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ProClin
®
300 Preservative
Features & Benefits
KVS04770-5031200049
ProClin
®
Preservatives
Mechanisms and Stabilityfor Diagnostics Reagents
Table 13. Sensitization Tests
TestConcentration
 
Effect
Skin Sensitization
Guinea Pigs(Buehler Method)SensitizerEC
50
induction90ppm a.i.Guinea Pigs(Magnusson-Kligman Method)56ppm a.i. inEC
50
elicitation429ppm a.i.No sensitizationaqueoussolution
Photosensitiza-tion/Phototoxicity
Guinea Pigs1, 3, and 10%Not phototoxic;irritant at thehigherconcentration0.3% a.i. insultdosage
Nophoto-sensitization
0.3% and 1% a.i.challenges
Safe Handling Information
ProClin 300 preservative presents notoxicological problems or health hazardsat recommended use levels. However, thefollowing precautions must be observedwhen handling the neat product.Danger As supplied, ProClin 300 preservative iscorrosive, and can cause irreversible eyedamage and skin burns. These effects maynot manifest themselves for several hoursafter contact. ProClin 300 preservativecan also cause allergic skin reactions insusceptible individuals. It may be harmful if swallowed or absorbed through the skin.Personal Protection
Do not get in eyes, on skin, or on clothing.
Plant personnel handling ProClin 300preservative should wear splash gogglesor face shield, rubber gloves, and animpervious apron and boots.Laboratory personnel should wear a labcoat, safety glasses, and rubber gloves. Avoid breathing vapor or mist, andwash hands thoroughly after use toprevent contamination of food and drink.Special care should be taken to avoidcontamination of surfaces that may laterbe handled by unprotected personnel.First Aid MeasuresEye Contact –FLUSH IMMEDIATELY withwater for at least 15 minutes. Get promptmedical attention, but FLUSH FIRST!Skin Contact –WASH IMMEDIATELY  AND THOROUGHLY with soap and water.Consult a physician if irritation develops.If Inhaled –Move victim immediately to
fresh air. If not breathing, initiate articialrespiration. If breathing is difcult, give
oxygen. Call a physician.If Swallowed –Dilute by giving water todrink. Never give an unconscious personanything to drink.Note to Physician –Corrosive material.Probable mucosal damage maycontraindicate the use of gastric lavage.Measures against circulatory shock,respiratory depression, and convulsionsmay be needed.Neutralizing SolutionProClin 300 preservative is quickly
neutralized by solutions of sodium bisulte
at acidic pH. To prepare a neutralizing
solution, dissolve 1lb. of sodium bisulte
in 1 gallon of water (120g/L). The solutionshould have a pH of 4.0-5.0 for maximumeffectiveness. If a neutralizing solutionis permanently maintained, it shouldbe replaced weekly. Keep containers of neutralizing solution covered to reducethe release of sulfur dioxide and maintainsolution activity.Tests indicate that ProClin 300 preservativeis completely deactivated within 30 minuteswhen treated with at least 2 volume equiva-lents of neutralizing solution.Note:Neutralizing solution should notbe used to treat ProClin 300 preservativespilled on skin.
SAFC Supply Solutions
3050 Spruce St., Saint Louis, MO 63103800-244-1173 (US Toll Free)+1-314-534-4900 (International)Global Email:safcglobal@sial.com
SAFC
®
, SAFC Supply Solutions
®
and Sigma-Aldrich
®
are registered trademarksof Sigma-Aldrich Biotechnology L.P. and Sigma-Aldrich Co.ProClin
®
is a registered trademark of Rohm and Haas Company.© 2009 SAFC All rights reserved.
www.safcsupplysolutions.com/proclin
 
www.safcsupplysolutions.com/proclin
ProClin
®
300 Preservative
Features & Benefits
for Diagnostics Reagents
 
ProClin
®
300 Preservative
Features & Benefits
23
for Diagnostics Reagents
ProClin 300 preservative is a highly effective biocide offered for the control of microorganisms in reagents and products intended for
 in vitro
diagnostic use. With broad-spectrum activity, excellent compatibility and stability, and low toxicity at use levels, ProClin300 is the ideal choice for an effective preservative in diagnostic reagents. At unusually lowconcentrations, ProClin 300 preservative eradicates bacteria, fungi, and yeasts in reagentsfor prolonged periods, thereby increasing a product’s shelf life. The water solubility permitseasy incorporation into reagents. Further, ProClin 300 preservative does not affect thefunctionality of most enzyme- or antibody-linked reactions, and will not interfere with assayindicators. The information presented in this bulletin is designed to help you predict andevaluate the performance of ProClin 300 preservative in your products.
Features and Benets•
Broad-spectrum antimicrobial activity
•
Compatible with key enzymes
•
Does not inhibit antibody binding
•
No interference with ISE electrodes
•
Excellent stability
•Effective at pH ≤ 3.5•
No interfering inerts
•
No color imparted to reagents
•
No health hazards at recommendeduse levels
•
Safe disposal at recommendeduse levels
Chemical Identication
Structural FormulasThe active ingredients of ProClin 300preservative are two isothiazolones
identied by the IUPAC system of 
nomenclature as:5-Chloro-2-methyl-4-isothiazolin-3-one and
2-Methyl-4-isothiazolin-3-one (see Figure A).
Chemical PropertiesProClin 300 preservative is a preciseformulation of active ingredients and an
organic stabilizer in a modied glycol
(see Table 1).Table 1. Chemical Composition of ProClin 300 Preservative
Acive Ingredients
5-Chloro-2-methyl-4-isothiazolin-3-one2.30%2-Methyl-4-isothiazolin-3-one0.70%
Inerts
Modifed glycol
93-95% Alkyl carboxylate2-3%
Table 2. Typical Physical Properties
(do not constitute specications)
PropertyDescription
 AppearanceClear LiquidColorColorless to Pale YellowOdorMild (sweet)Density (lbs/gal)8.59
Specifc gravity (22 °C)
1.03pH (10% aqueous solution)4.1
Viscosity 25 °C
58cPsFlash Point (Seta Closed Cup)
224 °F (118 °C)
 Absorbance Maximum275nmFluorescenceNoneEvaporation Rate (Bu acetate = 1)<1
Table 3. Typical Metals Analysis
(do not constitute specications)
MetalVolume (mg/L)
 Arsenic<1.0Selenium<1.0Barium<1.0Chromium<1.0Lead<1.0Mercury<0.01 Antimony<1.0Copper<1.0Calcium1.7Iron1.0Magnesium<1.0Manganese<1.0Nickel<1.0Tin<2.0Nitrate<5.0Nitrite<5.0Insoluble ResidueND (<5.0)
ND = Not Determined
Solubility
ProClin 300 preservative is 100% solubleand completely miscible in water. It is alsosoluble in a wide range of oxygenatedsolvents.
Stability
ProClin 300 preservative is formulatedfor long product shelf life. Stability testsindicate that after two years storage at
25 °C, there is no detectable loss of active
ingredients. Additional stability data inTable 4 show that the material is stable forseveral weeks at elevated temperatures(prolonged storage at high temperatures isnot recommended).Table 4. Stability of ProClin 300Preservative (as formulated)
Temperature (°C)Weeks% Active Remaining
2536>984036995581005512100
Stability in ReagentsThe active ingredients in ProClin 300preservative have an established history of successful use as preservatives. However,there are some circumstances under which
we would advise potential users to conrm
biocide stability prior to use:Temperature: As a general rule, a risein temperature accelerates the rate of degradation of chemicals. Temperatures in
excess of 55 °C should be avoided during
manufacturing once the biocide hasbeen incorporated into the reagent. Amines:The presence of amines,particularly secondary amines, has adeleterious effect on the stability of ProClin300 preservative. This can be avoided byreducing the pH of a regent containingamines below 7, which converts amines totheir acid salts.Reducing Agents:Some reducing agentsare detrimental to isothiazolone stability.We have found that use levels of ProClin300 preservative are stable in the presence
of up to about 50ppm bisulte (expressed
as SO2).pH:ProClin 300 preservative is stable andeffective over a wide range of pH. At pHlevels below 9, ProClin 300 preservativeis generally stable over the lifetime of thereagent. The stability of the preservative isreduced when pH exceeds 9.Serum:The presence of strongnucleophiles such as glutathioneand cysteine in blood and serum canhave a deleterious effect on ProClin300 preservative, but this reaction isdependent on concentration, pH, andother components of the reagent. Werecommend that the manufacturer test theperformance of ProClin 300 preservative in
a specic product before its use.
Stability in Biological BuffersBiological buffers (e.g. TRIS, TES, andHEPES) may contain amines that areaggressive to ProClin 300 above pH7. Thedegradation of ProClin 300 preservativein these buffers can be minimized byreducing their pH to 7 or lower, whichprotonates free amines and converts themto a less aggressive acid salt. Short-termstability data are summarized in Figure B1.
SNCOCH
3
COClSCH
3
N
5-Chloro-2-methyl-4-isothiazolin-3-oneCAS number 26172-55-4Empirical formula C
4
H
4
ClNOS2-Methyl-4-isothiazolin-3-oneCAS number 2682-20-4Empirical formula C
4
H
5
NOS
Figure A.Active Ingredients of ProClin 300 Preservative
Table of Contents
Page No.
Chemical Identication
2Structural Formulas2Chemical Properties2
Physical Properties
2
Solubility
3
Stability
3In Reagents 3In Biological Buffers3In Enzyme Conjugates 4Lyophilization 4
 
Testing
 
4
Degradation Products
4
Biocidal Efcacy
4In Enzyme Conjugates 5
Directions for Use
5
Compatibility with Enzymes
5Short-Term Effects 6Long-Term Effects 6
Effect on Antibody Binding
6
Toxicology Properties
7
Safe Handling Information
8Personal Protection 8First Aid Measures 8Neutralizing Solution 8
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ProClin
®
300 Preservative
Features & Benefits
45
for Diagnostics Reagents
Organism (ATCC# Number)ActiveIngredient(ppm)Gram-Negative Bacteria

*
Bacillus cereus mycoides (R&H L5*)2Bacillus subtilis (R&H B2*) 2Brevibacterium ammoniagenes (6871) 2Cellulomonas sp. (21399) 6Sarcina lutea (9341) 5Staphylococcus aureus (6538) 2Staphylococcus epidermidis (155)2Streptococcus pyrogenes (624) 9Streptomyces albus (3004) 1
Fungi – Yeasts *
 Aspergillus foetidus (16878)9 Aspergillus niger (9642) 9 Aspergillus oryzae (10196) 5 Aureobasidium pullulans (9348) 5Candida albicans (yeast; 11651)5Chaetomium globosum (6205)9Cladosporium resinae (11274) 5
Gliocladium fmbriatum (QM7638)
9Lentinus lepideus (12653) 4Lenzites trabea (11539)6Mucor rouxii (R&H L5-83*)5Penicillium funiculosum (9644)5Penicillium variabile glaucum (USDA**)2Phoma herbarum pigmentivora (12569)2Rhizopus stolonifer (10404) 5Rhototorula rubra (yeast; 9449) 2Saccharomyces cerevisiae (yeast; 2601)2Trichophyton mentagrophytesinterdigitale (95332)5
Bacteriostatic and fungistatic tests performed by serially diluting test compounds in trypticase soy broth and 1:100Inoculation with 24-hour broth cultures of test bacteriumor a fungal spore suspension prepared from 7-to-14 day culture slants washed with 7 mL of deionized water.Minimum inhibitory concentration levels determined visually after 2-4 days’ incubation at 30-37 °C for bacteria and 7 days’ incubation at 28 °C to 30 °C for fungi.
Efcacy in Enzyme Conjugates
The antimicrobial activity of ProClin 300 inHRP is summarized in Table 6. Preservedand control solutions were challenged at 0,2, 4, and 6 weeks with an inoculum consist-ing of 107 CFU/mL Ps. aeruginosa ATCCstrain 15442. A concentration of only 15ppmProClin 300 preservative (0.05% as supplied)completely eradicated this Pseudomonassp. in both 1% and 10% HRP solutions afterevery challenge. This antimicrobial activitywas superior to that sodium azide.
Table 6. Efcacy of ProClin 300
Preservative in Enzyme Conjugate
StorageTime (Weeks)10% HRPControlProClin 300(15ppm)SodiumAzide (0.1%)
0 9.5 x 10
6
1.1 x 10
7
1.8 x 10
7
2>1.0 x 10
9
<10<1048.9 x 10
7
<10<1064.4 x 10
8
<10 <10
1% HRP
01.2 x 10
6
3.5 x 10
6
2.3 x 10
6
2 7.2 x 10
8
<10 1.9 x 10
3
4 4.4 x 10
8
<10 <106 4.4 x 10
8
<10 <10
*
Colony-forming units.
Directions for Use
Long-term microbial protection in reagentsis accomplished with levels of ProClin 300preservative between 6 and 20ppm activemicrobicide. Typical use levels fall in therange of 9-15ppm active (0.03-0.05% assupplied). It is usually advisable to treat areagent with a higher level than the mini-mum effective dose to extend its shelf life.Some reagents are sold in concentratedform, and require a limited shelf life afteropening and dilution by the user. Reagentssuch as these can incorporate higher levelsof ProClin 300 preservative, but speciallabeling may be required to inform theuser of sensitization effects. For guidelinesunder these circumstances, contact SAFCTechnical Service at 800-244-1173.Since the components of diagnosticreagents vary considerably, and may
impact on the stability and efcacy of 
preservatives, we urge each manufacturer
to conrm the efcacy of ProClin 300
preservative in use.
Compatibility with Enzymes
The effect of ProClin 300 preservative onboth the short-term and long-term activityof HRP and AP was determined usingstandard assays.Data from laboratory studies (Table 7)indicate that ProClin 300 preservativedoes not affect the functionality of mostkey enzymes, used for diagnostic testing,including HRP and AP (Figure C).Stability in Enzyme ConjugatesProClin 300 preservative is also unaffectedby the presence of enzymes. Therewas virtually no loss of active biocide inhorseradish peroxidase (HRP) or alkalinephosphatase (AP) treated with 250ppmProClin 300 after six months’ storage at
temperatures ranging from -4 °C to 18 °C
(see Figure B2).Lyophilization StabilityThe use of ProClin 300 preservative inlyophilized products is not recommended.Data from several trials indicate that theactive ingredients in ProClin 300 are volatil-ized during the lyophilization process, andare not available for preservation once theproduct is reconstituted.Stability TestingThe level of active ingredients in ProClin300 preservative, as supplied and informulated products, can be determined byHPLC analysis. See Determination of ppm
Levels yer for analytical methods. Working
standards can be obtained from SAFC.
Degradation Products
ProClin 300 may degrade in the presenceof high pH, high temperature, reducingagents, and aggressive nucleophiles.The degradative pathway for each of these mechanisms is similar. Degradationproducts include small organic acids(acetic, formic), carbon dioxide, chlorideion, and elemental sulfur. None of these
degradation products have a signicant
impact on the environment.
Biocidal Efcacy
ProClin 300 preservative is an outstand-ing antimicrobial agent with unique cidalmechanisms. Within minutes after contactwith microorganisms, ProClin 300 inhibitsgrowth and respiration. Intracellular energylevels decline rapidly and macromolecu-lar synthesis is inhibited. Accumulation of damage to cellular constituents, possible
induced both by direct covalent modica
-tion of protein and via radical-mediatedevents, results in the death of the cell. FlyerMechanisms and Stability describes themechanisms of ProClin 300 preservativebiocidal action in greater detail.Table 5 lists the minimum levels of activeingredient that inhibit the growth of variousmicroorganisms. These results weregenerated under standard laboratoryconditions in nutrient-rich growth mediaand are intended to demonstrate thebroad-spectrum activity of ProClin 300preservative. Actual use concentration willvary according to application.
Table 5. Minimum InhibitoryConcentration of ProClin 300 Preservative
Organism (ATCC# Number)ActiveIngredient(ppm)Gram-Negative Bacteria
■
 Achromobacter parvulus (4335)2 Alcaligenes faecalis (8750) 2 Azotobacter vinelandii (12837) 5Enterobacter aerogenes (3906) 5Escherichia coli (11229) 8Flavobacterium suaveolens (958) 9Nitrobacter agilis (14123) 0.1Proteus vulgaris (8427) 5Pseudomonas aeruginosa (15442) 5Pseudomonas cepacia (Gibraltar 165) 0.75
Pseudomonas uorescens (13525)
2Pseudomonas oleoverans (8062) 5Salmonella typhosa (6539) 5Shigella sonnei (9292)8
   %   a .   i   /  r  e  m  a   i  n   i  n  g
Di H
2
OTrisTes HepesBuffers
020202020100
Figure B1. Stability of ProClin 300Preservative in the Presence of 50mMPhysiological Buffers After Three Weeks
 
   %   a .   i .  r  e  m  a   i  n   i  n  g
Temperature-4 °C
010050150
+4 °C18 °C
Figure B2. Stability of ProClin 300Preservative in the Presence of HRP andAP After Six Months
�
ProClin 300
�
ProClin 300 + HRP
�
ProClin 300 + AP
 
-
Pseudomonas aeruginosa
(CFUs /mL)
*
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