j. Soc. Cosmet. Chem.

,38, 307-319 (September/October 1987)

Determination shampoo of preservative stability and apparent activation energies the linearregression by method preservative of efficacy testing
D. S. ORTH, C. M. LUTES, S. R. MILSTEIN, and

J. J. ALLINGER, TheAndrew Jergens Company, 2535 Spring Grove

Avenue,Cincinnati, OH 45214.

Received OctoberO, 1986. 3

Synopsis

Preservative efficacy testswereperformed stabilitysamples a shampoo on of during storage 18 mo. at for

3, 20, 38, and 49C.The shampoo preservative system deteriorated increasing with storage time and
temperature, measured the "linearregression as by method."Thus, the decimalreduction time (D-value), whichwasusedas the measure the rateof deathfor Escherichia Pseudomonas of coli, aeruginosa, Bacillus and

sp., increased from about4 hr to over30 hr afteraccelerated agingat 49Cfor 3 mo. The D-values for
$taphylococcus increased aureus moreslowly.

Apparent activation energies (Ea') for the change shampoo in preservative potency werecalculated fromthe D-values different at temperatures. Ea' values The decreased from -7, -6, and -4 Kcal/mole 1 mo. at to -16, -10, and -9 Kcal/moleat 12 mo. for shampoo challenged with E. coli, P. aeruginosa, and Bacillus sp., respectively. This loss preservative of system potency appeared followfirst-order pseudo to or first-order reaction kinetics, whilethe magnitude the Ea' variedwith the lengthof storage the test of and
organisms used.

This workillustrates need using the for different microorganisms conducting when preservative efficacy tests cosmetic of products shows thequantitative and how values obtained thelinearregression by method arewell-suited monitoring to stability tests. The useof this method determining apparent for the molar concentration a preservative of from D-values and for predicting stabilityof cosmetic the preservative
systems from Ea' values discussed. is

INTRODUCTION

Stabilitytestingis the final stepin the development cosmetic of products. The objective

of thistesting to demonstrate a product is that does change not significantly duringits expected shelflife. Product stabilityis necessary because several months years or may elapse between time a cosmetic manufactured usedup by the consumer. the is and Recognizing current this, Good Manufacturing Practices (cGMP)forhuman OTC drug products stipulate that products mustbe stable at least3 yr. andthat this mustbe for supported appropriate by stability data(1). In manyinstances, cGMP areused a basis as

forsupporting documentationcosmetic for products. Thus,stability tests bepermay

3O7

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formedby storage products various of at temperatures up to 5 yr. to demonstrate for

satisfactory shelflife.

Microbialspoilage may occurin aqueous products and in anhydrous products that are exposed water;consequently, to cosmetic preservatives includedin formulations are to inhibit the growthof bacteria,yeasts, and moldswhile products in tradechannels are and in the hands the consumer. of The principles preservative of efficacy testinghave beenreviewed (2,3), includingthe needfor demonstrating products that haveadequate stability(2).

Althoughseveral methods preservative of testingare available (4-6), our laboratory uses "linearregression the method"because provides rapid, quantitative it a expression
of the rate of death of specifictest organisms a product when using definedtest in conditions (4). The rate of death determined the linear regression by methodis expressed the decimalreduction as time (D-value),which is the time requiredfor inactivationof 90% of the populationof test organisms. The rationale usingD-valuesis that everyorganism a characteristic of death for has rate when subjected a specific to lethal treatment (4). This enables laboratory provide a to quantitative results the rate of inactivation specific on of test organisms a product. in Thus, the linearregression methodcanbe usedto determine effectof formulation the changes component and interaction the stabilityof the preservative on system.

Sincethe linearregression methodwasadopted, haveobserved we changes preservain tive efficacy someformulations of duringthe course stabilitystudies. of This report illustrates the value of performingpreservative efficacy testson stability samples by showing how a shampoo preservative system deteriorates with age.

EXPERIMENTAL
TEST ORGANISMS

The testorganisms usedin these studies weretakenfrom theJergens culturecollection andconsisted Staphylococcus (FDA 209 strain),Pseudomonas of aureus aeruginosa (PRD 10 strain),Bacillus (isolated sp. from a contaminated cosmetic product),andEscherichia coli (ATCC 8739). These organisms were cultured and used for challengingthe test samples, described a previous as in report(4).
TEST SAMPLES

The test samples consisted a proprietary of formulation a shampoo high-density of in

polyethylene containers. shampoo The contained ammonium laurylsulfate, cocamidopropylbetaine, propylene glycol,polysorbate hydrolyzed 20, animal collagen, tetrasodium EDTA (and other ingredients), waspreserved and with methylparaben IMP],
chloromethylisothiazolinone [CMIT], andmethylisothiazolinone [MIT].
STABILITY TEST

One bottle of freshlyprepared shampoo usedfor the initial determinations was (i.e., 0-mo.). Several bottles the testsamples stored refrigerator of were at temperature, room

SHAMPOO

PRESERVATIVE

TESTING

309

temperature, 100F,and 120F(i.e., 3, 20, 38, and 49C, respectively)for the durationof the stabilitystudy.One bottleof product stored eachtemperature at was removed specified at timesand subjected preservative to efficacy testing. After sampling, the bottleswereplaced the sample in storage archives.
TEST PROCEDURE

A 0.1-mL aliquotof test organism suspension, containing about 108organisms/mL, wasaddedto ca. 50=mLportions eachtest sample 100=mL of in screw-capped bottles. Aerobicplate counts weredetermined, andD-values werecalculated described an as in earlierreport(4).
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Test samples shampoo of weredilutedto 1% (wt/vol)in mobilephase. After filtration, the samples were assayed injecting250-1zLaliquotsonto a 250 x 4.6 mm i.d. by LiChrosorb RP-18 (5 Izm) columnwith a 40 X 4.6 mm i.d. guardcolumncon tainingPerisorb RP-18, 30-40 Izm. Chromatographic conditions wereas follows: mobilephase= water:methanol (45:55) with 0.2% acetic acid,flow rate0.5 mL/min, temperature 25C,anddetector range 0.05 AUFS. The column effluent monitored was at a UV wavelength 275 nm. of
CALCULATION OF APPARENT ACTIVATION ENERGY

The apparent activation energy (Ea') for the shampoo preservative system deter= was minedfor each organism each test at time period the stability of study (i.e., 1, 3, 6, or
12 mo.). A plot of Log 2.303/D-valuevs 1/T (whereT is the absolute temperature in K) wascalculated each for testorganism, the slope each and of line wasdetermined by linearregression. Ea' hasa valueof - 2.303R (slope), The whichis the same calculation as for Ea (17). Preservative efficacy testswere not performed samples on stored beyond mo. at 49Cor 12 mo. at 38C.The D-values 6 obtained samples on stored for
18 mo. at 3 and 20C were not used in calculation of Ea' values becauseD-values at

the highertemperatures not available. were

RESULTS

Figure ! shows changes preservative the in efficacy that occurred duringstorage the of

shampoo 3, 20, 38, and49Cfor 18 mo. whenusing coliasthe challenge at E.

organism.It is apparentthat the preservative systemwas not stableand that the Dvalues increased with increasing storage time and temperature. Thus, the D-valuesfor E. colichanged from <4 hr at the outset the studyto 35 hr afterstorage 1 mo. at of for

49C.Thepreservative efficacy decreased slowly samples more in stored 38C at thanat 49C,andsamples stored 3 and20Cwereaffected at muchless thanthose stored at 38C,asindicated smaller by changes D-values in duringthe test. The preservative efficacy results test obtained whenP. aeruginosa inoculated was into shampoo samples that had beenstored to 18 mo. at differenttemperatures up are presented Figure2. The findings in obtained with P. aeruginosa similarto those were

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

lOO

go

80

70

60

50

4.0

3o

20

lO

10

12

14

16

18

MONTHS ON STABIUTY TEST

Figure 1. D-values E. coliin shampoo for afterstorage 3, 20, 3 , and49Cfor l, 3, 6, 12, and 18 at mo. Explanation symbols: of . i,__i,, shampoo stored 49C;O--O, shampoo at stored 38 I--I, at C; I--, shampoo stored 20C; at and }--{, shampoo stored 3C. at

obtained with E. coli;however, comparison Figures1 and2 reveals of that the teststrain of E. coli wasslightlymoreresistant the preservative to system thanthe teststrain P. of aeruginosa in thesestudies. used
lOO

90

80

70

60

50

30

20

lO

o
0 2 6

10

12

14

16

18

MONTHS ON STABILITY TEST

Figure 2. D-values P. aeruginosashampoo storage 3, 20, 38 o, and49Cfor 1, 3, 6, 12, and for in after at 18 mo. Explanation symbols: of i,__i,, shampoo stored 49C; at O--, shampoo stored 38C; at --, shampoo stored 20C;and [--I, shampoo at stored 3C. at

SHAMPOO PRESERVATIVE TESTING

311

Figure3 illustrates stabilityprofileof the shampoo the whenchallenged with Bacillus sp. The D-values obtained with this gram-positive spore formerat several pointsin time weresimilarto thoseobtained with P. aeruginosa. AlthoughS. aureus much was lessresistant the preservative to system than the othertest organisms, storage the of shampoo decreased potency the preservative the of system this organism. for Thus, the D-values changed fromaround hr at the outset the stabilitystudy 7.6 hr after 4 of to

storage 6 mo. at 49C to 9.8 hr afterstorage 12 mo. at 38C for and for (Figure 4).
The preliminaryHPLC analyses MP, CMIT, and MIT revealed for that the concentration of MP was unchanged and that the concentration isothiazolinones of appeared to havedecreased all shampoo samples in test that had beenusedin the stabilitystudy. A shampoo spikedwith MP, CMIT, and MIT and assayed HPLC gavethe chromatoby gramshown Figure5. Here, the CMIT andMIT peaks clearly in are evident.This may be contrasted with the chromatogram obtainedfrom a shampoo aged for 18 mo. at

38C(Figure in whichno MIT peakis evident. 6), Similarfindings wereobtained with

shampoo samples storedat the highertemperatures.

The Ea' values the change the shampoo for in preservative system werecalculated by substituting 2.303/D-valuesfor the reaction rate constants in the Arrhenius (k) equation (7). The results presented Table I showthat the Ea' values the shampoo in for preservative system increased negatively from - 7, - 6, and - 4 Kcal/mole 1 mo. to at - 16, - 10, and -9 Kcal/mole 12 mo. whenperforming at preservative efficacy tests
with E. coli,P. aeruginosa Bacillus and sp., respectively. Insufficient datawereavailable

for calculating change Ea' values a in duringthe first6 mo. of the study whentesting
with S. aureus because this organism was inactivated quickly in the shampoo. so The

1 oo

8o

7o

i 60
-.., 50
!

3O

20

8
MONTHS

lO

12

14

16

18

ON STABILITY TEST

Figure3. D-values Bacillus in shampoo storage 3, 20, 38 , and49C 1, 3, 6, 12, and for sp. after at for

18mo.Explanation ofsymbols: &--&, shampoo at49C; stored O--O, shampoo at 38C; stored I--I,
shampoo stored 20C; at and }--{, shampoo stored 3C. at

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

lOO

90

80

70

60

50

40

30

20

10

10

12

14

16

18

MONTHS ON STABILITY TEST

Figure4. D-values S. aureus shampoo storage 3, 20, 38,and49C 1, 3, 6, 12, and 18 for in after at for mo. Explanation symbols: of &--&, shampoo stored 49C;O--O, shampoo at stored 38C ; I--I, at shampoo stored 20C; at and }--{, shampoo stored 3C. at
Ea' values obtained with S. aureus were calculated to be -2 and -4 Kcal/mole from

D-values obtained afterthe shampoo stored 6 and 12 mo. respectively. was for

Regression for the plot of Ea' vsmonths storage lines of werecalculated the test for
organisms (Figure7). The slopes these of regressions showed similarprogression all a for of the testorganisms, exhibitingvalues - 0.8, - 0.4, - 0.6, and - 0.3 Kcal/mole/ of month and the corresponding correlation coefficients of -1.00, -0.93, -0.75, (r) and - 1.00 for E. coli,P. aeruginosa, Bacillus sp., andS. aureus, respectively. Thesedata suggest that the lossof preservative system potency the shampoo in followed first-order or pseudo first-orderreaction kinetics.

DISCUSSION

Accelerated aging, or storage elevated at temperatures, beenused studythe shelf has to life of cosmetic products and/orto determine effectof physical chemical the and parameters the formulaonpreservative of stability(8,9). Several reports indicate that cosmetic preservative systems interact may with components the formula packaging of or materials, with concomitant of preservative loss potency (9-14).

In the present study,thepreservative system the shampoo found be unstable, of was to as indicatedby the changein D-valueswith time and temperature. Higher storage temperatures produced fairly rapid deterioration shampoo in preservation, deteras minedwith E. coli,P. aeruginosa, Bacillus This inactivation the preservative and sp. of system wasnot as obvious whenusingS. aureus the challenge as organism (Figure4)
because net antibacterialeffectof the shampoo the (discussed below) inactivatedS.

SHAMPOO

PRESERVATIVE

TESTING

313

Figure 5. HPLC chromatogram shampoo of spikedwith methylparaben (MP), chloromethylisothiazolinone (CMIT), and methylisothiazolinone (MIT).

aureus rapidly. The differences the ratesof inactivation the preservative in of system occurring 3 to 49Cwerenot dueto differences the shampoo at in samples, because all samples usedat eachtime/temperature periodcamefrom the same bottle.

Theshampoo stored 49Cforover monthwas least at one the resistant contamination to by E. coli, as evidenced the largerD-valuesobtained comparison by in with those observed when usingthe othertest organisms. This may be due, in part, to limited
activityof MP against gram-negative organisms and, in part, to the ability of coli(2) forms growin thepresence anionic to of surfactants. example, ! % sodium For 0.0 lauryl sulfateis used to increase selectivityfor coliformsin enrichmentmedia such as the LaurylTryprose Broth (16).
Thesefindingsshowthat differentmicroorganisms not respond do monotonically the to preservative system the shampoo. in This is why it is essential that preservative efficacy testingbe performedusingtest organisms with all of the physiological characteristics expected be a potentialproblemin the formula. As a minimum, gram-negative to organisms with diverse metabolic capabilities, suchasP. aeruginosa, a representative of the coliform group, suchas E. coli, a gram-positivecoccus, suchas S. aureus, and a gram-positive, spore-forming (i.e., Bacillus rod sp.) shouldbe includedin preservative testing.Thesebacteria,in additionto selected yeasts and/ormoldsnot discussed the in

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

Figure 6. HPLC chromatogram shampoo storage 3Cfor 18 months. of after at

currentwork, providesufficiently diverse morphological physiological and characteristicsto give reasonable assurance the test results that offera goodindication the of

resistancetheproduct contaminants could of to that come contact theproduct into with

duringproduction while in the hands the consumer. or of Other considerations the in selection testorganisms of havebeendiscussed (2).

It is knownthat some cosmetic materials raw affect preservative efficacy. addition, In it should recognized the preservative be that system a cosmetic of product mayinvolve
Table I

Change Apparent in Activation Energies of Shampoo (Ea') DuringStability Testing

Ea' (Kcal/mole)*
Test Organism
E. coli P. aeruginosa

0 mo
-** -

1 mo
-7 - 6

3 mo
-9 - 6 - 3

6 mo
- 1! - 9

12 mo
- 16 - 10

Bacillus sp.
S. aureus

....

- 4

- 10
2

- 9
- 4

* Calculated substitution 2.303/D-value k in Arrhenius by of for equation equation (see {3}).

** Calculation performed not due to lack of sufficient data.

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PRESERVATIVE

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315

-1 -2 -3
-4.

-5 -6
-7 -8 -9 -lO 11 -12

-13 -14.

-15
-16
-17

-18, -19
-20

lo

11

12

13

14

MONTHS ON STABIUTY TEST

Figure 7. Change apparent in activation energies (Ea') for the shampoo preservative system during the stabilitystudy, determined usingtest organisms. Explanation symbols: of I--I, E. coli;O--O, P.

aeruginosa; --,

Bacillus andl__,, S. aureus. sp.;

more than chemicals with known antimicrobialactivity. Factorssuch as pH, water activity, nutrientavailability,surfactant concentration, sequestering agents,and other interferences determine extentto whichpreservative will the actionis manifested any in given formulation.Thus, the preservative systemof a productinvolvesboth specific preservative chemicals the physicochemical and constitution the entireformulation. of

The inhibition of bacterial growth in a shampoo may conceivably due to more than be one mechanism (i.e., surfactant destabilization cell membranes, of preservative action on cellular metabolism,sequestration divalent metal ions by tetrasodiumEDTA, of unavailability nutrients,etc.). One would not expect of organisms with differentmetabolic capabilities be inactivated the samerate in any cosmetic to at product. In the currentstudy,it wasfoundthat the four testorganisms responded differentlyto the net antibacterialeffectof the shampoo.

Although the goal of this investigation not to determinethe cause changein was of preservative efficacy,the HPLC resultsrevealed presence all three preservatives the of initially, but only MP wasunchanged after oneweekof storage room temperature at (Figures and6). This suggests CMIT and/orMIT reacted 5 that with some component in the formula--possiblyhydrolyzed animal collagen,because theseisothiazolinones
are known to react with amines (15).

It is knownthat the rateconstants chemical for reactions influenced temperature, are by

asrepresented theArrhenius by equation (17)

k = Ae-r'RT
wherek is the reactionrate constant,A is the pre-exponential factor, Ea is the activa-

tion energy,R is the gasconstant, T is the absolute and temperature. Differentiating

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

equation withrespect temperature integrating (1} to and between limitsyields equation {2} (17):

log - 2.303R - T,) k2 Ea (TT 2) (T2 k

values, andT, asin equation Ea {3}:

{2}

Stumbo(7) reported that the D-value = 2.303/k; consequently, = 2.303/D-value. k Substituting2.303/D-values for k enablesone to use an expression that relatesD-

D-value (T - T) _ 2.303R2) Ea log D-value2 2 (TT

{3}

The ratesof reactions biological in systems affected are similarlyby temperature, that so the rate of inactivation a givenorganism the presence preservative of in of chemicals (or other physicochemical conditionsthat are bacteriocidal) increases with temperature.
Since D-values becomesmaller as the rates of microbial inactivation increase,it would

be expected that the D-valuefor a given organism a testsample in would decrease with an increase preservative in efficacy test temperature--as long asthe preservative system wasnot alteredby the assay conditions. The situationis differentin the currentstudysincethe shampoo preservative potency wasevaluated constant at conditions (i.e., by performing preservative all efficacy tests at room temperature) after test samples beenstoredfor specified had times at different temperatures. The preservative systemwas found to be unstablewhen testedby the linearregression method.This decrease preservative in potency with time and temperature of storage resulted decreases the slopes the survivor in in of curves and corre(4) sponding increases D-valuesfor eachtest organism. in

Althoughthe slope the Arrhenius of activation energy plot is negative whenk increases with temperature, decrease ratesof bacterialinactivation the in with temperature observedin this studygivesa positiveslopein the Arrheniusactivationenergyplot, asis illustrated the dataobtained for with the testsamples stored 12 mo. andchallenged for with E. coli (Figure The Ea calculated 8). fromthese results negative; are consequently,

theyaredesignated Ea'. The Ea' values calculated shampoo for preservative potency duringthe first 12 mo. of the stabilitystudyappear TableI. The progressive in decrease Ea' for all testorin
ganisms reflects decrease preservative the in system potency. Here, the rateof change of preservative system potencywasgreatest E. coli(i.e., the organism for most resistant to the shampoopreservative system)and smallestfor S. aureus (the test organismleast resistantto the preservative system).

Although negative values the Ea' appear becontradictory conventional to to systems in

which Ea is determined,oneshouldnote that the parameter beingmeasured--preservative system potency--decreased with increasing temperature, determinedby the as kineticsof inactivation the test organisms. of

In general, the rate of a chemicalreaction,as expressed k, is a functionof the by concentration the reactants. the concentration a reactant of If of (i.e., preservative) is changed a resultof storage elevated as at temperatures different for times,determining D-valuesand using2.303/D-valueenables one to determine at differenttemperak tures. Thesek valuesmay be compared with k valuesobtainedin systems known of

SHAMPOO PRESERVATIVE TESTING

317

3.0

3.2
1000/T

3.4

3.6

Figure 8. Arrhenius activation energy plot of shampoo preservative system potency determined using E. colias the challenge organism preservative in efficacy tests samples on stored 3, 38, and 49Cfor 12 at
months.

preservative molar concentration, that one may determinethe molar concentration so (or the apparent molar concentration) activepreservative the formulaafter any of in given time and temperatureof storage.Calculations this type may be useful in of systems containingonly one preservative chemical.Obviously, calculations will be quite complexin products containingmultiple surfactants preservative and chemicals

because loss preservative the of potency mayfollowhigher-order reaction kinetics.It is believed that determining D-values andEa' values may be usefulin studying kithe neticsof bacterialdeath, in determiningthe apparent molar concentration a preserof vative, and in monitoring the performance cosmetic of preservative systems during stability studies.
In discussing accelerated stabilitytesting,PopeusedEa values 10-20 Kcal/molefor of predicting goodprobability formulation of stability,whichhe definedasonethat degradedno morethan 10.5% in 3 mo. at 45C(18). He notedthat formulations that degrade throughsolvolysis haveEa values 10-30 Kcal/mole of and that systems with Ea values thismagnitude of showmarked increases reaction in rates elevated at temperatures.The findings thisstudyrevealed the unstable in that shampoo preservative system hadEa' values - 2 to - 16 Kcal/mole, of depending the time periodof the determion
nation and the test organismused. It is believedthat the differencebetween the Ea

values proposed Popeand the Ea' values by observed this study(includingboth in

absolute magnitudeand algebraic sign) may be due to 1) the difference k for the in solvolysis reactions citedby Popeandcomplexation reactions, such those as involved in the interaction isothiazolinones amines of and (15); and2) the wayin whichEa andEa'
were defined and derived.

The shampoo preservative system wassatisfactory whenexamined the outsetof these at studies (i.e., at 0 mo.), but it deteriorated during the agingstudy. Hence, the Ea'

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

valuesfor the shampoodecreased with time. By way of contrast,determinationof

D-values and Ea' values the testorganisms a proprietary for in lotionpreserved with MP andQuaternium-15 revealed changes these no in values testsamples for stored 3 at to 49C for the durationof the stabilitystudy. This indicated that the preservative
system the lotion wasnot changing of detectably,asevaluated the linear regression by

method.These examples illustrate useful a caveat: Ea' values the should deviate little
from the initial, acceptable values a productis to havesatisfactory if shelflife. Thesestudiesdemonstrate that the linear regression methodis usefulfor quantitating the ratesof inactivation bacteria of inoculated into stabilitytest samples and illustrate the valueof this methodfor monitoringpreservative efficacy stability test samples. of To our knowledge, this is the first reporton the useof a quantitative method,which provides the kinetics bacterial via of death,a methodto monitorpreservative potency at various timesduringstabilitystudies cosmetic of products.

In mostinstances, preservative efficacy testingdemonstrates the cosmetic that preservative system inactivates organisms test rapidlyand that the D-values positive.The are preservative system judgedto be inadequate is whenD-values greaterthan accepare tancecriteria(4) or whentheyarenegative. example, preservative For the system fails when test organisms grow in the sample.The slopeof the survivorcurveis positive whengrowthoccurs, whichmeans that the D-valueis negative. NegativeD-valuesare rarely,if ever,reported published in literature because indicate they preservative system
failure and the need for reformulation.

Althoughnegative values Ea' appear be somewhat to anomalous, must keepin one

mind that they represent decrease potency the preservative a in of system that occurs on storage and that the rate of preservative deterioration accelerated increases is by in temperature. noted As above, negative D-values obtained are whentestorganisms grow

in thetestsamples. Negative D-values cannot used calculating values be in Ea' because

onecannottake the log of a negative number.

This reportshows that the linearregression method may be usedfor predicting the stability theapparent and molar concentrationa preservative of system addition its in to already-documented in determining cosmetic utility the preservative efficacy. Thus, examination thepreservative of efficacy a formula of after storage 49C 1 to 3 mo. at for may indicate system unstable that reformulation necessary. is recomthe is and is It mended that formulation chemists quantirate preservative system potency duringaccelerated agingstudies determine likelihood preservative to the of system failure.
ACKNOWLEDGEMENT

The authors express their appreciation Mr. W. E. Dickmanfor his assistance to with
stability testing of the samples.

REFERENCES

(1) U.S. Food& Drug Administration, Human and Veterinary Drugs, CurrentGood Manufacturing Practice Manufacture, in Processing, Packing, Holding,Fed.Register, 45014-45087 (1978). or 43, (2) D. S. Orth, Principles preservative of efficacy testing,Cosmet. Toilet.,96(3), 43-52 (1981).

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PRESERVATIVE

TESTING

319

(3)

H. N. Bhargava A. Anaebonam, and Essentials cosmetic of preservation, Cosmet. Soap Chem. Special-

ties, 59(10), 39-41, 125 (1983). (4) D. S. Orth, Linear regression method rapiddetermination cosmetic for of preservative efficacy, Soc. J. Cosmet. Chem., 30, 321-332 (1979).

Anon, Microbiological tests,antimicrobial preservatives--effectiveness, UnitedStates Pharmacopeia XIX, United States Pharmacopeial Convention, Rockford,MD, pp. 587-592 (1975). (6) Preservation subcommittee the CTFA microbiological of committee,A guidelinefor the determination of adequacy preservation cosmetics toiletry formulations, of of and TGA Cosmet. 2, 20-23 J.,
(1970). (7) C. R. Stumbo,Thermobacteriology processing, infood Academic Press, Inc., New York, p. 59. (1965). (8) I. Matsuura,Methodsfor the prediction shelflife, Cosmet. of Toilet., 96(1), 39-44 (1981). (9) G. Jacobs, M. Henry,andV. F. Cotty, The influence pH, emulsifier, S. of andaccelerated aging

(5)

uponpreservative requirements O/W emulsions, Soc. of J. Cosmet. Chem., 26, 105-117 (1975).
(10) M. M. Rieger, Current aspects cosmetic of science. The inactivationof phenolicpreservatives I. in

emulsions,Cosmet. Toilet., 96(5), 39-43 (1981).

(11) S. Tanenbaum, Pseudomonads cosmetics,J. in Soc. Cosmet. Chem.,18, 797-807 (1967). (12) R. Bhadauria and D. G. Ahearn,Loss effectiveness preservative of of systems mascaras age, of with

Appl. Environ. Microbiol., 665-667 (1980). 39,

(13) T. J. McCarthy, Microbiological controlof cosmetic products, Cosmet. Toilet., 95(8), 23-27 (1980).

(14) P. A. Berke,D.C. Steinberg, andW. E. Rosen, Germaben a complete II, preservative system in

clearliquid form, Cosmet. Toilet., 97(11), 89-93 (1982).

(15) S. M. Henry and G. Jacobs, Cosmetic preservatives--1982update,Cosmet. Toilet., 97(11), 31-52 (1982).

(16) J. Cowls,American WaterWorks Assoc., 979 (1938), Cited in: DifcoManual. Dehydrated 30, Culture

MediaandReagents Microbiology, ed. Difco Laboratories, for 10th Detroit, p. 499. (1984)
(17) F. Daniels and R. A. Alberty, Physical Chemistry, ed. JohnWiley & Sons, 3rd Inc., New York, p. 342. (1967). (18). D. G. Pope,Accelerated stabilitytestingfor prediction drugproduct of stability,DrugCosmet. Ind.,

127(6), 48ff (1980).