- to five-day-old cultures of
112
JOURNAL OF ENDODONTICS I VOL 2, NO 4, APRIL 1976
Test
I
I
I
J
,i ,?be, sup.... tant I
~L I Centrifugation~ ~ (t-Samples)'-~ 2
I == -- =~oRelease
0
I
}
r-.~mples I
f
Fig 1--Spangberg method /or testing cytotoxicity o/ a solid material in vitro.
Cells in culture flask are labeled with sodium chromate. Test material is placed
in slide culture chamber. Labeled cells are added on top o/ material, followed
by incubation at 37 C. A t conclusion of experiment, fluid in culture chamber is
transferred to test t u b e s / o r / i n a l centri/ugation, placed in a planchet, dried, and
counted in gamma counter.
KB cells derived from a carcinoma of supplied as sodium chromate in
the pharynx were used. During growth isotonic saline solution. The isotope
the medium was changed every other was added to the target cells in about
day and the day the cells were labeled 1.5 to 2 microcuries per 106 ceils 20
(the day before the experiment). hours before the experiment. After
The cells were harvested with ATV, the labeling period, the cells were
a balanced saline solution of harvested with A T V and suspended
ethylenediaminetetraacetic acid with in the experimental medium. The cell
trypsin. suspension was washed and centri-
fuged (500 g) in medium five times.
Medium
The synthetic medium was Mc- Experimental Procedures
Coy's 5A modified medium with The procedure is shown schemati-
glutamine. The medium was supple- cally in Figure 1. A sample of ma-
mented with fetal calf serum (1- to terial (a cylinder 2 mm with a 4.76
6-month-old calves) to a final con- mm diameter) to be tested was placed
centration of 1.0%. Added to the into the center of the culture chamber.
medium was 500 international units Then, 1.0 ml of the labeled cell sus-
(IU) benzylpenicillin, 300/xg strepto- pension (2.5 x 10 ~ cells per milli-
mycin, and 0.25/zg Fungizone per liter) was added to cover the material
milliliter. The culture medium was sample. The materials had hardened
buffered with 2.2 mg sodium bicar- for periods of 24 hours, 72 hours, one
bonate per milliliter. week, two weeks, one month, two
months, three months, five months,
L a b e l i n q of C e l l s six months, and seven months. The
Radioactive chromium (51Cr) was chambers were then incubated for
 JOURNAL OF ENDODONTICS ] VOL 2, NO 4, APRIL 1976

Table .e Percent release of 51Cr (mean 1 error).

Material 24 hr 72 hr 1 week 2 weok 1 mo 2 mo 3 mo 5 mo 6 mo 7 mo
4-hour cell-material contact
SRC 6.3+_0.9 6.8+_0.9 6.9+_1.6 6.6+_0.3 7.4_+0.7 5.4+_0.9 6.8 9.5--.0.8 8.5_+0.8 7.8+_0.3
G 66.0_+5.0 19.0+_2.2 12.6+_1.3 14.9_+2.6 19.6_+1.8 12.3 16.1-+2.3 11.4-+1.7 11.2+_1.3 18.2-+1.8
N2 22.4-+2.2 13.9+_2.7 13.7_+2.0 14.6__.2.3 16.2-+1.9 12.4+_3.2 15.6-+2.0 29.0-+1.4 31.2_+1.9 21.0
R 20.9_+2.8 9.3_+2.6 8.2+_1.1 8.5 10.2+1.7 6.3_+0.7 9.9_+0.5 11.3_+1.0 13.8_+1.9 14.0_+1.9
C 5.3_+1.3 15.8+-4.0 6,4___1.6 7.8_+1.3 19.0+-3.0 5.2_+1.0 10.9 19.2+_2.7 10.8+_0.9 14.1_+1.0
24-hour ceil-material contact
SRC 21.5+_.0.4 20.5+-0.4 18.8+-0.6 22.5_+0.9 23.0+_1.3 18.8+_1.4 21.8+-1.7 23.6 23.2+_1.3 22.2-+0.9
G 70.0_+1.8 66.2+_2.6 56.7_+4.4 76.4__.2.3 74.4_+2.1 67.9_+0.8 67.2-+1.4 49.8+_2.5 61.6-+2.7 67.2_+2.2
N2 77.5_+1.7 56.7_+5.1 58.5+-1.2 64.0__.2.5 49.9_+1.6 35.0_+1.3 34.1_+2.6 45.2_+3.4 59.3_+2.2 42.0_+1.9
R 59.0+1.5 31.9_+1.6 31.6_+0.9 38.5_+2.6 38.2_.+2.6 31.3_+1.8 27.3+-0.8 33.4+-1.8 30.9_+1.6 47.0_+2.9
C 16.8_.+1.7 22.0_+1.9 16.5_+1.8 20.5_+2.0 46.0_+1.8 30.6_+1.3 37.2+_2.8 36.0_+4.2 35.7_+_1.9 45.4_+0.6

Note: Six experiments were made for each material and control in each time concentration group.
SRC, spontaneous release controls; G, Grossman's sealer; R, Rickert's sealer; C, Cavit.

4-hour and 24-hour periods at 37 C. Approximately 585 experiments were RESULTS

In all experiments, 0.5-ml samples
were withdrawn randomly during dis-
persion of the labeled cells into the
chambers with the material. These
samples (r samples) were used as
reference points for calculating the
nlCr release in the experiments. In
each experiment, empty culture cham-
bers were prepared with cell sus-
pension and were used as spontaneous
release controls.
At the conclusion of the experi-
ment, the medium in the culture
chambers was withdrawn, transferred
to test tubes, and centrifuged (500 g)
for ten minutes. Then 0.5 ml of
supernatant (t samples) was trans-
ferred to planchets, dried under a
heat lamp, and counted for ten
minutes in a gamma particle counter.
The percentage of 5~Cr release was
calculated on .the basis of the total
amount of label incorporated in the
target cells; that is:
release % =51Cr in t samples
X 100.
5~Cr in r samples
performed in this way.
The amount of toxicity displayed
by the materials is determined by the
amount of cell lysis and subsequent
release of radioactive material.
Sanderson 5 showed that 100%
lysed cells resulted in about 60% to
70% 51Cr release. It is, therefore,
reasonable to ,assume that, indepen-
dent of any changes or variations
produced by experimental conditions,
any release of 51Cr exceeding 70%
would indicate that cytolysis has oc-
curred in aU cells.
Consequently, an approximation
of degree of toxicity can be deter-
mined based on the percent release
the materials have displayed. A n y
material near the 70% release could
be considered highly toxic. Materials
near the percent release of the spon-
taneous release control would be con-
sidered mild in toxicity. Materials
falling in between these extremes
would range from mild to moderate
to highly toxic, depending on the per-
cent of 5xCr released.
The results of the cell-material in-
teractions .and subsequent percent
release of radioactive material are
listed in the Table. The same informa-
tion is shown graphically in Figures
2 and 3.
The four-hour cell-material contact
showed a relatively stable picfure. The
initial 24-hour responses of Gross-
man's sealer, N 2 (permanent), and
Rickert's sealer were significantly
more toxic than the spontaneous re-
lease control ( P < 0 . 0 0 1 ) . As the ex-
periments progressed in time the
three-, five-, six-, and seven-month
samples of N 2 (permanent) proved
to be significantly more toxic than the
spontaneous release control. This
would indicate that N2 (permanent)
is slightly more toxic, with some
significance at the older ages, than
the other materials. Cavit showed
somewhat erratic results.
The 24-hour cell-material contact
showed significant toxicity for all
materials when compared to the spon-
taneous release control ( P < 0 . 0 0 1 ) .

113

Fig 2 - - P e r c e n t release over speci-
/ied time intervals, /our-hour con-
tact.
Cavit initially showed a low toxicity,
but this increased at the one-month
time period and maintained a high
level of toxicity thereafter. Although
some fluctuation existed with each
material, the toxic effect was present
throughout all age levels and thus dis-
played lasting toxicity.
A n overall analysis of the data
was done using a general linear
hypothesis. A comparison of all ma-
terials with the spontaneous release
control showed a significant difference
in all materials ( P ( 0 . 0 0 1 ) . A n over-
all ranking of relative toxicity
showed Grossman's sealer to be the
most toxic, followed by N2 (perma-
nent), Rickert's sealer, and Cavit.
Grossman's sealer was significantly
more toxic when compared directly
with N2 (permanent) ( P ( 0 . 0 0 1 ) ,
and Rickert's sealer was more toxic
than Cavit.
An individual comparison of the
relative toxicity of all the materials
was made by use of the q test of
multiple confidence intervals using a
comparison of means. These compari-
sons showed, as can be seen in Figure
2, that the results of the four-hour
contact were virtually nonsignificant,
114
JOURNAL OF ENDODONTICS [ VOI. 2, NO 4, APRIL 1976
Fig 3---Percent release over speci-
/ied time intervals, 24-hour contact.
with a few exceptions which have been
mentioned.
A n analysis of the 24-hour cell-
material contact seemed to correlate
with the results seen in the general
linear hypothesis analysis. Grossman's
sealer was the most toxic, followed
by N2 (permanent), Rickert's sealer,
and Cavit. There was some over-
lapping of the various materials, as
can be seen in Figure 3.
When compared with the spontane-
ous release controls at the 4-hour and
24-hour levels, the materials show a
greater degree of toxicity at the 24-
hour level.
DISCUSSION
A comparison of the toxicity of the
sealers used in the current study
(except Cavit) with the study of
Spangberg a n d . Langeland 6 shows
some agreement. Spangberg and
Langeland showed that all of these
materials were highly toxic, that is,
total cell lysis, when the material
was set and tested at 1-hour, 4-hour,
and 24-hour cell-material contact
times. 6 In the current study the 24-
hour sample (set material) showed
total lysis when the cells were in con-
tact with the material for 24 hours.
The 4-hour cell-material contact,
however, did not display as much
toxicity as that reported by Spangberg
and Langeland. 6 Only Grossman's
sealer displayed total lysis. Rickert's
sealer and N2 (permanent) showed
approximately 50% lysis.
Preliminary examination of the
biologic effect of alien substances can
be carried out conveniently in tissue
or cell culture. The advantages of the
in vitro method lies not only in the
possibilities of control and observa-
tion of reaction, but also in the pos-
sibility of standardizing experimental
conditions. In establishing a measure
of cytotoxicity, Spangbe.rg 4 has used
a relatively quantitative in vitro sys-
tem that has already been proved to be
of considerable value in immunologic
studies for the determination of cell
lysis. His new method for testing
dental materials is rapid, highly sensi-
tive, allows adequate cell-material
contact, and permits objective quanti-
tation of cell damage with good ac-
curacy.
It is apparent, however, that cau-
tion must be exercised in attempting
to apply the results obtained by .this

method to the clinical situation. The
results of in vitro studies .are quite
different from those of in vivo ex-
periments. It is difficult, if not
impossible, to extrapolate the results
of an in vitro assessment of cyto-
toxicity involving tissue culture cells
to connective ceils in vivo.
Rappaport, Lilly, and Kapsimalis 3
showed some degree of correlation
between tissue culture testing and in
vivo implantation studies. They found
that the materials tested were fairly
consistent from study to study. Some
variables were noted, but for the most
part the materials exhibited a fairly
uniform response within each test
group when all materials were com-
pared.
Spangberg 1 tested several materials
with the tissue culture test, implanta-
tion with polyethylene tubes, and use
in dogs' teeth. He said that there is
an adequate correlation between in
vitro and in vivo tests.
Kawahara 2 recognized differences
in the results of in vitro and in vivo
studies. He further stated that it was,
however, necessary to investigate
analytically biologic responses to
various dental materials by means of
in vitro studies before clinical use.
He felt with the development of tissue
culture techniques in recent years that
the in vitro study may become .one of
the important methods for the biologic
standardization of dental materials.
Spangberg's 4 refinements of the tech-
nique may well have solidified this
prediction.
One deviation in the current study
from the technique of Spangberg and
Langeland 6 involved the material
sample size. They spread the material
over the entire bottom of the culture
chamber. This allowed complete cell-
material contact. Preliminary experi-
ments showed that the material that
was spread over the bottom of the
chamber and allowed to set for more
than 24 hours produced a cracking,
JOURNAL OF ENDODONTICS [ VOL 2, NO 4, APRIL 1976
peeling, dried-out product unsuitable
for cell-material contact. To elimina.te
this problem, the material was placed
in polyethylene tube pieces 2-mm
thick with an inside diameter of
4.76 mm. This resulted in material
samples with uniform surface area.
Kawahara 2 felt that one important
problem of the biologic testing of
cytotoxicity of dental materials is to
control strictly the active surface area
of the test piece that is in contact with
the culture medium. The use of the
tubing satisfies this requirement. The
tubing was removed from the test
sample before addition of the tissue
culture cells.
This experiment was conducted
over a seven-month period. It was,
therefore, necessary to use several
batches of cells, since various groups
of aged materials were grouped and
evaluated at different times. Although
the numbers of cells used for each ex-
periment can be approximated, exact
numbers cannot be obtained. The cells
are quite temperamental and some-
times are difficult to grow. To ensure
proper labeling and subsequent satis-
factory experimental results, cells that
look and act healthy and normal
should be used. The strength of the
51Cr is altered by time and great care
must be taken in getting the proper
amount of labeling into the cell
samples. These factors may be respon-
sible for the fluctuation seen in the
individual materials in this experiment
from the 24-hour to the seven-month
time periods. Ideally, the experiment
should be conducted at one time on
all groups of aged material using a
common supply of cells labeled with
the same strength of ~lCr.
Another variable that must be con-
sidered is material degeneration or
alteration during aging. All materials
were mixed according to the manu-
facturers' instructions in sterile con-
ditions, placed into the polyethylene
tubing, and allowed to set. The ma-
terials were then placed into sterile
test tubes with a damp cotton roll
and incubated at 37 C. It was thought
that these conditions approximated the
atmosphere found in the oral cavity.
All materials held up well except
Cavit, which showed some deteriora-
tion, This seemed to be due to its
absorption of the moisture content
within the test tube. This could
somewhat explain Cavit's fluctuant
behavior, although it proved to be the
least toxic material.
Tissue reaction to a material re-
flects a combination of the toxic
effect of the material per se and the
response to some of its physical pro-
perties. A material that is toxic in
vitro can always be expected to cause
tissue irritation. Low toxicity in vitro,
however, is not equal to low tissue
irritation. This depends on variations
in resorbability, solubility in tissue
fluids, or fragmentation, by which
inflammatory reactions may be
caused.r, s
Materials that have aged up to
seven months may show tissue toxi-
city in vitro. However, in human
tissues these materials may have been
affected by physiologic processes to
the point where they are being con-
tairred by the body and no longer pre-
sent a problem. Even though one ma-
terial may prove to be more toxic in
vitro than another, it m a y be removed
by the body defenses more readily
and thus do less damage than one that
displayed less toxicity.
Because of the highly toxic nature
of Grossman's sealer and N2 (perma-
nent), and to a lesser degree Rickert's
sealer, it is not recommended that
these materials be used as total root
canal filling materials. Use of these
materials as sealers in conjunction with
a solid or semisolid filling material
should be done judiciously to prevent
extrusion of gross amounts into
periapical tissues during obturation of
the canal.
115

SUMMARY
This study was directed toward
determining, by in vitro means, the
lasting and relative toxicity of four
root canal sealing agents. The method
used involved radioactively labeled
tissue culture cells. These cells were
allowed to contact the material sam-
ples. This resulted in cell lysis and
release of the radioactive particles.
The particles were counted in a
gamma counter. Comparisons of the
amount of release gave an accurate
quantitative comparison between the
materials tested and, thus, a relative
degree of toxicity. Performing the
experiment over periods of from 24
hours to 7 months and comparing the
amounts of release gave an indication
of lasting toxicity. The four sealing
materials were Grossman's sealer,
N2 (permanent), Rickert's sealer, and
Cavit.
CONCLUSIONS
The use of radioactive-labeled
tissue culture cells as a method of test-
ing dental materials in vitro is rapid
and highly sensitive; it allows ad-
equate cell-material contact and per-
mits objective quantitation of cell
116
JOURNAL OF ENDODONTICS
damage with accuracy.
All four sealing materials--Gross-
man's sealer, N2 (permanent),
Rickert's sealer, and Cavit--possess
some degree of lasting tissue toxicity.
The relative toxicity of these ma-
terials in descending degree of toxicity
is Grossman's sealer, N2 (permanent),
Rickert's sealer, and Cavit. Both
Grossman's sealer and N2 (perma-
nent), should be considered highly
toxic, with Rickert's displaying moder-
ate toxicity. Cavit should be con-
sidered mild to moderate in toxicity.
*Walser Drugs, San Marino, Calif.
tPatterson Dental Supply Co., Ana-
heim, Calif.
~tSteri-Kem, Inc., Whittier, Calif.
w Dental Products Co.,
Philadelphia.
The author acknowledges the tech-
nical assistance of Ms. Robin Hill.
The opinions or assertions contained
herein are those of the author and are
not to be construed as official or as
reflecting the views of the Department
of the Navy or of the Department of
Defense.
Dr. Antrim, formerly a graduate
student, division of endodontics, School
of Dentistry, Loma Linda University, is
VOL 2, NO 4, APRIL 1976
a Commander, DC, USN. Reprint re-
quests should be directed to Cdr. D. D.
Antrim, 1615 E Mission Rd, Fallbrook,
Calif 92028.
RefeFence et
1. Spangberg, L. Biological effects of
root canal filling materials. Odontol
Rev 20:123, 133, 283, 289, 427, 1969.
2. Kawahara, H.; Yamagami, A.; and
Nakamura, M., Jr. Biological testing of
dental materials by means of tissue cul-
ture. Int Dent J 18:443 June 1968.
3. Rappaport, H.M.; Lilly, G.E.; and
Kapsimalis, P. Toxicity of endodontic
filling materials. Oral Surg 18:785 Dec
1964.
4. Spangberg, L. Kinetic and quantita-
tive evaluation of material cytotoxicity
in vitro. Oral Surg 35:389 March 1973.
5. Sanderson, A.T. Applications of iso-
immune cytolysis using radiolabeled
target ceils. Nature 204:250 Oct 1964.
6. Spangberg, L., and Langeland, K.
Biologic effects of dental materials. 1.
Toxicity of root canal filling materials
on HeLa cells .in vitro. Oral Surg
35:402 March 1973.
7. Langeland, K.; Guttuso, J.; Lange-
land, L.K.; and Tobon, G. Methods in
the study of biologic responses to
endodontic materials; tissue response to
N2. Oral Surg 27:522 April 1969.
8. Spangberg, L. Biological effects of
root canal filling materials. 7. Reaction
of bony tissue to implanted root canal
filling material in guinea pigs. Odontol
Tidsk 77:133 April 15, 1969.