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ProClin 300 Preservative ProClin 300 Preservative

®
®

Features & Benefits


Features & Benefits
Table 13. Sensitization Tests First Aid Measures
Eye Contact – FLUSH IMMEDIATELY with
Test Concentration Effect
water for at least 15 minutes. Get prompt for Diagnostics Reagents
Skin Sensitization Sensitizer medical attention, but FLUSH FIRST!
Guinea Pigs EC50 induction
(Buehler Method) 90ppm a.i. Skin Contact – WASH IMMEDIATELY
AND THOROUGHLY with soap and water.
EC50 elicitation
Consult a physician if irritation develops.

ProClin Preservatives
Guinea Pigs 429ppm a.i. ®
(Magnusson- 56ppm a.i. in No sensitization If Inhaled – Move victim immediately to
Kligman Method) aqueous
fresh air. If not breathing, initiate artificial
solution
respiration. If breathing is difficult, give Mechanisms and Stability
Not phototoxic;
Photosensitiza-
irritant at the
oxygen. Call a physician.
tion/Phototoxicity
Guinea Pigs
1, 3, and 10%
higher
If Swallowed – Dilute by giving water to for Diagnostics Reagents
concentration
drink. Never give an unconscious person
No anything to drink.
0.3% a.i. insult
photo-
dosage
sensitization Note to Physician – Corrosive material.
0.3% and 1% a.i. Probable mucosal damage may
challenges contraindicate the use of gastric lavage.
Measures against circulatory shock,
Safe Handling Information respiratory depression, and convulsions
ProClin 300 preservative presents no may be needed.
toxicological problems or health hazards
Neutralizing Solution
at recommended use levels. However, the
following precautions must be observed ProClin 300 preservative is quickly
when handling the neat product. neutralized by solutions of sodium bisulfite
at acidic pH. To prepare a neutralizing
Danger solution, dissolve 1lb. of sodium bisulfite
As supplied, ProClin 300 preservative is in 1 gallon of water (120g/L). The solution
corrosive, and can cause irreversible eye should have a pH of 4.0-5.0 for maximum
damage and skin burns. These effects may effectiveness. If a neutralizing solution
not manifest themselves for several hours is permanently maintained, it should
after contact. ProClin 300 preservative be replaced weekly. Keep containers of
can also cause allergic skin reactions in neutralizing solution covered to reduce
susceptible individuals. It may be harmful if the release of sulfur dioxide and maintain
swallowed or absorbed through the skin. solution activity.

Personal Protection Tests indicate that ProClin 300 preservative


is completely deactivated within 30 minutes
Do not get in eyes, on skin, or on clothing.
when treated with at least 2 volume equiva-
Plant personnel handling ProClin 300 lents of neutralizing solution.
preservative should wear splash goggles
Note: Neutralizing solution should not
or face shield, rubber gloves, and an
be used to treat ProClin 300 preservative
impervious apron and boots.
spilled on skin.
Laboratory personnel should wear a lab
coat, safety glasses, and rubber gloves.

Avoid breathing vapor or mist, and


wash hands thoroughly after use to SAFC Supply Solutions
prevent contamination of food and drink. 3050 Spruce St., Saint Louis, MO 63103
Special care should be taken to avoid 800-244-1173 (US Toll Free)
contamination of surfaces that may later +1-314-534-4900 (International)
be handled by unprotected personnel. Global Email: safcglobal@sial.com

SAFC®, SAFC Supply Solutions® and Sigma-Aldrich® are registered trademarks


of Sigma-Aldrich Biotechnology L.P. and Sigma-Aldrich Co.
ProClin® is a registered trademark of Rohm and Haas Company.
© 2009 SAFC All rights reserved.

KVS
04770-503120
0049 www.safcsupplysolutions.com/proclin www.safcsupplysolutions.com/proclin
ProClin 300 Preservative
®

Features & Benefits for Diagnostics Reagents

ProClin 300 preservative is a highly effective biocide offered for the control of Table 3. Typical Metals Analysis Stability in Reagents
microorganisms in reagents and products intended for in vitro diagnostic use. With broad- (do not constitute specifications) The active ingredients in ProClin 300
spectrum activity, excellent compatibility and stability, and low toxicity at use levels, ProClin Metal Volume (mg/L) preservative have an established history of
300 is the ideal choice for an effective preservative in diagnostic reagents. At unusually low successful use as preservatives. However,
Arsenic <1.0
concentrations, ProClin 300 preservative eradicates bacteria, fungi, and yeasts in reagents there are some circumstances under which
for prolonged periods, thereby increasing a product’s shelf life. The water solubility permits Selenium <1.0
we would advise potential users to confirm
easy incorporation into reagents. Further, ProClin 300 preservative does not affect the Barium <1.0 biocide stability prior to use:
functionality of most enzyme- or antibody-linked reactions, and will not interfere with assay Chromium <1.0
indicators. The information presented in this bulletin is designed to help you predict and Temperature: As a general rule, a rise
Lead <1.0 in temperature accelerates the rate of
evaluate the performance of ProClin 300 preservative in your products.
Mercury <0.01 degradation of chemicals. Temperatures in
5-Chloro-2-methyl-4-isothiazolin-3-one and excess of 55 °C should be avoided during
Table of Contents Page No. Antimony <1.0
2-Methyl-4-isothiazolin-3-one (see Figure A). manufacturing once the biocide has
Chemical Identification 2 Copper <1.0 been incorporated into the reagent.
Structural Formulas 2 Calcium 1.7
Chemical Properties Amines: The presence of amines,
Chemical Properties 2
ProClin 300 preservative is a precise Iron 1.0 particularly secondary amines, has a
Physical Properties 2
Solubility 3 formulation of active ingredients and an Magnesium <1.0 deleterious effect on the stability of ProClin
Stability 3
organic stabilizer in a modified glycol Manganese <1.0 300 preservative. This can be avoided by
In Reagents 3
(see Table 1). reducing the pH of a regent containing
Nickel <1.0
In Biological Buffers 3 amines below 7, which converts amines to
Table 1. Chemical Composition of Tin <2.0 their acid salts.
In Enzyme Conjugates 4 ProClin 300 Preservative
Lyophilization 4 Nitrate <5.0
Reducing Agents: Some reducing agents
Testing 4 Figure A. Nitrite <5.0 are detrimental to isothiazolone stability.
Active Ingredients of ProClin 300 Preservative
Degradation Products 4 Insoluble Residue ND (<5.0) We have found that use levels of ProClin
Biocidal Efficacy 4 O O
C C 300 preservative are stable in the presence
In Enzyme Conjugates 5
ND = Not Determined
of up to about 50ppm bisulfite (expressed
Directions for Use 5 N CH3 N CH3 as SO2).
Compatibility with Enzymes 5 Cl Solubility
S S
Short-Term Effects 6 5-Chloro-2-methyl-4-isothiazolin-3-one 2-Methyl-4-isothiazolin-3-one ProClin 300 preservative is 100% soluble pH: ProClin 300 preservative is stable and
Long-Term Effects 6
CAS number 26172-55-4
Empirical formula C4H4ClNOS
CAS number 2682-20-4
Empirical formula C4H5NOS and completely miscible in water. It is also effective over a wide range of pH. At pH
soluble in a wide range of oxygenated levels below 9, ProClin 300 preservative
Effect on Antibody Binding 6
solvents. is generally stable over the lifetime of the
Toxicology Properties 7
Acive Ingredients reagent. The stability of the preservative is
Safe Handling Information 8
Stability reduced when pH exceeds 9.
Personal Protection 8 5-Chloro-2-methyl-4-isothiazolin-3-one 2.30%
First Aid Measures 8 ProClin 300 preservative is formulated Serum: The presence of strong
2-Methyl-4-isothiazolin-3-one 0.70%
Neutralizing Solution 8 for long product shelf life. Stability tests nucleophiles such as glutathione
Inerts indicate that after two years storage at and cysteine in blood and serum can
Features and Benefits Modified glycol 93-95% 25 °C, there is no detectable loss of active have a deleterious effect on ProClin
• Broad-spectrum antimicrobial activity ingredients. Additional stability data in 300 preservative, but this reaction is
Alkyl carboxylate 2-3%
• Compatible with key enzymes Table 4 show that the material is stable for dependent on concentration, pH, and
several weeks at elevated temperatures other components of the reagent. We
• Does not inhibit antibody binding
Table 2. Typical Physical Properties (prolonged storage at high temperatures is recommend that the manufacturer test the
• No interference with ISE electrodes
(do not constitute specifications) not recommended). performance of ProClin 300 preservative in
• Excellent stability a specific product before its use.
Property Description
• Effective at pH ≤ 3.5 Table 4. Stability of ProClin 300
Appearance Clear Liquid Preservative (as formulated)
• No interfering inerts Stability in Biological Buffers
Colorless to Pale
Color Temperature (°C) Weeks % Active Remaining
• No color imparted to reagents Yellow Biological buffers (e.g. TRIS, TES, and
• No health hazards at recommended Odor Mild (sweet) 25 36 >98 HEPES) may contain amines that are
use levels 40 36 99 aggressive to ProClin 300 above pH7. The
Density (lbs/gal) 8.59
• Safe disposal at recommended degradation of ProClin 300 preservative
Specific gravity (22 °C) 1.03 55 8 100
use levels in these buffers can be minimized by
pH (10% aqueous solution) 4.1 55 12 100 reducing their pH to 7 or lower, which
Viscosity 25 °C 58cPs protonates free amines and converts them
Chemical Identification
to a less aggressive acid salt. Short-term
Structural Formulas Flash Point (Seta Closed Cup) 224 °F (118 °C)
stability data are summarized in Figure B1.
The active ingredients of ProClin 300 Absorbance Maximum 275nm
preservative are two isothiazolones Fluorescence None
identified by the IUPAC system of
2 nomenclature as: Evaporation Rate (Bu acetate = 1) <1 3

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ProClin 300 Preservative ®

Features & Benefits for Diagnostics Reagents

Stability in Enzyme Conjugates Degradation Products Active Table 6. Efficacy of ProClin 300
ProClin 300 preservative is also unaffected ProClin 300 may degrade in the presence Organism (ATCC# Number) Ingredient Preservative in Enzyme Conjugate
(ppm)
by the presence of enzymes. There of high pH, high temperature, reducing
Gram-Negative Bacteria * Pseudomonas aeruginosa (CFUs /mL)
was virtually no loss of active biocide in agents, and aggressive nucleophiles. Bacillus cereus mycoides (R&H L5*) 2
horseradish peroxidase (HRP) or alkaline Storage
The degradative pathway for each of ProClin 300 Sodium
Bacillus subtilis (R&H B2*) 2 Time (Weeks) Control
phosphatase (AP) treated with 250ppm these mechanisms is similar. Degradation (15ppm) Azide (0.1%)
10% HRP
ProClin 300 after six months’ storage at products include small organic acids Brevibacterium ammoniagenes (6871) 2
0 9.5 x 106 1.1 x 107 1.8 x 107
temperatures ranging from -4 °C to 18 °C (acetic, formic), carbon dioxide, chloride Cellulomonas sp. (21399) 6
(see Figure B2). ion, and elemental sulfur. None of these 2 >1.0 x 10 9
<10 <10
Sarcina lutea (9341) 5
degradation products have a significant 4 8.9 x 107 <10 <10
Staphylococcus aureus (6538) 2
Figure B1. Stability of ProClin 300 impact on the environment. 6 4.4 x 108 <10 <10
Preservative in the Presence of 50mM Staphylococcus epidermidis (155) 2
Physiological Buffers After Three Weeks 1% HRP
Biocidal Efficacy Streptococcus pyrogenes (624) 9
100 0 1.2 x 106 3.5 x 106 2.3 x 106
ProClin 300 preservative is an outstand- Streptomyces albus (3004) 1
ing antimicrobial agent with unique cidal 2 7.2 x 108 <10 1.9 x 103
20 Fungi – Yeasts *
mechanisms. Within minutes after contact Aspergillus foetidus (16878) 9 4 4.4 x 108
<10 <10
% a.i/ remaining

20 with microorganisms, ProClin 300 inhibits Aspergillus niger (9642) 9 6 4.4 x 108 <10 <10
growth and respiration. Intracellular energy
20 Aspergillus oryzae (10196) 5 *
Colony-forming units.
levels decline rapidly and macromolecu-
lar synthesis is inhibited. Accumulation of Aureobasidium pullulans (9348) 5
20
damage to cellular constituents, possible Candida albicans (yeast; 11651) 5
Directions for Use
0 induced both by direct covalent modifica- Long-term microbial protection in reagents
Chaetomium globosum (6205) 9
Di H2O Tris Tes Hepes tion of protein and via radical-mediated is accomplished with levels of ProClin 300
Buffers Cladosporium resinae (11274) 5
events, results in the death of the cell. Flyer preservative between 6 and 20ppm active
Mechanisms and Stability describes the Gliocladium fimbriatum (QM7638) 9 microbicide. Typical use levels fall in the
Figure B2. Stability of ProClin 300 range of 9-15ppm active (0.03-0.05% as
mechanisms of ProClin 300 preservative Lentinus lepideus (12653) 4
ProClin
Preservative in the Presence of �HRP and300
� ProClin 300 + HRP biocidal action in greater detail. supplied). It is usually advisable to treat a
AP After Six Months Lenzites trabea (11539) 6
� ProClin 300 + AP reagent with a higher level than the mini-
150 Table 5 lists the minimum levels of active Mucor rouxii (R&H L5-83*) 5 mum effective dose to extend its shelf life.
ingredient that inhibit the growth of various
Penicillium funiculosum (9644) 5
microorganisms. These results were Some reagents are sold in concentrated
% a.i. remaining

generated under standard laboratory Penicillium variabile glaucum (USDA**) 2 form, and require a limited shelf life after
100
conditions in nutrient-rich growth media Phoma herbarum pigmentivora (12569) 2 opening and dilution by the user. Reagents
and are intended to demonstrate the such as these can incorporate higher levels
Rhizopus stolonifer (10404) 5
broad-spectrum activity of ProClin 300 of ProClin 300 preservative, but special
50 Rhototorula rubra (yeast; 9449) 2
preservative. Actual use concentration will labeling may be required to inform the
vary according to application. Saccharomyces cerevisiae (yeast; 2601) 2 user of sensitization effects. For guidelines
Trichophyton mentagrophytes under these circumstances, contact SAFC
0 interdigitale (95332) 5
-4 °C +4 °C 18 °C Table 5. Minimum Inhibitory Technical Service at 800-244-1173.
Temperature Concentration of ProClin 300 Preservative
* Bacteriostaticand fungistatic tests performed by serially Since the components of diagnostic
� ProClin 300 Active diluting test compounds in trypticase soy broth and 1:100 reagents vary considerably, and may
� ProClin 300 + HRP Organism (ATCC# Number) Ingredient Inoculation with 24-hour broth cultures of test bacterium
impact on the stability and efficacy of
� ProClin 300 + AP (ppm) or a fungal spore suspension prepared from 7-to-14 day
150 culture slants washed with 7 mL of deionized water. preservatives, we urge each manufacturer
Gram-Negative Bacteria■
Lyophilization Stability Minimum inhibitory concentration levels determined to confirm the efficacy of ProClin 300
Achromobacter parvulus (4335) 2 visually after 2-4 days’ incubation at 30-37 °C for bacteria
% a.i. remaining

preservative in use.
The use of ProClin 300 preservative in Alcaligenes faecalis (8750) 2 and 7 days’ incubation at 28 °C to 30 °C for fungi.
100
lyophilized products is not recommended. Azotobacter vinelandii (12837) 5
Data from several trials indicate that the Compatibility with Enzymes
Enterobacter aerogenes (3906) 5 Efficacy in Enzyme Conjugates
active ingredients in ProClin 300 are volatil- The effect of ProClin 300 preservative on
50 Escherichia coli (11229) 8 The antimicrobial activity of ProClin 300 in
ized during the lyophilization process, and both the short-term and long-term activity
HRP is summarized in Table 6. Preserved
are not available for preservation once the Flavobacterium suaveolens (958) 9 of HRP and AP was determined using
and control solutions were challenged at 0,
product is reconstituted. Nitrobacter agilis (14123) 0.1 standard assays.
0 2, 4, and 6 weeks with an inoculum consist-
-4 °C +4 °C 18 °C
Proteus vulgaris (8427) 5 ing of 107 CFU/mL Ps. aeruginosa ATCC
Temperature Stability Testing Data from laboratory studies (Table 7)
Pseudomonas aeruginosa (15442) 5 strain 15442. A concentration of only 15ppm indicate that ProClin 300 preservative
The level of active ingredients in ProClin
Pseudomonas cepacia (Gibraltar 165) 0.75 ProClin 300 preservative (0.05% as supplied) does not affect the functionality of most
300 preservative, as supplied and in
Pseudomonas fluorescens (13525) 2 completely eradicated this Pseudomonas key enzymes, used for diagnostic testing,
formulated products, can be determined by
sp. in both 1% and 10% HRP solutions after including HRP and AP (Figure C).
HPLC analysis. See Determination of ppm Pseudomonas oleoverans (8062) 5
every challenge. This antimicrobial activity
Levels flyer for analytical methods. Working Salmonella typhosa (6539) 5
was superior to that sodium azide.
4 standards can be obtained from SAFC. Shigella sonnei (9292) 8 5

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ProClin 300 Preservative ®

Features & Benefits for Diagnostics Reagents

Table 7. ProClin 300 Preservative Table 8. Effect of ProClin 300 Preservative binding were determined in the presence of Toxicological Properties
Short-Term Enzymes Compatibility♦ on the Km and Vmax of HRP and AP 0-300ppm active isothiazolones, and long-
Table 10. Acute Toxicity
No Inhibition Inhibition Observed Vmax term inactivation of the reporting antibody
Km (mM) (A/min/mg was determined by incubating aliquots of Test Results
Acid phosphatase α-Ketoglutarate
protein) Oral Toxicity (Rats)
Alkaline phosphatase Dehydrogenase the concentrated antibody reagent with
male >500<2000mg/kg
Horseradish peroxidase 6.9 904 0-50ppm active isothiazolones for a period
Catalase Lactate dehydrogenase female >500<2000mg/kg
+15ppm ProClin 300 6.8 861 of 35 days.
Chymotrypsin Pyruvate dehydrogenase Dermal Toxicity (Rabbits)
Horseradish peroxidase 7.9 520 male >500<2000mg/kg
b-Galactosidase Succinate dehydrogenase +250ppm ProClin 300 8.3 525 The data in Table 9 demonstrate that female <2000mg/kg
Glyceraldehyde-3-phosphate Alkaline phosphatase 0.69 138 concentrations of ProClin 300 preservative
dehydrogenase Primary Skin Irritation (Rabbits)
+15ppm ProClin 300 0.56 131 up to 300ppm active (1% as supplied) had Product as supplied (30,000ppm a.i.) Corrosive
Horseradish peroxidase
Alkaline phosphatase 1.40 * 153 no effect on the extent of antibody-antigen Product diluted to 18.6% (5600 ppm a.i.) Severe irritant
Malate dehydrogenase
+250ppm ProClin 300 1.20 * 142 binding. Incubation of HRP-linked antibody Product diluted to 9.3 (2800ppm a.i.) Moderate irritant
Monoamine oxidase
Results for AP were generated separately, using
*
reagent with 50ppm active ProClin 300 Product diluted to 1.8% (560ppm a.i.) Non-irritant
NADH dehydrogenase different enzyme preparations in each test. preservative for 35 days also had no effect Primary Eye Irritation (Rabbits)
3-Phosphoglycerate
phosphokinase on the performance of the reagent when Product as supplied (30,000ppm a.i.) Irreversible damage
Long-Term Effects
diluted for use in the binding assay. An Product diluted to 18.6% (5600ppm a.i.) Moderate irritant
Pyruvate kinase Enzymes that are compatible with ProClin
Trypsin 300 preservative in the short term also examination of the effect of 300ppm active Product diluted to 9.3 (2800ppm a.i.) Slight irritant
Tyrosine carboxylase retain their activity for extended periods of ProClin 300 preservative on the rate of Product diluted to 1.8% (560ppm a.i.) Non-irritant
time. The rate of color development with AP color development by HRP-linked antibody Inhalation Toxicity (Rats)
*
30 minute assays using 7500 ppm (25% as
indicated that there was no inhibition 4-hour aerosol (LC50)• 0.7-1mg/L product
supplied) of ProClin 300 preservative. remained constant over a 6-month period = 0.2mg/L a.i.
in the presence of 250ppm active ProClin (Figure E).
4-hour saturated vapor (LC50)• >2.31mg/L product
Figure C. Effect of 250ppm ProClin 300 300 preservative (Figure D1). Under the = 0.65mg/L a.i.
Preservative on a Substrate vs. Velocity Plot
These results demonstrate that ProClin 300
� HRP - No biocide same conditions, HRP activity increased has no effect on the activity of HRP-linked
• Air saturated with a solution containing ten
of HRP and AP
� HRP + ProClin 300 steadily with time (Figure D2). times the level of active ingredient that is present
� HRP - No biocide antibodies and is suitable for use with in ProClin 300 preservative.
500
� HRP + ProClin 300 antibody-based diagnostic assays.
500 Figure D1. Effect of ProClin 300 Preservative
400 on the Activity of AP at 4 °C and 37 °C
Table 11. Subchronic Toxicity
( OD/min)

400 Table 9. Effect of ProClin 300 Preservative


125 Active
300 on Short-Term Antibody Binding Test Response
( OD/min)

Enzyme Activity ( OD/min)

AP + ProClin 300 at 4 °C Ingredient


300 �
200 � � 100
AP + ProClin 300 at 37 °C 90-day dietary:rats 30mg/kg/day No mortalities, no
� ProClin 300 Concentration Percent Binding
Velocity


� pathological findings
200 �
� 75 (ppm active) vs. control
90-day dietary:dogs 27mg/kg/day No mortalities, no
Velocity

100 �


� 7.5 101.9 pathological findings
100 �
� �

50 90-day dermal:rabbits 0.4mg/kg/day No treatment-related
0 �
� 15.0 100.4
0.5 1 2 3 4

� pathological effects
0
mM Pyrogallol 22.5 104.9
0.5 1 2 3 4 25 90-day drinking 20mg/kg/day Slight gastric irrita-
mM Pyrogallol
37.5 102.5 water, tion, no adverse effect
0 75.0 101.5 one generation on reproduction, fetal
� AP + ProClin 300
� AP - No biocide
0 4 8 12 16 20 24
300.0 100.7 reproduction:rats health or survival
� AP + ProClin 300 Weeks
150 (values represent the average of three independent
4-week eye irritation: 8mg/kg/day No effect level
� AP - No biocide rabbits (8 applications/ 56ppm Non-irritant
150 experiments with duplicate determinations)
Figure D2. Effect of ProClin 300 Preservative day, 5 days/week)
on the Activity of HRP at 4 °C and 37 °C 13-week 0.34mg/m3 No observable effect
100
( OD/min)

500 inhalation:rats at that dose (6 hours/



� �
� Figure E. Effect of ProClin 300 Preservative
� day, 5 days/week)
Enzyme Activity ( OD/min)

100 � �
( OD/min)

� �
� on Antibody-Bound HRP Activity

� �
� �


� �
� 400 Teratology:rats 15mg/kg/day Not teratogenic

� �

50 � � 300ppm ProClin 300 � Control Teratology:rabbits 13.3mg/kg/day Not teratogenic
Velocity


� �

� 300 0.8
50 � �
Velocity


� �

0.7
200
0 �
� Table 12. Chronic Toxicity

Abs — 405nm
0.5 0.8 1.2 1.6 1.8 2 2.5 3 0.6
0 HRP + ProClin 300 at 37 °C
0.5 0.8 1.2 mM1.6
NPP 1.8 2 2.5 3 100 0.5 �
HRP + ProClin 300 at 4 °C �
Active
0.4 Test Response
mM NPP �
Ingredient
0 �
0.3
Short-Term Effects 0 4 8 12 16 20 24 Dermal Oncogenicity, 400ppm Slight skin irritation,no
Weeks 0.2 30-month skin painting: other treatment-related
ProClin 300 preservative has no significant 0.1 treatment mouse pathology, no increase
effect on either the Km or Vmax of HRP �
(3 times/week) in tumor frequency
0 �
and AP, even at 15 times the maximum Effect on Antibody Binding 0 7 10 15 30 60

recommended use levels (Table 8). A slight The effects of ProClin 300 preservative Minutes Genetic Toxicity
decrease in the Km of AP was observed on on antibody binding were studied using Based on a battery of genotoxicity tests, as
the presence of 250ppm active biocide, but purified mouse lgM as the antigen, and well as the oncogenicity studies, ProClin
the affinity of the enzyme for its substrate goat antimouse lgM linked to HRP as the 300 preservative has been determined not
was unaffected at the typical use level of reporting antibody. Direct effects on to pose any mutagenic or carcinogenic
6 15ppm. hazards to humans under normal conditions. 7

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