Additives to tampons

Absorbent products, especially catamenial tampons, for absorbing body fluids, such as menstrual fluid, blood and wound exudates, comprise an amount of a compound effective to inhibit the production of toxic shock syndrome toxin-1 by Staphylococcus aureus bacteria when the products are brought into contact with the bacteria. The compound is selected from the group consisting of monoesters of a polyhydric aliphatic alcohol and a C.sub.8 -C.sub.18 fatty acid; diesters of a polyhydric aliphatic alcohol and a C.sub.8 -C.sub.18 fatty acid; and mixtures thereof. The monoesters and diesters have at least one hydroxyl group associated with their aliphatic alcohol residue.

FIELD OF THE INVENTION 
The present invention relates to absorbent products and especially to 
absorbent products such as tampons, sanitary napkins, wound dressings and 
the like which are adapted to absorb body fluids like menstrual fluid, 
blood and wound exudates. More particularly, the invention relates to 
catamenial tampons which, owing to the presence therein or thereon of 
certain inhibitory agents, reduce the amount of toxins produced by 
bacteria coming into contact therewith. 
BACKGROUND OF THE INVENTION 
Menstrually occurring toxic shock syndrome (TSS), a severe and sometimes 
fatal multi-system disease associated with infection or colonization by 
Staphylococcus aureus (S. aureus) bacteria, has been linked to the use of 
tampons during menstruation. The disease is believed to be caused by toxic 
shock syndrome toxin-1 (TSST-1), the toxin produced by the majority of 
Staphlococcal strains isolated from menstrual TSS patients. 
Subsequent to the publication of reports associating toxic shock syndrome 
with the use of tampons, a number of investigators undertook studies 
designed to evaluate the effect of tampons on growth of S. aureus bacteria 
as well as the effect of tampons on the production of TSST-1 by that 
bacteria. Early efforts to elucidate the role of tampons in TSS yielded 
conflicting data. Schlievert et al. (Obstet. Gynecol., Vol. 64, pp. 
666-670, November 1984) studied the effect of tampons on S. aureus to 
evaluate whether or not tampon components increase growth of S. aureus and 
production of toxic shock syndrome toxin-1. It was concluded that, under 
the test conditions of their study, tampon components provide neither 
nutrients for growth of toxic shock syndrome S. aureus nor factors that 
induce production of toxic shock syndrome toxin-1 above control levels. 
After six hour incubation, some commercially available tampons which were 
tested were inhibitory to bacterial growth and suppressed toxin 
production. Others suppressed toxin production but did not inhibit cell 
growth. One tampon inhibited cell growth but increased the amount of toxin 
produced. On the other hand, Tierno and Hanna (Contraception, Vol. 31, pp 
185-194, 1985) reported that in their experiments tampons did stimulate S. 
aureus to produce TSST-1. 
Reiser et al. (J. Clin. Microbiol., Vol. 25, No. 8, pp 1450-1452, August 
1987) thereafter reported the results of tests they conducted to determine 
the effect of four brands of tampons on production of toxic shock syndrome 
toxin-1. The amount of air available to the tampons which were tested was 
limited to that contained in sacs (made from cellulose sausage casing with 
a molecular weight cut-off of less than 10,000) in which the tampons were 
enclosed during testing. This method was deemed advantageous in that the 
limited amount of available air was thought to mimic more closely than 
previously used methods the in vivo condition in the vagina during 
menstruation with a tampon in place and in that the tampons which were 
tested were not altered prior to testing. The results of the tests 
conducted by Reiser et al. indicated that tampons provide increased 
surface area for the S. aureus bacteria to grow and adequate oxygen for 
toxin production. No significant inhibition of growth of the staphylococci 
bacteria or TSST-1 production by any of the tampons tested was noted. 
Robbins et al., publishing in J. Clinical Microbiol., Vol. 25, No. 8, pp. 
1446-1449, August 1987 at the same time as Reiser et al., reported the 
effect of 17 commercially available tampons on TSST-1 toxin production 
using a disk-membrane-agar (DMA) method, with incubation at 37.degree. C. 
for 19 hours under 5% CO.sub.2 in air. Filter membranes overlaying agar 
medium (with or without blood) in small petri dishes were spread 
inoculated with a TSST-1 producing strain of S. aureus. Robbins et al. 
concluded that the main role of tampons in TSS may be that of providing a 
fibrous surface for heavy colonization and sufficient air for TSST-1 
production. In addition, they found evidence of inhibition of TSST-1 
production by additives such as the deodorant/surfactant used in a 
commercially available deodorant tampon and a decrease in TSST-1 
production by inhibiting growth of S. aureus as was observed in the case 
of a different commercially available tampon. It was thought that both 
inhibition of TSST-1 production and inhibition of S. aureus growth might 
prove to be important in reducing the risk of TSS. 
U.S. Pat. No. 4,405,323 to Auerbach discloses a tampon designed to 
eliminate the hazards of toxic shock syndrome and dysmenorrhea. The tampon 
has incorporated therein an antibacterial agent which is said to disperse 
on contact with body fluids and prevent development of the organisms which 
produce the toxins which cause toxic shock syndrome. Among the 
antibacterial materials disclosed for use are povidone-iodine compound, 
mercury, zinc, penicillin, erythromycin and nitrofurazone. 
Patent Cooperation Treaty Publication No. WO 86/05388 (published Sep. 25, 
1986) to Kass teaches that the inclusion of a salt of a nontoxic divalent 
cation in absorptive pads, e.g. catamenial tampons, inhibits production of 
toxic shock syndrome toxin-1 and other staphylococcal products during use 
of said absorptive pad. Suitable salts include those of magnesium, barium, 
calcium or strontium (preferred) or of other divalent cations such as 
zinc, manganese, copper, iron, nickel and the like. The anionic portion of 
the salt is not critical. Magnesium stearate and magnesium acetate are 
particularly preferred salts for use in the invention. 
In U.S. Pat. No. 4,374,522 to Olevsky it is stated that patterns of use of 
catamenial tampon seem to indicate that high absorptive capacity with the 
concomitant extended period of use of certain tampons are factors which 
contribute to the formation of toxic shock syndrome. The invention 
theorizes that tampons having limited absorptive capacity and requiring 
relatively more frequent changes may be desirable. The Olevsky patent 
provides a tampon made of conventional cellulosic materials, such as rayon 
fibers, which have been compressed into a bullet-shape with an open bottom 
surface sealed by a fluid impermeable sheet. The fluid impermeable bottom 
and the traditional bullet shaped pledger define a hollow core central 
reservoir area which is said to serve as a reservoir for excess menstrual 
fluid. 
U.S. Pat. No. 4,431,427 to Lefren et al. discloses menstrual tampons 
comprising physiologically safe, water-soluble acids in their monomeric, 
oligomeric or polymeric forms. Citric, glycolic, malic, tartaric and 
lactic acids are disclosed as being useful in the practice of the 
invention. The presence of one or more of the above-noted acids in a 
tampon is said to inhibit the growth of bacteria responsible for toxic 
shock. Where an acid is used in its polymeric form, the tampon may 
additionally include an enzyme to hydrolyze the polymeric acid to its 
monomeric form. 
Canadian Patent No. 1,123,155 to Sipos discloses a catamenial tampon for 
preventing toxic shock syndrome during menstrual flow. The body of the 
tampon, which is open at the insertion end and is closed at the withdrawal 
end, is snugly surrounded in its expanded condition by a fluid proof, thin 
and flexible membrane. This membrane, which can be made of polyethylene 
sheet, is biased against the vaginal wall during use of the tampon, is 
neutral to the vaginal mucosa and is completely impermeable to bacteria, 
viruses and toxic decomposition products of the menstrual flow. 
Canadian Patent No. 1,192,701 to Bardhan discloses a tampon for the 
absorption of menstrual flow and comprising an inner layer of 
liquid-absorbent material and an outer layer which surrounds and encloses 
the inner layer. Menstrual discharge may flow inwardly to the inner layer 
but the outer layer is impervious to the passage of menstrual fluid 
outwardly from the inner layer. A plurality of liquid absorbent wicks 
extending from the inner layer through apertures formed in the outer layer 
serve as conduits for the flow of menstrual discharge from outside the 
tampon to the inner layer thereof. The disclosed structure is said to 
minimize the availability of discharge outside the tampon with a resulting 
reduction in the likelihood of growth of S. aureus and consequently its 
production of toxin. This patent also discloses that an antimicrobial 
compound which is bactericidal or bacteriostatic to S. aureus may be 
included in the inner layer. The antimicrobial agent may take the form of 
an antibiotic (such as penicillin, erythromycin, tetracycline or 
neomycin), a chemotherapeutic agent (such as a sulfonamide) or a 
disinfectant (such as phenol). The patent states that since the tampon is 
protected by its outer layer from contact with the vaginal wall, the risk 
of an allergic or other adverse reaction to the anti-microbial agent is 
minimized, and since the antimicrobial agent is also protected by the 
outer layer from contact with menstrual discharge, there is little risk of 
the destruction of commensal organisms in the vagina or development of 
resistance to the antimicrobial agent by S. aureus in any menstrual 
discharge outside the vagina. 
S. Notermans et al. (Journal of Food Safety, Vol. 3 (1981), pages 83-88) 
reported that glyceryl monolaurate, when used in the proportion of 5 g per 
kg. of meat slurry (pH 6.0-6.2) inhibited toxin. productions by 
Clostridium botulinum type A, type B and type E. This article does not 
mention Staphylococcus aureus nor any toxin(s) produced therefrom nor does 
it mention absorbent products or toxic shock syndrome. 
U.S. Pat. No. 4,585,792 to Jacob et al. discloses that L-ascorbic acid when 
topically applied to the vaginal area of a human female during manses will 
inactivate toxins known to contribute to Toxic Shock Syndrome. The 
ascorbic acid compound may be carried by a vaginal tampon. The disclosure 
of U.S. Pat. No. 4,722,937, is to the same effect. 
U.S. Pat. No. 4,413,986 to Jacobs discloses a sterilely-packaged tampon 
assembly for sterile insertion of a tampon into the vagina and having a 
guide tube telescoped around an insertion tube and a flexible sheath 
attached to the inner end of the guide tube and tucked into the inner end 
of the insertion tube. In use, as the insertion tube is pushed through the 
guide tube and into the vagina, the flexible sheath is pulled over the 
inner end of the insertion tube and extends along the exterior thereof. 
The portion of the insertion tube which is inserted into the vagina is at 
all times fully sheathed by the flexible sheath. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, it has now been discovered that 
an absorbent product comprising a compound selected from the group 
consisting of: 
a) a monoester of a polyhydric aliphatic alcohol and a fatty acid 
containing from eight to eighteen carbon atoms and wherein said monoester 
has at least one hydroxyl group associated with its aliphatic alcohol 
residue; 
b) diesters of a polyhydric aliphatic alcohol and a fatty acid containing 
from eight to eighteen carbon atoms and wherein said diester has at least 
one hydroxyl group associated with its aliphatic alcohol residue; and 
c) mixtures of the aforesaid monoesters and diesters unexpectedly reduces 
the amount of toxic shock syndrome toxin-1 produced in vitro when said 
absorbent product is exposed to Staphylococcus aureus bacteria. 
The fatty acid portion of the aforementioned monoesters and diesters may be 
derived from caprylic, captic, lauric, myristic, palmitic and stearic 
acids, which are saturated fatty acids whose chain lengths, respectively, 
are C.sub.8, C.sub.10, C.sub.12, C.sub.14, C.sub.16 and C.sub.18. The 
fatty acid portion of the aforementioned monoesters and diesters may be 
derived as well from unsaturated fatty acids having carbon chain lengths 
also ranging from C.sub.8 to C.sub.18, one example of such unsaturated 
fatty acids being oleic acid. The preferred fatty acid for use in the 
practice of the present invention is lauric acid, a saturated fatty acid 
whose chemical formula is C.sub.11 H.sub.23 COOH. 
As used in this specification and the appended claims, the term "aliphatic" 
has the meaning usually accorded it in organic chemistry, i.e. "aliphatic" 
refers to organic compounds characterized by straight--or branched--chain 
arrangement of the constituent carbon atoms. 
As used in this specification and the appended claims, the term 
"polyhydric" refers to the presence in a chemical compound of at least two 
hydroxyl (OH) groups. Thus, a polyhydric aliphatic alcohol is one which 
has at least two hydroxyl groups and in which the carbon backbone is 
either straight or branched. 
Polyhydric alcohols suitable for forming monoesters and/or diesters for use 
in the practice of the present invention are 1,2-ethanediol; 
1,2,3-propanetriol (glycerol); 1,3-propanediol; 1,4-butanediol; 
1,2,4-butanetriol and the like. The preferred polyhydric aliphatic alcohol 
for forming monoesters and diesters for use in the practice of the present 
invention is 1,2,3-propanetriol (commonly called glycerol) whose formula 
is HOCH.sub.2 CH(OH)CH.sub.2 OH. 
It will be observed that the esters which are useful in the practice of the 
present invention have at least one hydroxyl group associated with their 
aliphatic alcohol residue. Thus, it will be understood that the monoester 
of 1,2-ethanediol and one of the aforementioned fatty acids may be used in 
the practice of the present invention because said ester, whose general 
formula is 
##STR1## 
has at least one hydroxyl group (i.e. the hydroxyl group at the far 
right-hand side of the structural formula shown above) in that portion of 
the ester derived from the aliphatic alcohol 1,2-ethanediol. On the other 
hand, it will be understood that the diester of 1,2-ethanediol and one of 
the aforementioned fatty acids cannot be used in the practice of the 
present invention because said ester, whose general formula is 
##STR2## 
does not have at least one hydroxyl group in that portion of the ester 
derived from the 1,2-ethanediol. 
The monoester of glycerol and one of the designated fatty acids may be used 
in the practice of the present invention because that ester will have two 
hydroxyl groups associated therewith which are derived from the glycerol. 
The diester of glycerol and one of the designated fatty acids may also be 
used because that ester will have one hydroxyl group associated therewith 
which is derived from the aliphatic alcohol glycerol. Indeed, as will be 
seen hereinafter, blends of glycerol monolaurate and glycerol dilaurate 
have been found to be useful in the practice of the present invention. 
Finally, it will be understood that the triester of glycerol and one of 
the designated fatty acids cannot be used in the practice of the present 
invention because that ester does not have at least one hydroxyl group in 
that portion thereof which is derived from the aliphatic alcohol, i.e. 
glycerol. 
Preferred esters for use in the practice of the present invention are 
glyceryl monolaurate, glyceryl dilaurate and mixtures thereof. 
In accordance with the invention, the absorbent product contains an amount 
of the above-described ester which is effective to inhibit the formation 
of TSS toxin-1 when said product is exposed to S. aureus. For example, 
effective amounts have been found to be from about 0.1% and higher and, 
preferably, at least about 0.5% of the specified mono- or diester compound 
(or mixtures thereof), based on the weight of the absorbent material 
comprising the absorbent product. As used herein, the term "absorbent 
material" includes natural or synthetic fibers, films, foams, wood pulp, 
peat moss, superabsorbent polymers and the like which are capable, either 
inherently or by virtue of the manner in which they have been assembled, 
of absorbing liquids such as water, urine, menstrual fluids, blood, wound 
exudates and the like.

GENERAL PROCEDURE FOR PREING TAMPONS OF THE INVENTION 
During the course of conducting the investigations forming the basis for 
the present patent application, varying amounts of the aforementioned 
ester compounds (or mixtures thereof) were added to several different 
kinds of tampons. These tampons included ones prepared in the laboratories 
of the assignee of the present patent application as well as commercially 
available tampons made by several different manufacturers. Tampons of one 
manufacturer had different weights from those of another manufacturer and, 
indeed, no two tampons from a given manufacturer had identical weights. 
The ester compounds to be investigated were dissolved in isopropyl alcohol 
to form solutions which were then uniformly applied, by pipetting, to the 
outer surfaces of the various tampons, after which the isopropyl alcohol 
was evaporated to provide a tampon comprising the ester compound. 
In order to ensure that the absorptive capacity of the tampons was not 
exceeded, it was decided to fix the amount of isopropyl alcohol solution 
applied to each tampon at four (4) grams in all cases. In view of this 
desire to hold constant the weight of isopropyl alcohol solution applied 
to each tampon, it was necessary to vary the concentration of ester 
compound in the isopropyl alcohol solution in order to vary the level of 
the selected ester compounds in the tampon. Accordingly, the following 
general procedure was used to apply a given ester material to a tampon. 
Tampons were labeled for identification and weighed to the nearest 
one-tenth of a gram. The amount of ester required to give the desired 
concentration in the treated tampon was then calculated. Solutions of the 
ester in reagent grade isopropyl alcohol were prepared at concentrations 
such that four (4) grams of the solution contained the amount of ester to 
be included in the tampon to be tested. In this manner, the amount of 
ester in a given tampon could be varied while the total weight of the 
ester/isopropyl alcohol solution used to prepare each individual tampon 
was held constant at four (4) grams. For example, if the untreated tampon 
weighed 2.6 grams, then 0.26 gram of ester was required to provide a 
tampon comprising 10% of the ester based on the weight of the untreated 
tampon (i.e. 2.6 gram tampon weight.times.0.10=0.26 gram ester). In this 
instance, six and one-half (6.5) grams of ester were dissolved in 
ninety-three and one-half (93.5) grams of reagent grade isopropyl alcohol 
to give a solution containing six and one-half (6.5%) by weight of ester. 
Four (4) grams of this solution contained the required 0.26 gram of ester. 
As another example, if the untreated tampon weighed 2.8 grams, and it was 
desired that the concentration of ester be one percent (1%) based on the 
weight of the untreated tampon, a solution containing 0.70 grams of ester 
and 99.3 grams of isopropyl alcohol was prepared. Four grams of this 
solution then contained the required 0.028 grams of ester. 
As a third example, if the untreated tampon weighed 2.5 grams, and it was 
desired that the concentration of ester be 0.1% based on the weight of the 
untreated tampon, a solution containing 0.0625% by weight of ester in 
isopropyl alcohol was prepared. Four (4) grams of this solution contained 
the required 0.0025 gram of ester. 
All solutions of ester compound in isopropyl alcohol were thoroughly 
stirred to ensure uniformity. In addition, especially at higher 
concentrations, the rate of dissolution of the ester could be increased by 
warming the ingredients to about 60.degree. C., e.g. in a heated water 
bath. 
Once the tampon had been weighed and the appropriate solution of ester in 
reagent grade isopropyl alcohol had been prepared in the manner explained 
above, four (4) grams of the ester/isopropyl alcohol solution (at room 
temperature) were uniformly applied, by pipetting, to the outer surfaces 
of the tampon. Tampons were rotated during the application of the ester 
solution to ensure as uniform an application as possible. The isopropyl 
alcohol was then evaporated at 70.degree. C. in a hooded drying oven to 
yield a tampon comprising the desired level of ester compound. 
The foregoing procedure was used to prepare all the ester-containing 
tampons mentioned in the Examples of the present patent application. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In Example 1 which follows herein, the invention will be described in 
detail in connection with a catamenial tampon comprising an absorbent 
material, a liquid-pervious covering fabric, and an amount of a mixture of 
glycerol monolaurate and glycerol dilaurate which is effective to inhibit 
the production of toxic shock syndrome toxin-1 by S. aureus bacteria when 
said bacteria are brought into contact with the tampon. It will be 
understood that the principles of the invention apply as well to other 
absorbent products such as wound dressings, disposable diapers, sanitary 
napkins and other kinds of tampons, such as those intended for medical, 
surgical, dental and/or nasal use. 
Catamenial tampons comprising rayon fibers as their absorbent material were 
prepared as follows. The rayon fibers employed were 3-denier, vicose rayon 
staple fibers having a length of 11/8 inches (2.86 cm) and 11-25 crimps 
per inch (about 4.3-9.8 crimps per centimeter). The fibers were 100% 
vicose rayon, i.e. they were substantially free of all finishes and 
additives, such as surfactants and the like, commonly used in commercial 
production. 
Using commercially available carding equipment, the above-described rayon 
fibers were carded into a fibrous web weighing about 520 grains/yd.sup.2 
(33.6 grams/meter.sup.2). The carded web of rayon fibers was gathered into 
a tubular ribbon having a diameter of about one-inch (2.54 cm). This 
tubular ribbon was thereafter covered in a nonwoven fabric made from 
heat-fusible fibers and weighing about 0.25 oz/yd.sup.2 (7.08 gm/m.sup.2). 
The edges of the heat-fusible nonwoven fabric were overlapped slightly and 
subsequently heat treated to form a seal. The covered ribbon of rayon 
fibers was cut into blanks. A white rayon string was pierced and looped 
through each blank. The blank was then compressed in known fashion to 
provide a test tampon having a diameter of 0.47 inch (1.2 cm), a length of 
1.75 inches (4.44 cm), and a weight of about 2.6 grams. The dangling 
portion of the withdrawal string was cut from the tampon prior to testing. 
A mixture of glycerol monolaurate and glycerol dilaurate, commercially 
available under the tradename "Lauricidin", was obtained from Lauricidin, 
Inc. located in East Lansing, Mich., U.S.A. This mixture, which is 
hereinafter sometimes referred to as "Lauricidin", was analyzed and found 
to contain 93 percent by weight of glycerol monolaurate and 3.5 percent by 
weight of glycerol dilaurate. It is known that Lauricidin has 
antimicrobial properties and is non-toxic to humans. It has been suggested 
for use in anti-caries products, insecticides, cosmetic preparations and 
food compositions. 
EXAMPLE 1 
Tampons comprising, respectively, 0.1%, 1.0% and 10% by weight of the 
aforementioned Lauricidin mixture based on the weight of the untreated 
tampon were prepared using the above-mentioned test tampons. The 
Lauricidin mixture was applied to the test tampons according to the 
General Procedure for Preparing Tampons of the Invention described earlier 
in this application. The Lauricidin containing tampons were prepared in 
duplicate. The Lauricidin treated tampons were then tested according to 
the Tampon Sac Method reported by Reiser et al. in the Journal of Clinical 
Microbiology, Vol. 25, August 1987, pp. 1450-1452, the disclosure of which 
is hereby incorporated by reference. Staphylococcus aureus strain 
FRI-1169, obtained in lyophilized form from Dr. Merlin Bergdoll, Food 
Research Institute, University of Wisconsin, in Madison, Wisc., U.S.A., 
was employed in the tests. A S. aureus suspension was prepared by 
thoroughly mixing one (1) milligram of the lyophilized S. aureus strain to 
one (1) milliliter of Brain Heart Infusion (BHI) Broth (obtained from 
Difco Laboratories, Detroit, Mich., U.S.A.), transferring said mixture 
into a test tube containing five (5) milliliters of BHI Broth, thoroughly 
mixing again, and incubating for twenty-four (24) hours at 37.degree. C. 
prior to use. 
100 milliliters of brain heart infusion (BHI) agar (also obtained from 
Difco Laboratories in Detroit, Mich., U.S.A.) were put into each of ten 
3.8 cm.times.20 cm culture tubes. Cellulose sacs were made and sterilized 
in the manner reported by Reiser et al. The sterile cellulose sacs were 
inoculated with the aforementioned S. aureus suspension in an amount 
sufficient to provide at the beginning of the test a concentration therein 
of 1.times.10.sup.8 CFU/ml Staphylococcus aureus bacteria. 
Each Lauricidin treated tampon to be tested was inserted into a sterile 
cellulose sac containing the S. aureus bacteria and each sac was then 
inserted into a culture tube containing the BHI agar. Two controls, each 
in duplicate, were used. In one control (called the "inoculum control"), 
an inoculated sac (with no tampon therein) was placed in each of two 
culture tubes containing BHI agar. In the second control, two untreated 
tampons (i.e. tampons made in the described manner but with no Lauricidin 
in the isopropyl alcohol) were placed in cellulose sacs which in turn were 
placed in culture tubes containing BHI agar. Thus, ten culture tubes were 
used in this test, four containing the aforementioned controls (two with 
tampons; two without tampons) and the others containing the aforementioned 
Lauricidin-treated test tampons in duplicate. 
The concentrations of S. aureus strain FRI-1169 and toxic shock syndrome 
toxin-1 at the outset of the test (0 hours) and after incubation for 24 
hours at 37.degree. C. are shown in Table 1. 
TABLE 1 
__________________________________________________________________________ 
THE EFFECT OF LAURICIDIN TREATED TAMPONS ON TSST-1 FORMATION 
BY AND GROWTH OF STAPHYLOCOCCUS AUREUS 
INITIAL FINAL 
CONCENTRATION 
INITIAL CONCENTRATION 
FINAL FINAL 
OF S. AUREUS 
CONCENTRATION 
OF S. AUREUS 
CONCENTRATION 
CONCENTRATION 
CELLS OF CELLS OF S. AUREUS 
OF 
(CFU/ml) TSST-1.sup.a) 
(CFU/ml) CELLS.sup.b) 
TSST-1.sup.a 
SAMPLE (10.sup.8) (ng/ml) (10.sup.8) (/ml) ug/ml 
__________________________________________________________________________ 
No tampon 
1.0 ND 100 10.0 50 
(control) 
Untreated 
1.0 ND 100 10.0 31 
Tampon 
(control) 
Tampon 1.0 ND 794 10.9 15 
w/0.1% 
Lauricidin 
Tampon 1.0 ND 158 10.2 0.09 
w/1.0% 
Lauricidin 
Tampon 1.0 ND 316 10.5 0.08 
w/10.0% 
Lauricidin 
__________________________________________________________________________ 
ND = NOT DETECTED. Detection limit of the ELISA method of Reiser et al is 
0.5 ng/ml. 
.sup.a) = As determined by the ELISA method reported by Reiser et al. in 
Applied and Environmental Microbiology, December 1982, pp. 1349-1355, the 
disclosure of which is hereby incorporated by reference. 
.sup.b) = Expressed as log to base 10. 
All data above are mean determinations of duplicate samples. 
The data in Table 1 show that S. aureus bacteria in the presence of a 
tampon comprising 0.1% (w/w) Lauricidin produce 51% less toxic shock 
syndrome toxin-1 (TSST-1) than when exposed to a control tampon containing 
no Lauricidin, in spite of the fact that there was no reduction in the 
actual number of viable S. aureus cells in the presence of the 
Lauricidin-treated tampon. The data further show that S. aureus bacteria 
in the presence of tampons comprising 1.0% (w/w) and 10.0% (w/w) of 
Lauricidin produced 99% less TSST-1 than did the same number of S. aureus 
cells in the presence of a control tampon containing no Lauricidin, again 
in spite of the fact that there was no reduction in the number of viable 
S. aureus cells in the presence of Lauricidin-containing tampons. The data 
show that although the number of viable S. aureus cells does not decrease 
when the cells are exposed to tampons containing various levels of 
Lauricidin, the actual amount of TSST-1 produced by those cells is 
significantly reduced (as in the case of the tampon treated with 0.1% 
Lauricidin) or substantially eliminated (as in the case of tampons treated 
with 1.0% and 10% of Lauricidin). In other words, while Lauricidin does 
not significantly reduce the number of viable S. aureus bacteria cells, 
but it does significantly inhibit the production of toxic shock syndrome 
toxin-1 by those cells. 
Thus, it is believed that in vivo use of Lauricidin treated tampons in 
menstruating women would be beneficial in that the production of TSST-1 by 
any S. aureus bacteria normally present in the vagina would be 
significantly reduced. In addition, in preliminary studies conducted using 
the predominant microorganism present in the vagina, i.e. Lactobacillus 
acidophilus, it was found that said microorganism was not adversely 
affected when brought into contact with an Example 1 tampon comprising 
1.0% (w/w) Lauricidin. 
EXAMPLE 2 
A second experiment was conducted to evaluate growth of, and TSST-1 
production by, S. aureus cells in the presence of commercially available 
tampons treated with varying amounts of Lauricidin. Tampax* brand 
menstrual tampons (regular size, Lot No. 8L015Z) which had been purchased 
on the open market were employed in the experiment of this Example 2. 
These tampons were manufactured by Tambrands, Inc., Lake Success, N.Y., 
U.S.A. The Tampax* tampons comprised a cotton absorbent core, a rayon 
fabric covering, and a withdrawal string. The tampon withdrawal strings 
were cut from the tampons prior to testing. Tampons comprising 0.1%, 1.0% 
and 10% Lauricidin based on the weight of the untreated tampons were 
prepared in duplicate according to the aforementioned General Procedure. 
Two Tampax* tampons (with strings cut off), treated with isopropyl alcohol 
containing no Lauricidin and thereafter dried, were used as a control. The 
dried, Lauricidin-treated tampons and the control tampons, were then 
tested according to the procedure and conditions reported in Example 1. 
The test results are reported in Table 2. 
TABLE 2 
______________________________________ 
THE EFFECT OF LAURICIDIN TREATED TAMPAX 
TAMPONS (LOT NO. 8L015Z) ON TSST-1 FORMATION 
BY STAPHYLOCOCCUS AUREUS (FRI-1169) 
FINAL 
CONCEN- FINAL 
TRATION CONCEN- FINAL 
OF S. AUREUS 
TRATION CONCEN- 
CELLS OF S. AUREUS 
TRATION 
(CFU/ml) CELLS.sup.b) 
OF TSST-1.sup.a) 
SAMPLE (10.sup.8) (/ml) (.mu.g/ml) 
______________________________________ 
0% Lauricidin 
5,248 11.72 27.50 
Tampon (control) 
0.1% Lauricidin 
199 10.30 3.70 
On Tampon 
1.0% Lauricidin 
251 10.40 1.05 
On Tampon 
10.0% Lauricidin 
2.13 8.33 0.19 
On Tampon 
______________________________________ 
.sup.a) = As determined by ELISA method (Reiser et al.). 
.sup.b) = Expressed as log to base 10. 
All sample determinations were made after 24 hrs. incubation at 37.degree 
C. 
All data above are mean determinations of duplicate samples. 
The data in Table 2 show that S. aureus bacteria in the presence of Tampax* 
brand tampons treated with varying amounts of Lauricidin produce less 
TSST-1 than when exposed to the control tampon containing no Lauricidin, 
the extent of the reduction in toxin production being related to the 
amount of Lauricidin in the tampons. Tampax* tampons treated with 0.1% by 
weight of Lauricidin resulted in an 86% reduction in TSST-1 produced 
compared to the control, while the Tampax* tampons treated with 1% and 10% 
by weight of Lauricidin resulted in, respectively, a 96% and 99% 
reduction. The results with the Tampax* brand tampons also show a 
reduction in the total number of S. aureus cells; this effect is dependent 
upon the concentration of Lauricidin in the tampon. At the end of the 24 
hour incubation period, the log concentration of S. aureus cells in the 
presence of the control tampon was 11.72; the log concentration of S. 
aureus cells in the presence of the tampon containing 0.1% Lauricidin was 
10.30 (12% less); the log concentration of S. aureus cells in the presence 
of the tampon containing 1.0% Lauricidin was 10.40 (11% less); and the log 
concentration of S. aureus cells in the presence of the tampon containing 
10% Lauricidin was 8.33 (29% less). 
EXAMPLE 3 
A third experiment was conducted to evaluate growth of, and TSST-1 
production by, S. aureus cells in the presence of commercially available 
tampons treated with varying amounts of Lauricidin. Playtex* brand 
menstrual tampons (regular size, Lot No. 3496P) which had been purchased 
on the open market were employed in the experiment of this Example 3. 
These tampons were manufactured by International Playtex Inc., Dover, 
Del., U.S.A. The Playtex* tampons were made of all rayon fiber and had a 
withdrawal string but no cover fabric. The tampon withdrawal strings were 
cut from the tampons prior to testing. Treated tampons comprising 0.1%, 
1.0% and 10% Lauricidin based on the weight of the untreated tampon were 
prepared in duplicate according to the aforementioned General Procedure. 
Two Playtex* tampons (with strings cut off) without any Lauricidin 
treatment were used as a control. The dried Lauricidin-treated tampons and 
the untreated controls were then tested according to the procedure and 
conditions described in Example 1. The test results are reported in Table 
3. 
TABLE 3 
______________________________________ 
THE EFFECT OF LAURICIDIN TREATED PLAYTEX 
TAMPONS (LOT NO. 3496P) ON TSST-1 FORMATION 
BY STAPHYLOCOCCUS AUREUS (FRI-1169) 
FINAL 
CONCEN- FINAL 
TRATION CONCEN- FINAL 
OF S. AUREUS 
TRATION CONCEN- 
CELLS OF S. AUREUS 
TRATION 
(CFU/ml) CELLS.sup.b) 
OF TSST-1.sup.a) 
SAMPLE (10.sup.8) (/ml) (.mu.g/ml) 
______________________________________ 
0% Lauricidin 
3,388 11.53 10.86 
Tampon (control) 
0.1% Lauricidin 
213 10.33 2.69 
On Tampon 
1.0% Lauricidin 
131 10.12 0.38 
On Tampon 
10.0% Lauricidin 
3.23 8.51 0.29 
On Tampon 
______________________________________ 
.sup.a) = As determined by ELISA method (Reiser et al.). 
.sup.b) = Expressed as log to base 10. 
All sample determinations were made after 24 hrs. incubation at 37.degree 
C. 
All data above are mean determinations of duplicate samples. 
The data reported in Table 3 show that the amount of TSST-1 produced by S. 
aureus bacteria in the presence of Playtex* tampons treated with 0.1%, 
1.0% and 10.0% w/w Lauricidin was reduced 75%, 96% and 97%, respectively, 
when compared to the amount of TSST-1 produced in the presence of a 
control tampon containing no Lauricidin. On the other hand, compared to 
control values, the Playtex* tampons treated with 0.1%, 1.0% and 10.0% w/w 
of Lauricidin resulted in 10%, 12% and 26% fewer S. aureus cells at the 
end of the 24 hour incubation period. 
EXAMPLE 4 
A fourth experiment was conducted to evaluate growth of, and TSST-1 
production by S. aureus cells in the presence of another brand of 
menstrual tampons treated with varying amounts of Lauricidin. Rely* brand 
menstrual tampons (regular size, Lot No. 2060LC01A) which had been 
purchased prior to September 1980 on the open market were employed in the 
experiment of this Example 4. These tampons were manufactured by Procter & 
Gamble, Cincinnati, Ohio, USA. The Rely* tampons comprised carboxymethyl 
cellulose dispersed in a polyester foam which was wrapped in a nonwoven 
fabric made of spunbonded polyester fibers. They had the usual withdrawal 
strings. The tampon withdrawal strings were cut from the tampons prior to 
testing. Treated tampons comprising 0.1%, 1.0% and 10% Lauricidin based on 
the weight of the untreated tampon were prepared in duplicate according to 
the aforementioned General Procedure. Two Rely* tampons (with their 
withdrawal strings cut off) without any Lauricidin treatment were used as 
a control. The dried Lauricidin-treated tampons and the untreated control 
tampons were then tested according to the procedure and conditions 
described in Example 1. The test results are reported in Table 4. 
TABLE 4 
______________________________________ 
THE EFFECT OF LAURICIDIN TREATED RELY 
TAMPONS (LOT NO. 2060LC01A) ON TSST-1 
FORMATION BY STAPHYLOCOCCUS AUREUS (FRI-1169) 
FINAL 
CONCEN- FINAL 
TRATION CONCEN- FINAL 
OF S. AUREUS 
TRATION CONCEN- 
CELLS OF S. AUREUS 
TRATION 
(CFU/ml) CELLS.sup.b) 
OF TSST-1.sup.a) 
SAMPLE (10.sup.8) (/ml) (.mu.g/ml) 
______________________________________ 
0% Lauricidin 
12,303 12.09 64.32 
Tampon (control) 
0.1% Lauricidin 
2,818 11.45 6.92 
On Tampon 
1.0% Lauricidin 
1,995 11.30 1.54 
On Tampon 
10.0% Lauricidin 
1,096 11.04 0.09 
On Tampon 
______________________________________ 
.sup.a) = As determined by ELISA method (Reiser et al.). 
.sup.b) = Expressed as log to base 10. 
All sample determinations were made after 24 hrs. incubation at 37.degree 
C. 
All data above are mean determinations of duplicate samples. 
The data reported in Table 4 show that the amount of TSST-1 produced by S. 
aureus bacteria in the presence of the Rely* tampons treated with 0.1%, 
1.0% and 10.0% w/w Lauricidin was reduced by 89%, 97% and 99%, 
respectively, when compared to the amount of TSST-1 produced in the 
presence of a control tampon containing no Lauricidin. Whereas, at the end 
of the 24 hour incubation period, the total S. aureus cell concentration 
(expressed as log to the base 10) in the presence of the control tampon 
was 12.09, the total S. aureus cell concentration (expressed as log to the 
base 10) in the presence of the Rely* tampon treated with 0.1%, 1.0% and 
10% Lauricidin was, respectively, 11.45 (5.3% less), 11.20 (7.4% less) and 
11.04 (7.8% less). 
EXAMPLE 5 
A fifth experiment was conducted to evaluate growth of, and TSST-1 
production by S. aureus cells in the presence of commercially available 
tampons treated with varying amounts of Lauricidin. O.b.* brand menstrual 
tampons (regular size, Lot No. 0694T) which had been purchased on the open 
market were employed in the experiment of this Example 5. These tampons 
were distributed by Personal Products Company, Milltown, N.J., U.S.A. The 
o.b.* tampons comprise a blend of rayon and cotton. They included a 
withdrawal string but did not have an outer cover sheet. The tampon 
withdrawal strings were cut from the tampons prior to testing. Treated 
tampons comprising 0.1%, 1.0% and 10% Lauricidin based on the weight of 
the untreated tampon were prepared in duplicate according to the General 
Procedure described earlier herein. Two o.b.* tampons (with strings cut 
off) without any Lauricidin treatment were used as a control. The dried 
Lauricidin-treated tampons and the untreated control tampons were then 
tested according to the procedure and conditions described in Example 1. 
The test results are reported in Table 5. 
TABLE 5 
______________________________________ 
THE EFFECT OF LAURICIDIN TREATED O.b.* 
TAMPONS (LOT NO. 0694T) ON TSST-1 FORMATION 
BY STAPHYLOCOCCUS AUREUS (FRI-1169) 
FINAL 
CONCEN- FINAL 
TRATION CONCEN- FINAL 
OF S. AUREUS 
TRATION CONCEN- 
CELLS OF S. AUREUS 
TRATION 
(CFU/ml) CELLS.sup.b) 
OF TSST-1.sup.a) 
SAMPLE (10.sup.8) (/ml) (.mu.g/ml) 
______________________________________ 
0% Lauricidin 
3,388 11.53 13.46 
Tampon (control) 
0.1% Lauricidin 
158 10.20 3.26 
On Tampon 
1.0% Lauricidin 
316 10.50 0.28 
On Tampon 
10.0% Lauricidin 
95 9.98 0.19 
On Tampon 
______________________________________ 
.sup.a) = As determined by ELISA method (Reiser et al.). 
.sup.b) = Expressed as log to base 10. 
All sample determinations were made after 24 hrs. incubation at 37.degree 
C. 
All data above are mean determinations of duplicate samples. 
The data presented in Table 5 show that the amount of TSST-1 produced by S. 
aureus bacteria in the presence of o.b.* tampons comprising 0.1%, 1.0% and 
10% by weight Lauricidin was reduced by 75%, 98% and 98%, respectively, 
when compared to the amount of TSST-1 produced in the presence of a 
control o.b.* tampon containing no Lauricidin. The total S. aureus cell 
concentration (expressed as log to the base 10) in the presence of the 
control tampon was 11.53. The total S. aureus concentration (expressed as 
log to the base 10) in the presence of the o.b.* tampon treated with 0.1%, 
1.0% and 10% by weight of the Lauricidin was, respectively, 10.20 (11% 
less), 10.50 (8.9% less), and 9.98 (13% less). 
EXAMPLE 6 
A sixth experiment was conducted to evaluate growth of, and TSST-1 
production by S. aureus cells in the presence of commercially available 
tampons treated with varying amounts of Lauricidin. Kotex* Security* brand 
menstrual tampons (regular size, Lot No. 5C0907C) which had been purchased 
on the open market were employed in the experiment of this Example 6. 
These tampons were marketed by Kimberly-Clark Corporation, Neenah, Wisc., 
U.S.A. They comprised a blend of 60% cotton and 40% rayon, had the usual 
withdrawal string, and were covered with a nonwoven fabric made of 
polypropylene fibers. The tampon withdrawal strings were cut from the 
tampons prior to testing. Treated tampons comprising 0.1%, 1.0% and 10% 
Lauricidin based on the weight of the untreated tampon were prepared in 
accordance with the above-described General Procedure. Two Kotex* 
Security* tampons (with strings cut off) without any Lauricidin treatment 
were used as controls. The dried Lauricidin-treated tampons and the 
untreated control tampons were then tested according to the procedure and 
conditions described in Example 1. The test results are reported in Table 
6. 
TABLE 6 
______________________________________ 
THE EFFECT OF LAURICIDIN TREATED KOTEX 
SECURITY TAMPONS, LOT NO. 5C0907C ON TSST-1 
FORMATION BY STAPHYLOCOCCUS AUREUS (FRI-1169) 
FINAL 
CONCEN- FINAL 
TRATION CONCEN- FINAL 
OF S. AUREUS 
TRATION CONCEN- 
CELLS OF S. AUREUS 
TRATION 
(CFU/ml) CELLS.sup.b) 
OF TSST-1.sup.a) 
SAMPLE (10.sup.8) (/ml) (.mu.g/ml) 
______________________________________ 
0% Lauricidin 
1,698 11.23 10.19 
Tampon (control) 
0.1% Lauricidin 
194 10.29 4.90 
On Tampon 
1.0% Lauricidin 
426 10.63 0.09 
On Tampon 
10.0% Lauricidin 
426 10.63 0.05 
On Tampon 
______________________________________ 
.sup.a) = As determined by ELISA method (Reiser et al.). 
.sup.b) = Expressed as log to base 10. 
All sample determinations were made after 24 hrs. incubation at 37.degree 
C. 
All data above are mean determinations of duplicate samples. 
The data presented in Table 6 show that the amount of TSST-1 produced by S. 
aureus bacteria in the presence of Kotex* tampons comprising 0.1%, 1.0% 
and 10% by weight of Lauricidin was reduced by 52%, 99% and 99%, 
respectively, when compared to the amount of TSST-1 produced under the 
same experimental conditions in the presence of a control Kotex* tampon 
containing no Lauricidin. The total concentration of S. aureus cells 
(expressed as log to the base 10) in the presence of the Kotex* tampons 
containing 0.1%, 1.0% and 10% by weight of Lauricidin was, respectively, 
10.29 (8.4% less), 10.63 (5.3% less) and 10.63 (5.3% less). 
As can be seen from the preceding Examples 1-6, a variety of tampons, one 
of which was made by the inventors (Example 1), others of which were 
commercially available (Examples 2, 3, 5 and 6) and one of which had been 
commercially available but was subsequently withdrawn from commercial 
distribution (Example 4), have been treated with varying levels of 
Lauricidin, a commercially available mixture comprising 93% by weight 
glycerol monolaurate and 3.5% by weight glycerol dilaurate. The data 
reported in Tables 1-6 show that, depending on the levels of Lauricidin in 
the tampons, S. aureus bacteria produce significantly less TSST-1 or, in 
other words, are inhibited from producing significant amounts of TSST-1 
when compared to the amounts of TSST-1 produced, under the same 
experimental conditions, by S. aureus bacteria in the presence of control 
tampons containing no Lauricidin. 
EXAMPLE 7 
Test tampons of the kind used in Example I were used in this Example 7. 
Test tampons comprising 0.1%, 0.5%, and 1.0% Lauricidin based on the 
weight of the untreated test tampons were prepared according to the 
General Procedure described earlier herein and were tested according to 
the Tampon Sac Method described in Example 1. In this Example 7, however, 
the tampon sacs were inoculated with different strains of S. aureus 
bacteria prior to the insertion therein of the Lauricidin treated tampons. 
The respective strains of S. aureus tested are identified in Table 7. The 
concentration of S. aureus at the outset of the experiment was 
1.times.10.sup.8 CFU/ml. TSST-1 producing S. aureus strain FRI-1169 
utilized in this Example 7 was obtained from Merlin Bergdoll, Ph.D., Food 
Research Institute, University of Wisconsin, Madison, Wisc. U.S.A. TSST-1 
producing S. aureus strain designated 1169W was obtained from Fred Quimby, 
V. M. D., Ph.D., Cornell Medical School, New York, N.Y., U.S.A. A third S. 
aureus strain (specifically a substrain of FRI-1169) was isolated from the 
parent strain and designated TSS Isolate. This TSS Isolate can be obtained 
in a lyophilized state from S. K. Brown-Skrobot, Ph.D., Personal Products 
Company, Milltown, N.J., U.S.A. A fourth TSST-1 producing S. aureus strain 
designated Mn8, was obtained from Patrick Schlievert, Ph.D., University of 
Minnesota, Minneapolis-St. Paul, Minn., U.S.A. A fifth TSST-1 producing S. 
aureus strain designated 1187 was obtained from Keith T. Holland, Ph.D., 
University of Leeds, Leeds, England. All of the S. aureus strains tested 
in this example can be obtained from the aforementioned individuals. 
Suspensions of the various strains were prepared as described in Example 1 
and used to inoculate the sacs prior to insertion of the tampons which 
were then tested according to the Tampon Sac Method described in Example 
1. Duplicate test tampons without any Lauricidin were used as controls. 
The test results are reported in Table 7. 
The results shown in Table 7 show a reduction in TSST-1 formation with 
increasing concentration of Lauricidin. This was noted in all five strains 
which were tested. It was concluded from the test results that the 
beneficial effects of Lauricidin observed in Examples 1-6 were not 
specific to any particular TSST-1 producing S. aureus strain. 
TABLE 7 
______________________________________ 
THE EFFECT OF LAURICIDIN TREATED TAMPONS 
ON GROWTH OF AND TSST-1 PRODUCTION BY 
VARIOUS STRAINS OF S. AUREUS 
CONCENTRATION TOTAL AMOUNT 
TSST-1 OF TSST-1 
PRODUCING LAURICIDIN.sup.a) 
PRODUCED.sup.b) 
STRAIN (%) (.mu.g) 
______________________________________ 
1169W NONE 56.54 
(Quimby Strain) 
0.1 17.54 
0.5 0.35 
1.0 0.07 
FRI-1169 NONE 48.75 
(Bergdoll Strain) 
0.1 5.06 
0.5 0.04 
1.0 0.03 
TSS Isolate NONE 53.04 
(Substrain of 
0.1 5.25 
FRI-1169) 0.5 1.27 
1.0 0.48 
Mn8 NONE 66.30 
(Schlievert 0.1 0.66 
Strain) 0.5 0.12 
1.0 0.05 
1187 NONE 46.80 
(Holland Strain) 
0.1 6.63 
0.5 0.92 
1.0 0.58 
______________________________________ 
.sup.a) = based on weight of untreated tampon. 
.sup.b) = Total TSST1 per tampon after 24 hr. incubation at 37.degree. C. 
All samples were tested for total TSST1 produced using the ELISA method 
(Reiser et al.). 
EXAMPLE 8 
In this Example 8, tampons comprising various fatty acid esters were tested 
to determine their effect on growth of and TSST-1 formation by S. aureus 
bacteria (FRI-1169). Test tampons of the kind used in Example 1 were used 
for this Example 8. All of the test tampons weighed 2.6 grams. 0.65 gram 
of each fatty acid ester to be tested was dissolved in 99.35 grams of 
reagent grade or ester mixture isopropyl alcohol. Four (4) grams of each 
fatty acid ester solution were applied to the outer surfaces of each of 
two test tampons to provide treated tampons comprising 1% by weight of the 
ester or ester mixture based on the weight of the untreated test tampon. 
The alcohol was removed by evaporation at 70.degree. C., after which the 
treated tampons were tested according to the Tampon Sac Method described 
in Example 1. Following is a list of the fatty acid esters which were 
evaluated: 
Tampon No. 1--A mixture of glyceryl monocaprylate and glyceryl caprate. 
Caprylic acid is a saturated fatty acid containing 8 carbon atoms. Captic 
acid is a saturated fatty acid containing 10 carbon atoms. The mixture 
contained about 38.3% by weight of the caprylate ester, about 36.9% by 
weight of the caprate ester and about 0.6% free glycerine. The remainder 
of this mixture contained minor amounts of di- and triesters of the two 
fatty acids. 
Tampon No. 2--Glyceryl monolaurate of 90-95% purity and containing 0.2% 
free glycerine and minor amounts of the di- and triesters. Lauric acid is 
a saturated fatty acid containing 12 carbon atoms. 
Tampon No. 3--Glyceryl monomyristate of 90-95% purity and containing about 
0.2% free glycerine and minor amounts of the di- and triesters. Myristic 
acid is a saturated fatty acid containing 14 carbon atoms. 
Tampon No. 4--Glyceryl monopalmitate of 90-95% purity and containing 0.2% 
free glycerine and minor amounts of the di- and triesters. Palmitic acid 
is a saturated fatty acid containing 16 carbon atoms. 
Tampon No. 5--Glyceryl monostearate of 90-95% purity and containing 0.2% 
free glycerine and minor amounts of the di- and triesters. Stearic acid is 
a saturated fatty acid containing 18 carbon atoms. 
Tampon No. 6--Glyceryl monooleate of 90-95% purity and containing 0.2% free 
glycerine and minor amounts of the di- and triesters. Oleic acid is an 
unsaturated fatty acid containing 18 carbon atoms and one double bond. 
In this Example 8, two untreated tampons were used as controls. 
The results of the tests are shown in Table 8. The data show that there was 
a marked reduction in the amount of TSST-1 produced by S. aureus strain 
FRI-1169 in the presence of the tampons treated with the various fatty 
acid esters when compared to the amount of TSST-1 produced in the presence 
of the untreated control tampons. The reduction in the amount of TSST-1 
produced ranged from about 90% to 99%, except in the case of the tampon 
containing glyceryl monostearate. The 60% reduction in TSST-1 production 
observed in the case of the tampon containing glyceryl monostearate, 
though not as high as that obtained with the tampons containing other 
esters, was nevertheless quite substantial and is regarded as significant. 
No corresponding pattern of reduction in the number of viable S. aureus 
cells was observed. It should be noted, however, that at the end of the 24 
hour incubation period, there were fewer viable S. aureus cells on the 
treated tampons than on the tampons having no ester treatment. 
TABLE 8 
__________________________________________________________________________ 
IMT OF VARIOUS GLYCERYL ESTER COMPOUNDS ON GROWTH OF 
AND TSST-1 FORMATION BY STAPHYLOCOCCUS AUREUS (FRI-1169) 
FINAL FINAL TOTAL 
CONCENTRATION 
CONCENTRATION 
AMOUNT REDUCTION 
OF OF S. AUREUS 
TSST-1 IN TSST-1 
S. AUREUS CELLS 
CELLS.sup.b) 
PRODUCED.sup.a) 
FORMATION 
SAMPLE (CFU/ml) (/ml) (ug) (%) 
__________________________________________________________________________ 
Control 4.20 .times. 10.sup.9 
9.62 17.15 -- 
Tampon 
Tampon No. 1 
5.80 .times. 10.sup.6 
6.76 0.18 98.9 
Tampon No. 2 
6.56 .times. 10.sup.6 
6.81 0.14 99.1 
Tampon No. 3 
5.04 .times. 10.sup.8 
8.83 0.58 96.5 
Tampon No. 4 
1.44 .times. 10.sup.9 
9.15 1.75 89.7 
Tampon No. 5 
6.08 .times. 10.sup.8 
8.78 6.43 60.2 
Tampon No. 6 
6.86 .times. 10.sup.8 
8.83 1.01 94.0 
__________________________________________________________________________ 
.sup.a) As determined by ELISA method (Reiser et al.) 
.sup.b) Log to the base 10. 
All determinations were made after 24 hours at 37.degree. C. 
All data above are mean determinations of duplicate samples. 
EXAMPLE 9 
The mixture of glyceryl monolaurate and glyceryl dilaurate used in the 
experiments reported in Examples 1-7 hereof was obtained from Lauricidin, 
Inc. under the tradename of Lauricidin. As indicated earlier herein, this 
mixture was analyzed and found to contain 93% by weight of glyceryl 
monolaurate and 3.5% by weight of glyceryl dilaurate. Mixtures of glyceryl 
esters of lauric acid were obtained from two other sources. One such 
mixture was obtained from Stepan Chemical Company, Maywood, N.J. U.S.A. 
under the tradename Kessco. This mixture was analyzed and found to contain 
50% by weight of glyceryl monolaurate, and 37% by weight of glyceryl 
dilaurate. Another such mixture was obtained from Henkel Corporation under 
the name Monomuls 90-L12 and found to contain 96% by weight of glyceryl 
monolaurate. No glyceryl dilaurate was detected. Using the aforementioned 
General Procedure and the same test tampons as those used in Example 1, 
the following tampons were prepared in duplicate: 
Tampons comprising, respectively, 0.1%, 0.5% and 1.0% of Lauricidin based 
on the weight of the untreated test tampons; 
Tampons comprising, respectively, 0.1%, 0.5% and 1.0% of the Kessco ester 
mixture based on the weight of the untreated test tampons; and 
Tampons comprising, respectively, 0.1%, 0.5% and 1.0% of Monomuls 90-L12 
mixture based on the weight of the untreated test tampon. 
Tampons treated with isopropyl alcohol without any ester therein were 
employed as controls. All samples were prepared and tested in duplicate 
according to the Tampon Sac Method described earlier herein. Test results 
are set forth in Table 9. 
TABLE 9 
__________________________________________________________________________ 
IMT OF VARIOUS GLYCEROL MONOLAURATES 
ON GROWTH OF AND TSST-1 FORMATION 
BY STAPHYLOCOCCUS AUREUS (FRI-1169) 
FINAL FINAL TOTAL 
CONCENTRATION 
CONCENTRATION 
AMOUNT REDUCTION 
OF S. AUREUS 
OF S. AUREUS 
TSST-1 IN TSST-1 
% CELLS CELLS.sup.b) 
PRODUCED.sup.a) 
FORMATION 
SAMPLE ADD-ON 
(CFU/ml) (/ml) (ug) (%) 
__________________________________________________________________________ 
Control 
0.0 8.8 .times. 10.sup.7 
7.94 69.94 -- 
Kessco 0.1 1.28 .times. 10.sup.8 
8.10 45.24 35 
Glyceryl 
0.5 4.16 .times. 10.sup.7 
7.62 33.60 52 
Monolaurate 
1.0 3.44 .times. 10.sup.7 
7.53 1.40 98 
Lauricidin* 
0.1 1.4 .times. 10.sup.7 
7.15 4.49 93 
Glyceryl 
0.5 1.3 .times. 10.sup.7 
7.11 3.93 94 
Monolaurate 
1.0 3.2 .times. 10.sup.7 
7.50 0.39 99 
Monomuls 
0.1 1.28 .times. 10.sup.8 
8.10 2.41 96 
90-L12 0.5 4.16 .times. 10.sup.7 
7.62 0.36 99 
1.0 3.44 .times. 10.sup.7 
7.53 0.16 99 
__________________________________________________________________________ 
Kessco* contained 50% by weight of glyceryl monolaurate. 
Lauricidin* contained 93% by weight of glyceryl monolaurate. 
Monomuls 90L12 contained 96% by weight of glyceryl monolaurate. 
.sup.a) As determined by ELISA method (Reiser et al.) 
.sup.b) Log to the base 10. 
All determinations were made after 24 hours at 37.degree. C. 
All data above are mean determinations of duplicate samples. 
It can be seen from the test data set forth in Table 9 that, for any given 
concentration (i.e. add-ons of 0.1%, 0.5% or 1.0% w/w) of ester mixture in 
the tampon, the final amount of TSST-1 produced under the described test 
conditions is inversely proportional to the concentration of glyceryl 
monolaurate in the ester mixture. Thus, for example, where the amount of 
ester mixture in the test tampons was held constant at the 0.5% add-on 
level, the final amounts of TSST-1 declined from 33.60 ug when the ester 
mixture contained 50% by weight of glyceryl monolaurate (i.e. Kessco*), to 
3.93 ug when the ester mixture contained 93% by weight GML (i.e. 
Lauricidin*), to 0.36 ug where the ester mixture contained 96% glyceryl 
monolaurate (i.e. Monomuls* 90-L12). Similar reductions in the final 
amounts of TSST-1 produced were observed where the three ester mixtures 
were used at 0.1% and 1.0% by weight of the tampon. The results set forth 
in Table 9 suggest that glyceryl monolaurate (which contains two unreacted 
hydroxyl groups derived from glycerol) is more effective in inhibiting 
production of TSST-1 than glyceryl dilaurate (which contains a single 
unreacted hydroxyl group derived from glycerol). 
EXAMPLE 10 
In vivo Activity of Glyceryl Monolaurate-Impregnated Tampons 
Test tampons were made as follows. Avtex rayon (100%) #SN2587 three denier 
was used as the test fiber. The fiber was scoured to remove Tween 20 and 
either left as unfinished or coated with glycerol monolaurate (Henkel 
Monomuls L-90)(hereinafter referred to as "GML"). The analytical 
determination of the monolaurate content of the material was 96.0%, 2.0% 
of the 1-3 diestar and 2.0% unidentified material. The fiber was coated as 
follows. Seventy-five pounds of rayon fiber was loaded into a holding tank 
and the tank filled with water (120 gallons total). Ammonia (NH.sub.3) 
(29.4% v/v) was added to the water in the holding tank. The system was 
then heated to 200.degree. F. for 30 minutes. The fiber was then washed 
with hot water (150.degree. F.) three times, the wash water checked for 
any residual foam evidencing the presence of Tween 20. The fiber was then 
washed with cold water, 60.degree. F. 
The fiber was transferred to a centrifuge where it was spun while still hot 
for 5 minutes to remove excess water. The 75 lbs. of rayon initially 
contained 54 lbs. of water. The rayon was then opened by hand and placed 
back into the holding tank. Two restraining plates were placed over the 
fiber to reduce agitation and minimize foaming. Hot water 
(10.degree.-200.degree. F.) was added followed by four 5-pound samples of 
GML, each dissolved in five gallons of 170.degree. F. water. The system 
was pressurized and heated to 190.degree. F. and circulated for 30 
mintues. After the system was allowed to drain, the fiber was taken to the 
centrifuge and spun for 5 minutes. At this point, there was 52 lbs. of 
water (70%) remaining. The temperature of the outside fiber was 
160.degree. F. while the temperature of the inside fibers was 
175.degree.-180.degree. F. 
The moist fiber was placed in a belt oven, which was heated to about 
250.degree.-260.degree. F. This heat treatment further opened and dried 
the rayon fiber. The coated and unfinished fiber were then run through a 
Rando Webber followed by carding in order to form a workable ribbon from 
which to make tampons. 
After the fiber was scoured or coated, dried and carded the rayon ribbon 
was used in the production of 2.30 g tampons. The fiber was compressed 
bidirectionally and held in a compression puck for five seconds. After 
compression, the tampons were placed into 0.62" o.d. applicators. The 
tampons were wrapped in cellophane and sealed. Control tampons were 
labelled (y) while GML-coated tampons were designated (x). The tampons 
were made as follows: Blanks were made by cutting the rayon into sections 
2.75" in length by 3.0" in width. Fiber orientation for length was 
machine-direciton and for width, was cross-direction. The sections were 
either built up or torn down to obtain blank weights of 2.28 g. The rayon 
section was then hand-rolled and covered. For control blanks, the cover 
was 0.25 oz. Enka bicomponent fabric (2.75".times.4.75"). For GML blanks, 
the cover was 0.25 oz. Enka bicomponent fabric coated with 2.4% GML 
solution. The cover was heat sealed to itself using a hand iron. A 8/5 
White rayon string, available from Blue Mountain Industries, was cut to 
13.0" lengths. The string was pierced through one end of each blank at a 
distance of 5/8" from the end on the piercing unit and then looped. Each 
blank was tested for anchor strength by manually pulling the strig after 
looping. The blanks were compressed bidirectionally (side-to-side, then 
end-to-end) and held in the compression puck for 5 seconds. Immediately 
after compression, the tampons were placed in Reggie three-piece 
applicator (0.62" o.d.). The pull string was not knotted. The tampons were 
wrapped in white cellophane sleeves and sealed. 
The compressed tampons were analyzed for determination of the concentration 
of GML on the tampon fibers. The average concentration of GML on the 
tampons tested was 2.38% w/w. 
The in vitro impact of the GML tampons compared to that of the unfinished 
tampons was evaluated using both the Holland Shake Flask Method and the 
Reiser Tampon Sac Methods for evaluation on TSST-1 formation by S. aureus. 
The Reiser Tampon Sac Method has been described above in Examples 1 and 7. 
The results of the determination of the impact of the GML (2.38% w/w) 
coated tampons is set forth in Tables 10 and 11. Table 10 shows that 
greater than 99.9% reductions in TSST-1 formation were noted when the 
tampons were evaluated using the following Holland Shake Flask Method: two 
liter triple-baffle flasks were autoclaved containing 500 ml of Difco 
Brain Heart Infusion Broth. After sterilization, five ml of a 24-hour old 
culture of S. aureus strain identified as 1187 was added to the flasks. 
Either 25.0-gram quantities of test material or no material (in the 
control flasks) were added to the flasks in duplicate. All flasks were 
incubated at 37 C with shaking at 160 rpm for 24 hours, at which time 
TSST-1 concentration and total S. aureus cell count determinations were 
made. TSST-1 level was determined using the ELISA test, while total cell 
counts were made using Standard Plate Count procedures. 
Exposure of the GML and unfinished tampons to S. aureus using the Tampon 
Sac Method can be seen in Table 11. Reductions in TSST-1 formation ranging 
from 81.1% in media with blood to 95.9% without are demonstrated, while 
the impact on the total number of S. aureus cells was either none at all 
in the presence of blood or 9.1% in tubes without blood. 
The Holland Shake Flask Method showed higher antimicrobial activity with 
the shaking and agitation of the system. This in vitro test method was not 
predictive of the in vivo situation. In contrast, the Tampon Sac Method 
was found to be the preferred in predicting the in vivo situation. 
The in vivo evaluation of efficacy was performed as follows. Both control 
and GML tampons were mailed to the Southwest Research Institute in San 
Antonio, Tex., for evaluation for reduction to TSST-1 formation by S. 
aureus in the baboon vagina. Twelve female baboons were identified by 
immobilization with Ketamine HCl and vaginal examination for gross 
evidence of infection. 
Unfinished control tampons had 5 ml of a S. aureus toxigenic strain grown 
in Brain Heart Infusion Broth for 24 hours at 37.degree. C. absorbed onto 
their distal ends (ends distant from the string). Pre-weighed seed tampons 
were immediately introduced into the baboon vagina, without the use of a 
speculum, and the pull string cut. Rectal temperature and indirect 
systolic blood pressure was taken and recorded. Five milliliter blood 
samples were taken from the cephalic vein and the serum stored at 
-70.degree. C. until analysis for the presence of anti-TSST-1 antibody and 
clinical chemistry could be executed. 
Seed tampons were maintained intravaginally for twelve hours. After the 
first twelve hours, the baboon was immobilized with Ketamine HCl and the 
tampon was removed. The seed tampon was placed in a pre-weighed 4-oz. 
plastic cup. Tampon plus cup were weighed and the amount of tampon 
associated fluid was calculated. The tampon was transferred to a stomacher 
bag containing 50 ml of sterile saline (0.9% NaCl) and mixed for 60 
seconds. The stomacher fluid was then submitted for quantitative 
determination of total S. aureus cell count and TSST-1 concentration. The 
total cell count determinations were made using standard plate count 
method and TSST-1 concentrations were determined using radioimmunoassay 
(R.I.A.). 
All tampons inserted after the seed tampon were treated as previously 
described. After removal of the seed tampon, all baboons had control (y) 
tampons inserted intravaginally to allow for additional growth of S. 
aureus and TSST-1 production within the vaginal cavity. After twelve 
hours' additional incubation, the animals were divided in two sets of six 
whereby six baboons were tested with the control tampons and six with GML 
coated tampons. After the 48-hour exposure period, all animals had tampons 
inserted supplemented with 5.0 ml of their own blood serum because of 
diminished menstrual flow. Total viable S. aureus cell count and TSST-1 
level determination were made on all tampons. After all tampons were 
processed and both toxin and cell count determinations were made, four 
animals were excluded from the study (two control animals and two GML test 
animals). In these animals, either the organism was not transferred to the 
vaginal cavity to initiate an infection, or less toxin or cell levels than 
those known to have been applied to the seed tampon were found. Table 12 
sets forth the data of total toxin per milliliter of tampon associated 
fluid and total toxin on the tampons with the impact on cell count. Tha 
data shown is representative of four animals in each test group. 
The data set forth in Table 12 demonstrate considerable decreases in toxin 
formation in the four animals wearing the GML tampons over those wearing 
the control tampons alone. FIGS. 1-4 represent the data of the impact of 
the GML tampons. Although initially the toxin level in the test animals 
wearing control tampons had higher TSST-1 levels, the GML tampons brought 
the level of toxin down significantly over that observed in the controls. 
The data representing the total toxin produced per 10.sup.6 cells of S. 
aureus, thus normalizing the data with respect to individual cells, 
demonstrates the significant reduction in the tampons containing GML over 
the control tampons. A trend of increasing toxin in relation to bacterial 
cells was noted in the control animals after the addition of blood on the 
60-hour tampons. The trend noted in the control animals appears to be a 
direct impact on the cells themselves and the growth curve. This trend was 
observed in the animals wearing the GML tampons. 
The data set forth in Table 13 show a direct comparison of a percentage of 
the control of toxin on the tampon-associated fluid and total toxin formed 
in the tampons in comparison with the control tampons. 
TABLE 10 
______________________________________ 
IMT OF GML COATED TAMPONS ON TSST-1 
FORMATION USING HOLLAND SHAKE FLASK METHOD 
FINAL TOTAL 
CONC. AMOUNT 
TOTAL OF VIA- 
OF VIA- 
TSST-1 RE- BLE S. BLE S. 
PRO- DUC- AUREUS AUREUS 
DUCED TION CELLS CELLS 
SAMPLE MEDIUM (ug) (%) (cfu/ml) 
(cfu) 
______________________________________ 
S. aureus 
BHI 81.57 -- 2.4 .times. 10.sup.9 
1.20 .times. 10.sup.12 
Control 
BHI 1.62 -- 1.92 .times. 10.sup.9 
9.60 .times. 10.sup.11 
Tampon 
GML BHI &lt;0.001 99.93 &lt;10 &lt;5.00 .times. 10.sup.3 
(2.38%) 
Tampon 
S. aureus 
Blood* 66.89 -- 2.0 .times. 10.sup.9 
1.00 .times. 10.sup.12 
Control 
Blood 6.23 -- 2.40 .times. 10.sup.9 
1.20 .times. 10.sup.12 
Tampon 
GML Blood 0.004 99.92 1.92 .times. 10.sup.4 
9.60 .times. 10.sup.6.sup. 
(2.38%) 
Tampon 
______________________________________ 
*Denotes defribrinated Sheep Blood added to BHI at 1.0% v/v concentration 
TABLE 11 
______________________________________ 
IMT OF GML COATED TAMPONS ON TSST-1 
FORMATION USING THE