J. $oc. Cosmet. Chern.

29 3-24 (1978)

The activity and safety of the antimicrobial agent

Bronopol(2.bromo.2.nitropropan.1, $-diol)

D. M. BRYCE, B. CROSHAW, J. E. HALL, V. R. HOLLAND and

B. LESSEL The BootsCompanyLimited, NottinghamNG2 3.8.8

Synopsis

Recentwork on the microbiologicalactivity, chemistryand safetyof the antimicrobialagent Bronopolis

reported. Methods for the estimationof Bronopol are described,and the nature of its decomposition
productsis discussed.Animal metabolismand toxicologyresultsare reported,togetherwith animal and
human studieson irritancy and sensitisation.
The performanceof Bronopol in a number of experimental
formulations is described.

INTRODUCTION

Early work by Hodge,Dawkinsand Kropp (1) and by Zsolnai(2)suggestedthat geminal

bromonitroalkanes had antifungalactivity.The broad-spectrum
antibacterialproperties
of 2-bromo-2-nitropropan-l,3-diol (Bronopol) have been describedin a preliminary
communicationby Croshaw,Grovesand Lessel(3) andin comparisonwith othermembers
of a seriesof antimicrobialaliphatichalogeno-nitrocompoundsby Clark et al. (4).
Bronopol
is usedasa preservative
in various
cosmetic,
toiletryandhousehold
pre-
parationsparticularlybecauseof its high activity against Gram-negativebacteria,
especiallyPseudomonas aeruginosa and otherpseudomonads.Theseorganismsare com-
mon residentsin water and as suchcan causecontaminationand spoilageproblemsin
cosmetics andtoiletties(5, 6, 7, 8). Pseudomonadsare frequentlyimplicated,particularly
in oil-in-water emulsionswhich contain a significantamount of nonionic surfactants
(9, 7, 10).
Bronopolis an effectiveantibacterialpreservativeover a wide pH range.It is stable
at acid pH's and is also usefulas a labile antibacterialpreservativein alkaline media.
Becauseof its broad-spectrumantibacterialactivity Bronopol can also be used as an
activeagent,for example,in aerosolformulations.Bronopolhasbeenreportedto show
persistentactivityon the skinby Marplesand Kligman (11), this contrastswith the fact
that in vitroit hasbeenshownto havea weakgrowth-inhibitoryeffecton culturedhuman
skin cellsby Onoda and Saito (12).
A programmeof experimentalwork was begunsometime ago to extendour knowl-
edgeon the safetyof Bronopol.This work is now completedand it is appropriateto
reviewthe resultsof theseand otherhithertounpublishedstudieson the microbiology,
chemistry,analysisand formulationof this compound.
0037-9832/78/0100-0003$02.00 ¸ 1978 Societyof CosmeticChemistsof Great Britain
4 D.M. Bryceet al.

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 Activity
andsafety
ofBronopol 5

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6 D.M. Bryce et al.

RESULTS AND DISCUSSION

MICROBIOLOGY

AntibacterialActivity
The bacteriostaticactivity of Bronopolin comparisonwith that of a range of other
agentshasbeendeterminedby serialdilutionin 'Oxoid' nutrientagar (seeTablesI and
II). Plateswereinoculatedwith a multi-pointinoculator(13). The bacteriostaticactivity
againstsomepseudomonads, includingPs. aeruginosa,Ps.fluorescens
andpseudomonads
isolatedfrom paints,water,cosmetics and unpreserved pharmaceuticalformulationshas
been comparedusinga similartechnique.Of the antibacterialagentscomparedonly
Phenylmercuric Nitrate BP and Chlorhexidine AcetateBPChad similarbroad-spectrum
activityto that of Bronopol(TablesI andII). 2,4,4'-Trichloro-2'-hydroxydiphenylether
wasmoreactivethanBronopolagainstmostof the organisms tested,but thiscompound
wasmuchlessactiveagainstPs. aeruginosa. Of the agentstestedagainstpseudomonads
only Phenylmercuric Nitrate BP wasmore activethan Bronopol(seeTableIII).

Table 1II. Comparativeactivityof Bronopoland other agentsagainstPseudomonas spp.by agar-dilution.

Agar platesinoculatedwith 0.01 ml of 18 h culturesundiluted; incubatedfor 48 h at 32øC

No. of No. of strainswith m.i.c. (3tg/ml)of:

strains
Preservative tested 12.5 25 50 100 200 400 >400

Bronopol 23 5 18 0 0 0 0 0
Propyl HydroxybenzoateBP 23 0 0 0 0 0 0 23
Methyl HydroxybenzoateBP 23 0 0 0 0 0 0 23
PhenoxyethanolBPC 23 0 0 0 0 0 0 23
PhenylmercuricNitrate BP 23 19 2 2 0 0 0 0
PhenylethylAlcohol BPC 1963 23 0 0 0 0 0 0 23
Benzalkonium Chloride 23 0 0 0 0 0 19 4
Chlorocresol BP 23 0 0 0 0 0 23 0
Chlorbutol BP 23 0 0 0 0 0 0 23
Chlorhexidine Gluconate 23 0 0 3 20 0 0 0
Chlorhexidine Acetate BPC 23 0 4 18 1 0 0 0
6-Acetoxy-2,4-dimethyl-m-dioxane [1] 12 0 0 0 0 0 0 12
cis-isomerof 1-(3-chloroallyl)-3,5,7-triaza-
1-azonia-adamantanechloride [2] 12 0 0 0 0 5 3 4
Substitutedimidazolidinyl urea cpd. [3] 6 0 0 0 0 2 1 3
N-Trichloromethylthio-4-cyclohexene-l,2-
dicarboximide[4] 12 0 0 0 0 3 2 7
Zinc salt of 2-mereaptopyridine-l-oxide[5] 12 4 1 2 0 2 2 1
2,4,4'-Trichloro-2'-hydroxy-diphenyl ether [6] 6 0 0 0 0 0 0 6

[1] Giv-Gard DXN.

[2] Dowicil 200.
[3] Germall 115.
[4] Vancide89 RE.
[5] Zinc pyrithione.
[6] Irgasan DP 300.

The effectsof organicmatterand somepossibleantagonists are shownin TableIV.

Bryceand Smart(14) reportedthat nonionicsurfaceactiveagents,e.g. polysorbate80
and lecithin,havelittle or no effecton the antibacterialactivityof Bronopol,although
 Activity and safetyof Bronopol 7

suchagentsare known to antagonisethe action of many preservatives and Brown (15)

confirmedthat a plot of activityversus
the log phaseconcentrations
of Ps. aeruginosa
for
solutionsof Bronopol containing1• polysorbate80 showedthat activity did not de-
crease.Sulphydrylcompoundsare markedly antagonisticto the in vitro activity of
Bronopol(3). This has beenconfirmedby Strettonand Manson(I6).
Table IV. The effectof organicmatter and possibleantagonists
on the bacteriostaticactivity of Bronopol by Agar dilution
(strainsof Pseudornonas aeruginosatest organism)

Decrease (-fold) in
Additive bacteriostaticactivity*
10•o ox serum 0-2
50•o ox serum 4-8
10• human serum 2
50•o human serum 4
10• oxalatedhorseblood 4-8
50•o oxalatedhorseblood 32-64
10•o milk 0
1• polysorbate80 0
0.1Yolecithin 0
0.1•o cysteinehydrochloride 16-64
0'1•o sodiumthioglycollate 8-16
0'1•o sodiumthiosulphate 4-16
0.01% sodiummetabisulphite 8-16
* 2-fold serial dilution.

Using the filter paper strip technique(17) with Staphylococcusaureusas the test
organism,it has been shownthat there was no inhibition of the activity of Bronopol
by CetrimideBP, DomiphenBromideBP, BenzalkoniumChlorideBPC or trichloro-
carbanilide.
Furtherwork has confirmedthe reportby Croshawet al. (3) that thereis no evidence
of the developmentof Bronopol-resistant organismsafter passagein the presenceof
Bronopolfor 20 subcultures.In practiceBronopol-resistant
organisms havenot occurred.
Someinsightinto the modeof actionof Bronopolhasbeenobtained.SinceBronopol
is moreactiveagainstmetabolisingcellsthan restingcellsand its antibacterialactivityis
reversedby thiol-containingcompounds(3), thiol-containingenzymeswould appear to
be implicated.Bronopolforms disulphidebonds from thiol groupsand thesemay
accountfor the observedinhibitionof dehydrogenase activityby the compoundat con-
centrationsapproximatingthe minimuminhibitoryvaluefor eachorganism.Inhibition
of microbial membrane-bounddehydrogenaseenzymesmay causealterationsin mem-
branestructureand accountfor the cell leakageobservedon Bronopoltreatment(16).
Thus thiol-containingenzymesare involvedin the mode of action of Bronopolagainst
bacteria.The selectivityof the compoundfor micro-organisms,
indicatedby its very low
mammaliantoxicity, may be due in part to the rapid metabolismof Bronopolby the
body tissues.

CHEMICAL AND ANALYTICAL

Stabilityof Pure Bronopol

Experiments
wereconducted
to establish
thestabilityof Bronopolon storage
in thepure
8 D.M. Bryce et al.

solidstate.The results,shownin TablesV and VI, demonstrateno evidenceof instability

overa periodof oneyear'sstorageat temperatures up to 45ø, and at elevatedhumidity.
No photodecomposition wasobservedat roomtemperatureoverthisperiod.Samplesof
Bronopolstoredin the dark at room temperatureover periodsup to 2 yearsalso show
no evidenceof decomposition. All the assayswere carriedout by gas-liquidchromato-
graphy(g.l.c.) usingthe trimethylsilylationproceduredescribedlater in the sectionon
analyticalmethods.Both the internal standardand normalisationmethodswereutilised.
Initial assayswere by g.l.c. usingn-pentadecaneas the internal standardafter acetyla-
tion. Sincethat time, the trimethylsilylation
proceduredescribedlaterhasbeendeveloped
in orderto achievean improvementin precision.Assaysafter storagewere doneby this
latter method,usingthe internal standardtechnique.

Table V. Stability of Bronopol during storage

g.l.c. (internal
standardmethod) g.l.c. (normalisationmethod)

Bronopol Bronopol Impurity at RT(rel) 0.62

Storageconditions % % •o
Initial 100.0 99.6 0.45

weeks,
at 20 to 25øC
(a) at normal RH 99.5 99.6 0.43
(b) at 90•o RH 100.3 99.6 0.41
at 37øC 99.9 99.6 0.39
at 45øC 99.7 99.6 0.39
in north window 100.4 99.6 0.43

weeks
at 20 to 25øC
(a) at normal RH 99.7 99.6 0.40
(b) at 90•o RH 99.9 99.6 0.39
at 37øC 100.0 99.6 0.41
at 45øC 99.4 99.6 0.41
in north window 100.1 99.6 0.44

12 weeks
at 20 to 25øC
(a) at normal RH 99.8 99.5 0.49
(b) at 90•o RH 100-1 99.6 0.41
at 37øC 100.2 99.5 0.46
at 45øC 99.6 99.6 0.44
in north window 99.8 99.6 0.44

52 weeks
at 20 to 25øC
(a) at normal RH 100.2 99.5 0.50
(b) at 90•o RH 99-2 99-5 0.47
at 37øC 100.3 99.5 0.46
at 45øC 100-3 99.5 0.46
in north window 99.3 99.4 0.51

RH =relative humidity.
Rx(rel)--relative retention time.
 Activity and safetyof Bronopol

Table VI. Stabilityof Bronopolafter storageat room temperaturein

the dark

Initial assay Assayafter

Bronopol storage,Bronopol
•o Time of storage •
99.2 24 months 98.2
99.2

101.1 22 months 101.5

102-2
100.2 21 months 101.4
102.4

99.9 18 months 99.7

99.5

Stabilityin AqueousSolution
Bryceand Smart(14) have shownthat aqueoussolutionsof Bronopolare reasonably
stablewhenacid.To investigate
the stabilityof the compoundin moredetail,aqueous
solutionsof Bronopol(0.2• w/v) werepreparedat pH 4 and6 (McIlvainebuffer)and
pH 8 (phosphate buffer).Thesolution
at pH 4 wasstoredin thedarkat 50øC,the solu-
tion at pH 8 at 30øC,andthe solutionat pH 6 at 30, 40 and 50øC.At appropriatetime
intervalsaliquotswereremovedandexaminedmicrobiologically, polarographically,gas-
chromatographically and for bromideion, nitrateion, nitriteion and formaldehyde.
Aqueoussolutionsof Bronopol(10• w/v) at aboutpH 6 were storedat temperatures
rangingfrom 40 to 100øC,the pH beingmaintainedby the additionof 5N sodium
hydroxide.Aliquotswereremovedandexaminedby thin-layerchromatography (t.l.c.)
and bioautography.
Attemptsweremadeto isolatethe decomposition productsfrom
partiallydecomposed
solutionsby preparativelayerand Sephadexcolumnchromato-
graphy.
The decomposition of Bronopolwasfoundto be acceleratedby increasing the pH
or thetemperature of thesolution.
Theseeffectsareshowngraphically in Figs.I and2;
usinga factorof approximately4 astheincrease
or decrease
in therateof decomposition
per 10øCtemperature change,thetimesfor 50• decomposition extrapolated to 20øC,
and basedon the g.l.c. assayresults,are as shownbelow:

Time for 50Yo

pH decomposition
4 • 5 years
6 1« years
8 2 months

Theinitialprocessin thedecomposition
of Bronopolappears to bea retroaldol
reaction
with theliberationof formaldehyde
andthe formationof bromonitroethanol'
NO• NO,

I I
HOCH•-- C -- CH•OH•CH• + HOCH•-- CH
I I
Br Br
10 D.M. Bryceet al.

I00 PH
4(m•rob•ologi•al
assay._..•)

IO[
PH
•(G
LC)•og,½al assoy)
I 2 5 4 5 6 7 8 9 I0
Time of storoge(weeks)

Figure1. Effectof pH on stabilityof aqueoussolutionsof Bronopolat 40øC

(initially 0'2•o w/v)

100

8O

2O

10

II 2
•ß 5.I .-'•--•_
I 5I•500C
4
(G/C)
6
• I
7 8
I
9
I
I0
Time of storage(weeks)

Figure2. Effectof temperature

onstabilityof aqueous
solutions
of Bronopol
at pH 6
(initially0'2•o w/v)
 Activity
andsafety
ofBronopol 11

Bromonitroethanol
itself
isconsiderably
less
stable
thanBronopol
andintherangeof
conditions
investigated
themaximal
concentrations
didnotexceed
0'5•o
oftheinitial
Bronopolconcentrations.
Simultaneously
asecond-order
reaction
involving
Bronopol
and
formaldehyde
occurs
togive2-hydroxymethyl-2-nitro-
1,3-propanediol:

NO•
I H20 iH•
OH
HOCH2•C CH'•OH+
CH•O
• HOCH•C CH•OH
+HBr
I
Br
I
NO•

2_hydroxymethyl-2-nitro-l,3-propanediol
hasbeen
isolated
frompartially
decomposeu
10•ow/vsolutions
ofBronopol
bypreparative
layer
andSephadexcolumnchromato-
graphy.
Then.m.r.
andi.r.spectra
and
theelemental
analysis
support
theproposed
structure.
2_Hydroxymethyl-2-nitro-l,3-propanediol
itself
decomposeswiththeloss
of
formaldehyde.
This
reaction
isrelatively
slow,
however,
sothatafter
2-3Bronopol
half
lives
this
compound
accounts
for8-10•o
oftheorganic
material,
asshownbythethin
layerchromatogram
(Fig.3).
Solvent front

Bromonitroethanol
0 0 0 O 0 o o

OOoooooo Bronopol

o o o 0 0 0 o o
o o 0 0 0 0 o ø o
2-hy droxymet
hyl-2 -nitro-
oo oo000o0 1,3-propanediol

o o o o o 0 o o o
o o • o o---o 0 0 CL Origin
Timeof storageof solution
(min)
Figure
3.Thin-layer
chromatogram
ofBronopol
aqueous
solutions
(initially
10•ow/v)
storedat 100ø andmaintained
at pH 6

Inmore
dilute
solutions
thesecond
order
reaction
willbeless
important
andtheloss
ofbromine
follows
first
order
kinetics,
therate
ofloss
beingaboutone-half
oftheoverall
rateof decomposition
of Bronopol.
12 D.M. Bryce et al.

A number of reactionsinvolving formaldehyde occur simultaneously.The overall

result is that the formaldehydeconcentrationtends to a maximum which is lower than
an equimolarratio. The rate of formation of formaldehyderelativeto the rate of decom-
positionof Bronopolwas not markedlyaffectedby pH over the rangeinvestigated.
An additional mode of decompositionresults in the formation of nitrite but not
nitrate. The rate of formation of nitrite tends to follow second-order kinetics and is
slowerthan the overalldecompositionof Bronopol as measuredby g.l.c. No information
has been obtainedon the route by which the nitro group is lost and the final organic
productshavenot beenidentified.Their physicalpropertiessuggest, however,that some
may be polymeric. It should be noted that in the presenceof certain secondaryand
tertiary aminesand amides,nitrite can form nitrosamineswhich may be carcinogenic.
In the opinion of the authors,it is advisablethat formulatorsusingBronopol, or any
other substancegiving rise to nitrite, take stepsto ensurethat if nitrosaminesare pro-
duced,their presencedoesnot representa health hazard to the user.
The gaseousdecompositionproducts of Bronopol have been examined by mass
spectroscopy. Only threemajor peakswerefound the first of whichcouldbe attributed
to nitrogenplus a trace of ethyleneat m/e 28 and the secondto nitric oxideat m/e 30.
The only constituentof the third peak, at m/e 44, which could be identifiedwas the
radical CH•NO. Neither carbon monoxidenor carbon dioxide could be detected.
Thin-layerchromatograms of storedsolutionsof Bronopolweresprayedwith starch/
potassiumiodide solution,which would locate componentsincludingthosecontaining
the aliphatic nitro group. At least sevencomponentswere detected(Fig. 3), three of
which were identified.The origin containedsodium bromide and sodium nitrite. Bio-
autography,on the otherhand, showedonly two activezonescorresponding to Bronopol
and bromonitroethanol(Fig. 4).
It is difficultto explainthe g.l.c.and microbiologicalresultson the basisof the above
observationssincethe bromonitroethanoland formaldehydepresentare not sufficient
to account for the difference between them.

Analytical Methods
The methods described in this section have been used to obtain the results recorded in the
precedingsections,and to assayBronopolin the typesof formulationsin which it is
likely to be incorporated.
Pure Bronopolhasbeenassayed by the determinationof its brominecontent,by the
determinationof its nitrogencontentand by g.l.c.of the acetylatedand of the trimethyl-
silylated material, the methods using g.l.c. being the most specific.In formulations,
Bronopol has been estimatedby t.l.c., by a polarographicprocedure, by a micro-
biologicalprocedureand by g.l.c. The procedureby t.l.c. hasbeenappliedto ointments
(at a concentration of 0.1Yo),to barriercreams(at concentrations of 0.1 and 0'2Yo)and
to aerosolconcentrates (at a concentrationof 0.05•o). G.l.c. hasbeenappliedto aqueous
formulations(at concentrations of from 5 to 50 ppm). The polarographic procedurehas
been appliedto ointments,suppositories, creamsand gels(all at a concentrationof
0'2•o) and hasalsobeenusedto estimateBronopolin bufferedaqueoussolutionsand
in blood serum.The microbiological procedurehas beenappliedto creams,including
barrier creams(at concentrationsof 0.1 and 0'2•o), to liquid shampoosand also to
bufferedaqueoussolutions. The polarographic methodestimates thealkylnitrogroupand
therefore,althoughan acceptableprocedurefor freshly-prepared formulations,is not
 Activityandsafetyof Bronopol 13

Solvent front

Bromonitroethanol
t• O O O o o o o

Bronopol

Origin
0 2 4 6 8 I0 16 20 25 30

Time of storageof solution(rnin)

Fig•e 4. Autobiogram of Bronopol aqueoussolutions(initially 10•o w/v) storedat 100ø

and maintainedat pH 6 (test-organism:Pseudomonas aeruginosa)

very specificand is subjectto interferencefrom breakdownproducts.In the absenceof

interferingsubstances, the precisionof this procedureis about q-2•o. The methodusing
t.l.c. is more specific,but the spot-comparison procedurethat has beenusedis liable to
relativeerrors of about 15•o; errors of this magnitudemay be acceptable,however,at
the concentrationsin which Bronopol is usually incorporatedin formulations. The
microbiologicalmethodhad an error of about 4-10•o on aqueoussolutionsand about
4-10 to 20• on creams.

PolarographicAssay.The base electrolytewas Mcllvaine's buffer solution containing

2•o v/v of 0.2• v/v Triton X 200asa maximum-suppressor. Mcllvaine'sbuffersolution,
pH 4, was preparedby mixing 12.29 parts by volume of 0.1•t citric acid solutionand
7.71partsby volumeof 0'2•o disodiumphosphate(Na•HPOa) solution.The testsolution
waspreparedas follows.Aqueoussolutionsonly requireddilutionwith baseelectrolyte
to a Bronopol concentrationbetween 10-a and 10-SM. Bronopol in gels,creamsand
other fatty-baseformulationscouldbe extractedwith baseelectrolyteby warminggently
on a steam-bath,after whichany insolublematter in the aqueousphasewas removedby
centrifugingand the aqueousphase diluted to give appropriate concentrationsof
Bronopol.Other formulationswere more appropriatelytreatedby dissolvingin chloro-
form and extractingwith baseelectrolyte.
The determinationwas carried out by transferringa portion of the solutionin base
electrolyteto the cell of a suitablepolarograph.A streamof oxygen-freenitrogenwas
passedthroughthe solutionfor 10-15 min to removedissolvedoxygen.The heightof the
mercuryreservoirwasadjustedto givea constantdrop rate appropriateto the apparatus,
this drop rate beingidenticalwith that usedfor the preparationof the calibrationcurve.
14 D.M. Bryceet al.

The polarogramwas recordedover the range0 to -1 V relativeto the quiescent

mercurypoolusingthe appropriaterecorderor galvanometer sensitivity
to givea suitable
wave. The diffusioncurrentat -0.8 V relativeto the mercurypool was measured,and
the concentrationof Bronopolread from the calibrationcurve.
Sincethe polarographicresponsewasaffectedby the compositionof the testsolution,
it was necessaryto prepare a calibration curve for each formulation examined.Such
calibration curveswere obtained by adding known amounts of Bronopol to blank
formulationsand processingin the requiredmanner.
The followingare examplesof the assaymethodwhichhave beenused.

MicrobioloicalAssay.Bronopolcan be assayedmicrobiologically
by agardiffusionusing
Ps. aeruginosa
in agar of the followingcomposition:
% w/v
Dextrose 0.1
Lemco beef extract 0.15
Difco yeastextract 0.•3
Sodium chloride 0.5
Difco casitone 0.08
Magnesiumsulphate(7H•O) 0.004
Oxoid peptone 0.6
Davis agar 1.8
Distilled water to 100, pH adjustedto 5.3.
AlternativelyDifco AssayAgar No. 11 (pH 7.9) with BacillussubtillsNCIB 8054canbe
used.The minimumdetectable levelof Bronopolin waterwith Bacillussubtilisis0'005•o.
A rapid diffusionmethodusingBacillusstearothermophilushasbeendescribed by Kabay
(18).

Gas-liquidChromatographic Assay.AlthoughBronopolis a water-soluble compoundit

can be extractedfrom aqueoussolution into diethyl ether or ethyl acetateafter the
addition of sodiumchloride.The extractcan then be evaporatedto dryness,the residue
acetylatedandthe Bronopolestimatedby meansof g.l.c.with electron-capture detection.
This procedureoffersa meansof determiningBronopolin aqueousformulationsand
has beenappliedto Bronopolconcentrations down to 5 ppm.
In aqueousformulationscontainingconcentrations of Bronopoldownto 50 ppm,the
Bronopolhas beendeterminedby a similarprocedure,but usingn-pentadecane as the
internal standard,acetyl chloridein chloroformas an acetylatingreagent,carbondisul-
phideas the final solventand flame ionizationdetection.
The followingare examplesof the methodswhichhavebeenusedto assayBronopol
by g.l.c.
Basedon the acetylatedmaterial. The sample(about 0.15 g accuratelyweighed)was
dissolvedin 15ml of chloroformwith the aid of minimumheating,5 ml of a 2•o solution
of n-pentadecane
(asinternalstandard)in chloroformwasaddedandthesolutiondiluted
to 25 ml. To 1 ml of this solutionin a vial was added0.3 ml acetylchlorideand the vial
wassealedandthen heatedon a steambath for 3 h. The mixture(2 [tl) wassubjectedto
g.l.c. in a glasscolumn (183 cmx 3 mm) packedwith 10•o of siliconeJXR on Gas
ChromZ (70 to 80 mesh),operatedat 150øCwith nitrogen(20 ml min-x) ascarriergas
and flameionisationdetection.The ratio of the productof the peak heightandretention
 Activity and safetyof Bronopol 15

timefor the Bronopoldiacetate(relativeretentiontime= 1.00)to that for n-pentadecane

(relativeretentiontime= 1.54)wascalculatedand comparedwith the ratio for a standard
containingpurifiedBronopolwhich had been similarlytreated.The relative standard
deviation of the method was found to be 1'5•o.
Basedon thetrimethylsilylatedmaterial. The sample(about0.15g, accuratelyweighed
wasdissolvedin 15 ml of chloroformwith the aid of minimumheating,4 ml of a 1.4•o
solutionof n-tridecane(as internal standard)in chloroformwas addedand the solution
dilutedto 25 ml. To 1 ml of this solutionin a vial was added0.1 ml of silylatingreagent
(preparedby mixingtrifluoroacetic acid(1 part) andhexamethyldisilazane (2 parts)and
filteringthe mixturerapidlyunderdry conditions),the vial was sealedand then heated
on a steam bath for 1 h. The mixture (1 •tl) was subjectedto g.l.c. in a glasscolumn
(152 cm x 3 mm) packedwith 10•o of siliconeJXR on Gas Chrom Q (80 to 100 mesh),
operatedat 125ø C. with nitrogen(40 ml min-x) as carrier gas and flame ionisation
detection.The ratio of the productof the peak heightand retentiontime for Bronopol
di(trimethylsilyl)ether(relative retention time= 1.92) to that for n-tridecane (relative
retentiontime= 1.00) was calculatedand comparedwith the ratio for a standardcon-
tainingpurifiedBronopolwhichhad beensimilarlytreated.

Thin Layer Chromatographic Assay. 10}/oBronopol solutionwas examinedby t.l.c.

and bioautography using0.25 mm Kieselgel'G', with chloroform/methanol (4: 1) as
developingsolvent. 2 [tl aliquotsof the solutionwere spottedon the plates.A similar
method for ointmentformulationshas beendevisedusing an initial water:chloroform
extraction systemto remove excipients,followed by chromatographyon Kieselgel
GF•.5•usingisopropanolas the developingsolvent.

Determinationof Bromideion.Bromideion was determinedby potentiometrictitration.

Bronopol solution(5 ml) was acidifiedand titrated with 0.02M silver nitrate solution.

Determinationof Formaldehyde. Formaldehydewasdeterminedby reactionwith chromo-

tropic acid. 0'2•o Bronopolsolution(0.5 ml) was dilutedto 25 ml with 12N sulphuric
acid. To this solution(1 ml) was addeda 5•o solutionof chromotropicacid in 12•q
sulphuricacid(1 ml) and the mixtureheatedat 100øCfor 30 min. Concentratedsulphuric
acid (2 ml) was addedand the absorbanceat 570 nm measuredagainstthe appropriate
blank.

Determinationof Nitrite and Nitrate. Nitrite and nitrate were determinedby reaction
with 2,6-xylenol before and after decompositionof nitrite with sulphamicacid. This
methodwas not usedafter the preliminarywork as the resultswere in good agreement
with the polarographicestimationof alkyl nitro-groups.

TOXICOLOGY

Metabolism

After oral administration of [•C]Bronopol,radioactivitywasrapidlyabsorbedandevenly

distributedin tissuesof the rat and dog, Moore et al. (19). Excretionwas alsorapid, the
majority of the dosebeingexcretedwithin 24 h.
Bronopolwas rapidly and extensivelymetabolisedso that no unchangedcompound
wasdetectedin plasmaand urine. It hasbeenshownin vitrothat Bronopolis unstablein
16 D. M. Bryceet al.

plasma.The major urinarymetabolite,accountingfor more than 40}/oof administered

radioactivity, was 2-nitropropane-l,3-diol. Other minor metaboliteshave not been
identified.Completemetabolismwasdemonstrated by the findingof significantamounts
of radioactivityin expiredair and the appearanceof a smallamountof radioactivityin
the tissues of dosed animals.
When appliedin acetonesolutionto rat skin, a smallerproportionof the dosewas
absorbedthan when dosedorally, Moore et al. (20). This may in part be due to the
smallarea of skin to which the dosewas applied.
When p4C]Bronopolwas applied in acetonesolutionto the skin of rabbits, the
radioactivitywasmainlylocalisedon the epidermisaroundhair follicles,suggesting
that
limited percutaneous absorptionmay occurthroughthe hair follicles.
The patternof urinarymetabolites wassimilarwhenthe compoundwasadministered
orally or percutaneously,
indicatingno differencein metabolismrelatedto the route of
administration.

Acute Toxicity
Bronopoladministeredin singledosesby the oral and intraperitonealroutesto rodents
causedgastrointestinallesionsand peritonitis.The LD50 valuesare shownin Table VII.
A small number of rats were injectedsubcutaneously with Bronopol and thosethat died
hadhaemorrhage andoedemaat the siteof injection,stomachlesionsandlungcongestion
and oedema.The LD50 wasapproximately200 mg/kg.After dermalapplicationto rats
of acetonesolutionsof Bronopolusingthe procedureof Noakes and Sanderson(21),
death occurredat 160 mg/kg or more.
Oral administrationof singledosesof 40 or 100mg/kg to dogscausedgastricirrita-
ion but no permanentinjury.
No methaemoglobinaemia was observedin cats over a 24 h period following a
maximum singleoral doseof 25 mg/kg of Bronopol,whereasa marked rise in blood
methaemoglobin concentration followed20 mg/kgof acetanilide.

ChronicToxicity
In repeated-dose studies
theobservations andlaboratoryinvestigations
generally
included
signsof poisoning,body-weight,food consumption, haematology,bloodbiochemistry,
ophthalmoscopy, organweights,macroscopic appearanceat autopsyandhistopathology,
Gastrointestinallesions,respiratorydistress
and somedeathsresultedfrom dailyadmini-
strationof 80 or 160mg/kg of Bronopolby oral intubationto male and femalerats for
90 dayswhereasdosesof 20 mg/kgwerewell tolerated.When Bronopolwasgivenin the
drinkingwater, rats maintainedon 160 mg/kg/dayfor six weekshad a reducedwater
intake and slightlyenlargedkidneyswhile amongthosegiventhe highestdoselevelof
300mg/kg/daya few deathsoccurred.In dogsgivena maximumdaily doseof 20 mg/kg
by oralintubationfor 90 days,apartfrom somevomiting,therewereno significant toxic
reactions.
Aqueous2'5•o methyl cellulosesolutionscontaining0.2 or 0'5•o Bronopolwere
appliedoncedailyat a dosageof 1 ml/kg for 3 weeksto the clippedand abradeddorsal
skin of rabbits.The vehiclealone and the 0.2• Bronopolsolutionelicitedlocal skin
erythemaand the 0.5•o Bronopolsolutionproducedmoderateerythema,oedemaand
scabbing,otherwisethe rabbitsshowedno ill-effectsclearlyattributableto treatment.
 Activity and safetyof Bronopol 17

Table VII. Acute toxicity of Bronopol to mice and rats

Species Sex Route LD50 mg/kg

Mouse male oral 374
female oral 327

Mouse male i.p. 34.7

female i.p. 32.8
Rat male oral 307
female oral 342

Rat male i.p. 22.0

female i.p. 30.2

i.p. =intraperitoneal.

Carcinogenicity
A carcinogenicitystudywas carried out in mice by applicationof 0.3 ml of aqueous-
acetonesolutionscontaining0.2 or 0'5•o Bronopol to the shavedbacks three times
weekly for 80 weeks.The concentrationswere selectedafter a preliminary tolerance
studyshowedthat 1• or moreevokeda local skinreaction.Bronopoldid not alter the
spontaneous turnourprofileeitherlocallyor systemically.
A 2-yeartoxicityand tumorogenicity test,in whichratsreceived10, 40 or 160mg/kg
daily in the drinkingwater, providedno evidenceto suggestthat the administrationof
Bronopolaffectedtumourincidence. Therewasno indicationof toxicityat 10mg/kg/day,
whereasthe higherdoselevelsadverselyaffectedgrowth,food intake and survivalrate.
Renal changesassociated with diminishedwater intake, histologicalreactionsin stomach
andgastriclymphnodesprobablydueto irritancyfromprolonged exposure to Bronopol,
and an exacerbation
of spontaneous
morphological alterationsin the salivaryglandwere
also observedat the higher doselevelsin a dose-relatedmanner.

ReproductionStudies
The effectof Bronopolon reproductionwasinvestigatedin rats and rabbits.In rats
dosedfrom day 1 to 20 of pregnancywith 10, 30 or 100 mg/kg daily by oral intubation,
no embryotoxicor teratogeniceffectswereseeneventhoughthe damshad a dose-related
retardationin bodyweightgain and somedied from gastricand lung lesions.A slight
delayin calcificationof the foetalskeletonwasobservedat the highestdoselevel.Daily
application to theclippeddorsalskinof ratsof 0.5 or 2•o aqueoussolutions of Bronopol
thickenedwith 2'5•o methylcellulose in a doseof 1 ml/kg from day 6 to 15 of pregnancy
had no adverseeffectson the damsor foetusesapart from causinglocal skinreactionsat
the site of application.
Oral administrationof 1, 3.3 or 10 mg/kgdaily to rabbitsfrom day 8 to 16 of preg-
nancyalsofailed to produceembryotoxicor teratogeniceffectsthoughthe highestdose
level suppressed weightgainby the doesduringthe dosingperiod.
Bronopol,20 or 40 mg/kgdaily, givenorally to rats from day 15 of gestationand
throughout lactation did not affect parturition, litter size or postnatal survival and
developmentof the young.Fertility and generalreproductiveperformanceof rats were
unimpairedby thesedoselevelsgivento malesfrom 63 daysbeforematingand females
from 14 daysbeforematingup to day 12 of pregnancyor until the litterswere weaned
18 D. M. Bryceet al.

21 daysœostœartum.In this studybodyweightgain of the malesthat received40 mg/kg

daily was slightlyreduced.

Mutagenicity
Bronopol did not exhibit mutagenicactivity under in vitro or in vivo conditions.It was
tested using Salmonellatyphimuriumin the 'Ames' systemand in the host-mediated
assayin mice; in a dominantlethal assayin mice, the only noteworthyfindingwas anti-
fertility arisingfrom toxicityrather than dominantlethality.

Irritancy and ContactSensitivity

Animal Studies.Preliminary studies on irritancy and contact sensitisationhave been
reportedby Croshawet al. (3).
Bronopolwas testedfor local effectsto the mucousmembraneof the eye in rabbits.
A concentrationof 0'5•o in normal salinewas non-irritantwhen appliedoncedaily for
four successive days,whereassolutionsin polyethyleneglycol400 were irritant at 5•o
but not at 2•o followinga singleapplication.
Skin irritancywas investigatedby applicationof Bronopolin a variety of solventsto
the non-abraded,clippedand shavedbacksof rabbitsfor 6 h, with or without occlusion.
Acetonesolutionsof Bronopolwere non-irritant at 1•o whengivenas a singleapplica-
tion under occlusionthough highly irritant at 0'5•o on repeatedapplicationwithout
occlusion.Similarresultswereobtainedwith Bronopolin 2'5•o aqueousmethylcellulose
solutiontestedundertheseconditionsat 0'5•o concentrations. Bronopolin polyethylene
glycol300wasnon-irritantat 5•o asa singleapplicationwith occlusion. Thesefindings
indicatethat theirritancyof Bronopolto the skinis dependent
uponthevehicleemployed,
thus it would be advisableto test each new formulation containingBronopol for local
effectson topical application.
Bronopol was without skin-sensitisingactivity in the guinea pig when tested as a
1•o solutionin acetoneby the ear-flankmethod(Stevens(22)), whereasdinitrochloro-
benzenewas stronglypositive.

Human Studies.The skin irritant effectof Bronopolwas investigatedboth on volunteers

and on patientsattendinga contactdermatitisclinic.
The volunteerstudyshowedthat Bronopolis slightlyirritant to human skin at 1•o in
soft paraffin (petrolatum),and at 0'25•o in aqueousbuffer at pH 5.5. The studycon-
sistedof a closedpatch test using 1 cm lint squaresbackedwith Blendermsurgicaltape
on the forearmsof ten subjects.Concentrationsof 0, 0.5, 1 and 2•o Bronopolin soft
paraffinand of 0, 0'05, 0.1 and 0'25•o in aqueousbufferat pH 5-5 were used.Any skin
reactionafter 24 h was gradedfrom 0 (= normal skin) to 5 (= marked erythemawith
vesiclesand induration).The resultsare shownin Table VIII. The studycarried out on
patientsattendinga contactdermatitisclinic showedthat Bronopolis a mild irritant
when applied in yellow soft paraffin (yellow petrolatum)at 0'25•o. No evidenceof
sensitisatlonwas seenin this studynor was there any suggestion of cross-sensitisation
with any other substance,includingformalin. The compoundwas applied as one of a
battery of closedpatch testsusedin that clinic to screenthe patientsfor a potential
allergen.The patcheswereappliedfor 48 h and examinedon the secondandfourthdays
after the application.Of the 149patientsstudied,threeshoweda slighterythemaon the
 Activity and safetyof Bronopol 19

Table VIII. The irritancy of Bronopol to human skin

Bronopol Positive
Base concentration• skin response Degree of reaction
Soft
Paraffin 0 0/10 --
0.5 0/10 --
1 2/10 both slight erythema
2 4/10 all moderateerythema

Aqueous 0 0/10 --
buffer 0.50 0/10 --
pH 5.5 0.1 0/10 --
0.25 1/10 slight erythema

secondday which had faded by the fourth day, and one a moderateerythemaon the
secondday; this patient did not return for the secondexamination.
Marzulli and Maibach (23, 24) have studiedthe contactsensitisation in man of a
number of commonlyused biocides; and have concludedthat, under the conditionsof
a closedpatchtest,Bronopolin yellowsoftparaffinwas a potentialsensitizer.The chal-
lengeconcentrationin thesestudieswas 2'5•o which accordingto theseauthorswas a
non-irritantconcentration.However,the studiesreportedaboveare not consistentwith
thisview.The patchtestscarriedout by Marzulli and Maibachshoweda dose-response
relationship,and sincethe responsedecreased very rapidly to zero at an inductioncon-
centration of 2•o which is considerablygreater than that used in formulations, the
authorsinferredthat Bronopolmay be safelyusedin cosmeticformulations.In a further
study,Maibach (25), has confirmedthat Bronopolwas a direct irritant to human skin
at concentrations greaterthan 15/ounder theseconditions.A subsequentsensitisation
testincluded93 normalsubjects who wereinducedwith 10 applicationsof 5•o Bronopol
in yellowsoftparaffinunderan occlusivedressingover a period of 3 weeks.After a rest
periodof 2 weeksthe subjects werechallenged at 0'25•o Bronopolin yellowsoftparaffin
at a different site. No evidence of contact sensitisation was observed.

FORMULATION STUDIES

Bronopolhasbeenin usefor morethan 10 yearsat a levelof 0.01-0.02• or more in

conventionally formulatedshampoosbasedonsodiumlaurylethersulphates andalkanol-
amine alkyl sulphateswith 2• or more of a foam-boostingalkanolamide.When a
freshlypreparedformulationis challengedwith 1 x 10ø pseudomonads per ml the bac-
terial count is reducedto <10 per ml within 24 h. The inclusionof protein-derived
materials,e.g.0.1-0.$• CroteinC (hydrolysedcollagen,Croda ChemicalLtd) doesnot
affect this result.
The useof Bronopolin the preservationof shampooshas beendescribedby Bryce
and Smart(14), Schuster(26) and in proteinshampoosby Tuttle, Pharesand Chiostri
(27). Barnesand Denton (28) found Bronopolat 0.02• to be one of the most satis-
factorypreservatives againstGram-negativebacteriain a cream,suspension and solution
in their capacitytest.
Combinationsof preservativescan be justified on severalgrounds, one of these
being to increasethe spectrumof antimicrobialactivity. It is establishedthat the anti-
bacterialactivityof Bronopolis greaterthan its antifungalactivityand its spectrumcan
20 D.M. Bryce et al.

be increasedby the additionof parabens,Parker(29). Proserpio(30) and Jacobs,Henry

and Cotty (31) haveconsidered the combinationof Bronopolwith other agentsin cos-
metics and oil-water emulsions.

Medicated Skin Cream

Bronopolin an anhydrousbase or in an aqueousformulation of low pH may have

applicationsasan activeantibacterialagentin skincareproducts.Experimentalmedicated
skin creamscontainingBronopoland HexachlorophaneBP showedactivity on the skin
against Escherichiacoli whereasa cream without Bronopol did not. Although it is
theoretically possiblethat synergismbetween these compoundsis occurring, other
experiencesuggeststhat this is unlikely. It is concludedthereforethat Bronopol is
exhibitingantibacterialactivityper se in theseformulations.
The compositionof the creamwas as follows:
% w/w
HexachlorophaneBP 0.5
Bronopol 0.1 or 0.2
Sorbitol syrup 13.5
Arlacel 186 (ICI United StatesInc.) 1.5
Cosmolloidwax 70H (Astor Petrochemicals)* 7.5
Light mineral oil 20
Aqueouscitrate bufferpH 4.5 to 100
* The 70H grade is no longer available. Cosmolloid
wax 70 gradehas almostidenticalproperties.

The forearmsof eight subjectswere washed,dried and swabbedwith alcoholto remove

the transient skin flora. The creamswere applied in 0.05-g amountsto areas of 5 x 2.5
cm, and 0.025 ml of a 1 in 10 dilution in broth of an overnightculture of Esch. coli
NCTC 5934 was appliedon to eachcream. After 30 rain contactbetweencream and
organismeacharea was swabbedwith alginateswabs.The swabsweredissolvedin 10 ml
of quarterstrengthRingersolutioncontaining1•o of sodiumhexametaphosphate and
1•o of polysorbate80. Aliquots (1 ml) of the swab diluentswere plated in 'Oxoid'
MacConkeyagar No. 3 and plate countswere made after incubationfor 48 h at 37øC.
Resultsof a typical experimentare shownin TableIX.

Table IX. Antibacterial activity of medicated cream formulations on human skin. Viable
organisms(per ml) from subjects(A-H) after contacttime of 30 min

Subjects
Cream A B C D E F G H

Base (no active agen0 + + + + + + + +

Base+ 0'5•o HexachlorophaneBP + + + + + + + +
Base+ 0'5•o HexachlorophaneBP+
0.2•o Bronopol 7 2 1 1 2 0 3 9
Base+ 0'5•o HexachlorophaneBP+
0-1•o Bronopol 11 + + 6 20 20 0 29

+ = uncountable numbers.
 Activity and safetyof Bronopol 21

AlcoholicDeodorantSpray
An experimentalalcoholic deodorant spray formulation containing 0.12• w/w
Bronopolwasfound to be stableafter one-year'sstorageat room temperatureand 37ø
in a lacqueredtinplatecan, and showeda broad spectrumof antibacterialactivity.
Compositionof the spraywas as follows:
•o w/v
Diethyl PhthalateBPC 0.330 (v/v)
Propyleneglycol 0.167
DenaturedethanolB grade 750P 29.320
Perfume 0.083
Bronopol 0.100
Propellant11/12 (1: 1)to 100

This formulationwaspackedinto internallylacqueredtinplatecanssolderedwith solder

2/98 and fitted with a standardvalve.The can fill was 128g. Sampleswerethen placed
on storagetestat room temperatureand 37øC,and examinedat regularintervalsover a
period of one year.
Chemicalassayswerecarriedout usingan adaptationof the t.l.c. systemdescribed
earlier.The resultsshowedthat no decompositionof the Bronopolhad occurred.No
corrosion of the container was observed.
Microbiologicalassessments
were carriedout by sprayingthe formulationon to a
13 mm WhatmanA.A. discand allowingto dry. The discswerethen placedon to agar
seeded with various test bacteria and Candida albicans. The zones of inhibition were
recordedafter 18 h at 37øC.The resultsobtainedafter one-year'sstorageare shownin
Table X.
TableX. Antibacterialactivityof Bronopolin an alcoholicsprayformulation
after one year storageat room temperatueand 37øC
Diameter of zone of inhibition
(+ 13 mm disc) in mm

Sample1 Sample2
Test organism (containingBronopol (containing
initially at 0.12•o w/w) no Bronopol)
Temperatureof storage Room temperature 37ø 37ø
$taph. aureus8452 29 30 13
$taph. aureusFDA 35 35 13
Staph. albusNCTC 7944 28 29 13
Esch. coli NCTC 5934 26 26 13
ProteusvulgarisNCTC 4635 26 26 13
Ps. aeruginosa10S 28 28 13
Candida albicans 239 13 13 13

PersonalHygieneor Foot Spray

An experimentalhygienespray formulation containingBronopol was found to be
stablein a lacqueredaluminiumcan and showedmore antibacterialactivitythan a
similarformulationcontainingchlorhexidine,
evenafter 3 monthsstorageat elevated
temperature.
22 D.M. Bryce et al.

The followingformulationwaschosento representa typicalhygienespray:

% w/w
Talc 399 (Whittaker,Clark and DanielsInc.) 0.500
Bronopol 0.075
Perfume 0.100
Propellantl l (I.C.I. Ltd) 39.325
Propellant 12 (I.C.I. Ltd) to 100

CrystallineBronopolwaspassedthrougha micro-millto obtainthe necessary reduction

in particlesize,the material subsequently
passedthrougha 250 meshscreensieve.The
iron contentof the Bronopolwas 7 ppm and no evidenceof discolourationwasevident
after 5 months storageat room temperature.Particle size analysisof the talc showed
98.7% lessthan 15 I•m.
The aboveformulationwaspackedinto an internallylacqueredaluminiummonobloc
can fittedwith a standardvalve.The can fill was64 g. Sampleswereplacedon storage
testat room temperatureand 37øC,and wereexaminedat regularintervalsovera period
of 3 months.
Chemicalassayswere carriedout usingan adaptationof the t.l.c. systemdescribed
earlier.The resultsshowedthat no decomposition
of Bronopolhad occurred.No corro-
sion of the container was observed.
Microbiologicalassayswerecarriedout usingthe methoddescribedfor the alcoholic
deodorantsprayformulation.The resultsobtainedafterthreemonthsstorageare shown
in Table XI.

Table XI. Antibacterial activity of Bronopol and chlorhexidinein a hygienespray formulation after
3 monthsstorageat room temperatureand 37øC

Test organism Diameter of zone inhibition (+ 13 mm disc)in mm

Sample 1 Sample 2 Sample 3

(containingBronopol (containing chlorhexidine (containing
initially at initially at neitherbiocide)
0.075% w/w) 0.075% w/w) Room
Temperature of storage Room temperature 37ø Room temperature 37ø temperature 37ø
$taph. aureus8452 30 25 14 15 15 14
$taph. aureusFDA 32 22 15 15 13 13
$taph. albusNCTC 7944 31 24 14 14 13 13
Esch. coli NCTC 5934 21 21 13 13 13 13
Proteus vulgarisNCTC 4635 23 21 13 13 13 13
Ps. aeruginosa1OS 26 22 13 13 13 13
Candida albicans 239 13 13 16 16 13 13

Further microbiologicaltestswere carried out with sampleswhich had been storedat

room temperatureand 37øCfor 6 months.Althoughthesetestswere only qualitative,a
similarpatternof activityto that observedafter 3 monthswasobtained.

CONCLUSIONS

Bronopolhasbeenshownto possess a wide spectrumof antibacterialactivity.Its activity

againstGram-negativeorganisms,particularlyPs. aeruginosa, is greaterthan that of
most other antibacterialand preservativeagents.
 Activity and safetyof Bronopol 23

Bronopolis moststableunderacid conditions,althoughit demonstrates high anti-

bacterialactivityover a wide pH range.The modeof decomposition hasbeenstudiedin
detail, and a number of the decompositionproductsidentified.The assaymethods
describedare capableof estimatingBronopolin many formulationsat its normallyused
levels,the sensitivityof the methodsis dependentupon the natureof the formulations;
in certaincaseslevelsas low as 5 ppm can be assayed.
Bronopolis generallyusedas a preservative in formulationsat levelsbetween0.01
and0.1•o. Animal toxicitystudiesand humanpatchtestshavedemonstrated the safety
of Bronopolwhen usedat theseconcentrations.In particular, no evidenceof human
skin sensitisation has been obtained at these levels.
Bronopolhasbeenshownto be an effectiveantibacterialagentin a rangeof formula-
tions includingshampoos,skin creamsand spraysand bath products.Many of the
ingredientsusedin suchproductshave beenshownto have little or no effecton the
antibacterialactivity of Bronopol,althoughcompoundscontainingsulphydrylgroups
are antagonisticto its activity.

ACKNOWLEDGMENTS

The authors wish to acknowledgethe assistanceof the following staff of The Boots
CompanyLimitedin the productionof the data included:Dr D. F. Spooner(micro-
biology);Mr E. L. Crampton(chemistry);Mr D. A. Elvidge,the late Mr C. Vickers,and
Mr J. S Wragg (analysis);Dr P. C. Risdalland Miss M. M. Sutton(toxicology);and
Mr K. G. Jacksonand Dr D. P. Stokes(formulation).
In addition, the authorswish to thank Dr C. D. Calnan, Institute of Dermatology,
University of London; and ProfessorH. I. Maibach, Department of Dermatology,
Universityof Californiafor their help in the completionof the humanskin studies.

REFERENCES

1 Hodge, E. B., Dawkins, J. R. and Kropp, E. A new seriesof antifungalcompounds.J. Am. Pharrn.
Ass., Sci. Ed. 43 501 (1954).
2 Zsolnai,T, Versuchezur EntdecktmgneuerFungistatika.II. Nitro-verbindungen.Blochem.Pharmac.
s 287 (1961).
3 Croshaw, Betty, Groves, M. J. and Lessel,B. Some propertiesof Bronopol, a new antimicrobial
agentactiveagainstPseudomonas aeruginosa.
J. Pharrn.Pharmac.16 Suppl., 127T (1964).
4 Clark, N. G., Croshaw,Betty, Legetter, B. E. and Spooner,D. F. Synthesisand antimicrobial
activity of aliphaticnitro compounds.J. Med. Chem.17 977 (1974).
5 Morse, L. J. and Schonbeck,L. E. Hand lotions,a potential nosocomialhazard. New. Engl. J. Med.
278 376 (1968).
6 SykesG. and Smart, R. Preservationof preparationsfor applicationto the skin. Am. Perfurn.
Cosmet.84 45 (1969).
7 Smart, R. and Spooner,D. F. Microbiologicalspoilagein pharmaceuticals
and cosmetics.J. $oc.
Cosmet. Chem. 23 721 (1972).
8 Malcolm, S. A. and Woodroffe,R. C. S. The relationshipbetweenwater-bornebacteriaand shampoo
spoilage.J. Soc. Cosmet.Chem.26 277 (1975).
9 Tenenbaum,S. The significanceof pseudomonadsin cosmeticproducts.Am. Perfum. Cosmet.86
47 (1971).
10 Thomas, M. J. and Majors, P. A. Animal and human microbiologicalsafetytestingof cosmetic
products.J. Soc. CosmetChem.24 135 (1973).
11 Marples,R. R. and Kligman, A.M. Methodsfor evaluatingtopicalantibacterialagentson human
skin. Antimicrob.AgentsChernother.$ 323 (1974).
24 D.M. Bryce et al.

12 Onoda,T. and Saito,H. Influenceof a newantibacterialagent,Bronopol,uponthe growthof cultured

cells. Chemotherapy(Tokyo) 22 196 (1974).
13 Hale, L. J. and Inkley, G. W. A semiautomaticdevicefor multiple inoculationof agar plates.
Lab. Pract. 14 452 (1965).
14 Bryce,D. M. and Smart, R. The preservationof shampoos.J. $oc. Cosmet.Chem.16 187 (1965).
15 Brown, M. R. W. Turbidimetricmethodfor the rapid evaluationof antirnicrobialagents- Inactiva-
vation of preservativesby non-ionicagents.J. $oc. Cosmet.Chem. 17 185 (1966).
16 Stretton, R. H. and Manson, T. W. Some aspectsof the mode of action of the antibacterialcom-
pound Bronopol (2-bromo-2-nitropropane-l,3-diol).J. appl. Bact. 36 61 (1973).
17 King, M. B., Knox, R. and Woodroffe, R. C. S. Investigationof antituberculoussubstances-an
agar diffusionmethod usingMycobacteriumsmegmatis.Lancet i 573 (1953).
18 Kabay, A. Rapid quantitativemicrobiologicalassayof antibioticsand chemicalpreservatives of a
non-antibiotic nature. AppL Microbiol. 22 752 (1971).
19 Moore, D. H., Chasseaud,L. F., Lewis, J. D., Risdall, P. C. and Cramp(on, E. L. The metabolism
of the antibacterialagentBronopol (2-bromo-2-nitropropane-l,3-diol)givenorally to rats and dogs.
Fd. Cosmet.Toxicol. 14 183 (1976).
20 Moore, D. H., Chasseaud,L. F., Bucke, D. and Risdall, P. C. The percutaneousabsorptionand
dispositionof the antibacterial agent Bronopol in rats and rabbits. Fd. Cosmet. ToxicoL 14 189
(1976).
21 Noakes, D. N. and Sanderson,D. M. A method for determiningthe derreal toxicity of pesticides.
Brit. J. Indust.Med. 26 59 (1969).
22 Stevens,M. A. Use of the albino guinea-pigto detect the skin sensitizingability of chemicals.
Brit. J. Ind. Med. 24 189 (1967).
23 Marzulli, F. N. and Maibach, H. I. Antimicrobials: experimental contact sensitizationin man.
J. $oc. Cosmet. Chem. 24 399 (1973).
24 Marzulli, F. N. and Maibach, H. I. The useof gradedconcentrationsin studyingskin sensitizers:
experimentalcontactsensitizationin man. Fd. Cosmet.ToMcol.12 219 (1974).
25 Maibach, H. I. Dermal sensitizationpotentialof 2-bromo-2-nitropropane-l,3-diol (Bronopol).Con-
tact Dermatitis 3 99 (1977).
26 Schuster,
G. Die Conserviemng
von Shampoos
und Schaumbademitteln.
$eifen-Ole-Fette-Wachse
99 489 (1973).
27 Tuttle, E., Phares,C. and Chiostri, R. F. Preservationof protein solutionswith 2-bromo-2-nitro-
1,3-propanediol(Bronopol).Am. Perfum. Cosmet.85 87 (1970).
28 Barnes,M. and Denton, G. W. Capacitytestsfor the evaluationof preservatives in formulations.
Soap, Perrum. Costa.42 729 (1969).
29 Parker, M. S. Someaspectsof the useof preservatives in combination.Soap,Perfum.Costa.46 223
(1973).
30 Proserpio,G. Protectiondescosm6tiques par desm61anges synergiques de pr6servateurs
•i dosage
microbiocide.Parrum. Cosmet.$avons2 305 (1972).
31 Jacobs,G., Henry, S. M. and Cotty, V. F. The influenceof pH, emulsifier,and acceleratedageing
upon preservative requirementsof o/w emulsions.J. Soc. Cosmet.Chem.26 105 (1975).