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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
16 October 2008 (16.10.2008)
PCT
(21) International Application Number:
PCT/GB2008/001179
4 April 2008 (04.04.2008)
(25) Filing Language:
English
(26) Publication Language:
English
(30) Priority Data:
0706739.0
5 April 2007 (05.04.2007)
WO 2008/122773 A2
(74) Agents: MURGITRO YD & COMPANY et al; Scotland
House, 165-169 Scotland Street, Glasgow G5 8PL (GB).
(51) International Patent Classification:
AOlN 59/00 (2006.01)
AOlN 25/30 (2006.01)
AOlN 37/44 (2006.01)
AOlP 1/00 (2006.01)
AOlN 33/12 (2006.01)
(22) International Filing Date:
(10) International Publication Number
GB
(71) Applicant (for all designated States except US):
ALEXANDER
ROSS
HOLDINGS
LIMITED
[GB/GB]; North Main Street, Carronshore, Falkirk
FK2 8HT (GB).
(72) Inventor; and
(75) Inventor/Applicant (for US only):
ANDERSON,
Frances [GB/GB]; 44 Avonmill View, Linlithgow Bridge,
West Lothian EH49 7SH (GB).
(81) Designated States (unless otherwise indicated, for every
kind of national protection available): AE, AG, AL, AM,
AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA,
CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE,
EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID,
IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC,
LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN,
MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH,
PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV,
SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN,
ZA, ZM, ZW
(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,MC, MT, NL,
NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG,
CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
Published:
— without international search report and to be republished
upon receipt of that report
(54) Title: BIOCIDAL COMPOSITION
(57) Abstract: An composition which comprises a chelating agent such, as nitrilotriacetic acid or its derivatives, at least one biocide
such as a quaternary ammonium compounds and a metasilicate. The composition can be use as a disinfectant for work or hard
surfaces and/or body parts.
Biocidal composition
The invention relates to a biocidal composition which is advantageously
effective against infections and in particular nosocomial infections.
Background of invention
It has been estimated that as many as one in ten patients become infected
by nosocomial (or hospital acquired) infections. Such infections are
generally defined as those which occur as a result of hospital or hospitaltype treatment but are secondary to the patient's original condition. Such
infections are considered to be nosocomial if they appear either 48 hours
or later from admittance to hospital or within 30 days of discharge
therefrom. Nosocomial infections are becoming both more prevalent and
extensive as their resistance to a number of antibiotics increases.
Additional reasons for the increase in incidence of such nosocomial
infections is due to the high concentration and close proximity of patients;
the fact that such patients have suppressed or depressed immune
systems; the increased use of outpatient treatment means that inpatients
are sicker on average; and the fact that medical staff move from one
patient to another thereby providing a means for spreading pathogens.
Thorough hand washing by all medical personnel before and after each
patient contact is one of the most effective ways to combat transfer of
such infections. Further, washing hands as promptly and thoroughly as
possible between patient contacts and after contact with blood, body
fluids, secretions, excretions, and equipment or articles contaminated by
them is an important component of control and isolation procedures. In
addition, surface cleaning is also essential and has to be carried out
regularly and thoroughly with an appropriate anti-infectious agent.
More careful and considered use of anti-microbial agents, such as
antibiotics, is also considered vital to try to prevent further resistance to the
small number of active antibiotics still available. Alternatively,
investigations into new antibiotics are required. In the meantime, in order
to bridge the gap, in case resistance to all known antibiotics becomes a
reality and before new ones have been sufficiently developed,
investigations have been ongoing to provide biocidal compositions which
would be effective against such nosocomial infections.
A great variety of biocidal formulations are known. US 3,507,796
describes an antibacterial composition which comprises a chelating agent,
a zwitterionic detergent and an alkaline buffering salt. The alkaline
buffering can be chosen in a wide group of compounds such as
carbonates, bicarbonates, orthophosphates, tripolyphosphate,
metaphosphates, and silicates. Sodium carbonate and sodium
trypolyphosphate are the buffering agents preferred.
The general use of silicates as buffering or pH controlling agents is
generally known and is mentioned for example in US 2006/0128585,
US4597887, US 6,1 84,1 95 B 1 or US 2005/0055787. However sodium
metasilicate pentahydrate is seldomly used for biocidal composition
actually in the market.
However, these existing biocidal compositions have still been shown to be
insufficient in the fight against such infections as is demonstrated by the
current Methicillin-resistant Staphylococcus aureus (MRSA) epidemic in
UK hospitals which shows that there is still a need for more efficient
compositions effective against, in particular, nosocomial infections. Such
currently available biocidal compositions also have a number of
disadvantages such as their high level of toxicity (due mainly to the fact
that they are alcohol based and generally include a high level of
surfactant), their low biodegradability and their limited specificity. Such
limited specificity results in their efficiency often being limited to a
particular type of microbe (i.e. bacteria, viruses, spores, fungi) or even to a
particular genus or species.
Object of the Invention
Accordingly there is a need for a biocidal composition which would obviate
or mitigate at least one if not more of the above-mentioned disadvantages
and provide a biocidal composition which is effective when used against a
wide range of microbes of different types, including bacteria, virus, fungi
and spores and/or different species thereof.
Summary of the Invention
According to the present invention there is provided a biocidal composition
comprising a chelating agent, at least one bactericidal biocide, a surfactant
and metasilicate.
The chelating agent is preferably chosen from nitrilotriacetic acid (NTA),
and its derivatives, including but not limited to, its salts, its esters,
phosphonate or phosphonic acids thereof. The calcium, copper, iron and
sodium salts of NTA are preferred, amongst which the trisodic form of
NTA, or trisodium nitrilotriacetate, is particularly preferred.
Alternative chelating compounds which are similar or equivalent in
structure or characteristics thereto are also encompassed by the invention.
Amongst these compounds are ethylenediaminetetraacetic acid (EDTA),
ethylene glycol tetraacetic acid (EGTA), diethylene triamine pentaacetic
acid (DTPA), their salts, esters and their phosphonates or phosphonic
acids (NTMP, EDTMP and DTPMP). NTMP, EDTMP and DTPMP are
structural analogues to the known aminopolycarboxylates NTA, EDTA and
DTPA and are effective chelating agents. The major disadvantage of such
structural analogues is their relatively low biodegradability. EGTA has a
very high affinity of calcium ions and DTPA can chelate heavy metal ions
very efficiently as well, making both these chemical compounds possible
alternatives to NTA, though NTA is preferred.
Whilst the concentrations (% by weight) of both chelating agent and the
biocide can be altered as desired it is preferred according to one
embodiment that their respective concentrations be at least about 5%.
Alternatively the concentration (% by weight) of the chelating agent can
advantageously range from 3 to 10 % , and more preferably 4 to 8%.
The biocide should have anti-bacterial properties and can be chosen
amongst a wide variety of compounds. However the bactericide is
optimally selected from a group of quaternary ammonium compounds
and/or alkyl dimethyl betaines and a mixture thereof. Of course, more
than one biocidal agent may be suitable for inclusion in the composition.
Also, if more than one biocide is included in the composition, it is preferred
that they have a different mode of action such as, for example, different
efficacies for killing different forms of living organisms.
Advantageously the concentration (% by weight) of the biocide ranges
from 5-35%. However the optimal concentration depends upon the
potency of the particular biocide chosen. For example the concentration
of benzalkonium chloride in a ready-to-use composition advantageously
ranges from 5-25%, more preferably from 5-10%.
Sodium metasilicate, Na2SiO 3 (or Disodium trioxosilicate) and in particular
its pentahydrate form Na2SiO 3 5H2O are particularly preferred.
The concentration (wt %) of the metasilicate may range from 2-15%. For
example the concentration of metasilicate in a ready-to-use composition
advantageously ranges from 3 to 15%, more preferably from 4 to 11% and
even more preferably from 5 to 8 wt%. Advantageously the concentration
of metasilicate ranges from 3 to 6 wt%.
Additionally a surfactant may also be included in the composition but
preferably at a low concentration, for example less than 15 wt%.
According to a particularly preferred aspect of the invention the
composition is an aqueous (or water based) composition. In such a case
the chelating agent and/or the biocide are advantageously water-soluble.
According to another aspect of the invention the composition does not
include any substantial amount of alcohol and, in particular, does not
include alcohol(s) as a solvent.
Surfactants are preferably chosen by their ability to be soluble in water and
for their compatibility with the other components of the composition. They
can be selected from the group consisting of anionic, cationic, non-ionic
and ampholytic surfactants. Non-ionic surfactants are preferred as they
can show a weak bactericidal effect, especially on Gram positive bacteria.
Common surfactants used are: sodium dodecylsulfate (SDS), sodium
deoxycholate (DOC), lauroylsarcosine sodium salt, lauryldimethylamine
oxide (LSAO), cetyltrimethylammonium bromide (CTAB), sulfosuccinate
sodium salt, Craymul 7050 (an acrylic polymer dispersion), Licomet F3 (a
fluorinated anionic surfactant containing butyl glycol), liquid sodium lauryl
ether sulphate (SLES), Synperonic 9 1 10 ( a fatty alcohol ethoxylate) ,
Surfactant UN90/90 (C9-Cn alcohol ethoxylate with 9 MEO), Amphoteric
400 (an amphoteric surfactant), Synperonic LF/RA 280 (an alcohol
alkoxylate surfactant),Synperonic LF/RA 30 (an alcohol alkoxylate
surfactant), Synperonic 9 1 10:85% (a fatty alcohol ethoxylate 85%) and
Surfactant CDE/G (coconut diethanalamide). Synperonic 9 1 10, Surfactant
UN90/90, Synperonic LF/RA 280 and Synperonic LF/RA 30 are preferred.
Advantageously the chelating agent on the one hand and metasilicate on
the other hand are present in the composition in a ratio of 1: 1 (% by
weight).
According to a preferred embodiment of the invention the composition is
an aqueous biocidal composition which comprises a water-soluble
chelating agent in the form of NTA, a water-soluble bactericide in the form
of benzalkonium chloride together with sodium metasilicate, preferably
pentahydrate sodium metasilicate.
According to a preferred embodiment of the invention the composition
further comprises a surfactant in the form of Synperonic UN 90/90, C9-Cn
alcohol ethoxylate with 9MEO.
The invention also extends to the use of the composition of the invention
as a biocide or a disinfectant, particularly for use as a liquid disinfectant for
body parts (e.g. hand wash) or as a surface cleaner. The composition of
the invention is particularly useful in hospitals or other communal buildings
where hygiene is important such as care homes and schools.
According to the invention it is also disclosed as a method of disinfecting
and/or cleaning which comprises the step of applying the composition of
the invention onto a surface and/or body part to be disinfected.
Detailed Description of the Invention
Whilst the concentrations of each component of the formulation of the
invention can be modified as desired, the following concentrations (% by
weight) are preferred:
Biocide: 3%, 5%, 7%, 10%, 11% 15%, 23%, 25%, 27%, 35%.
Chelating Agent: 3%, 4%, 5%, 6%, 7%, 10%
Metasilicate: 3%, 4%, 5%, 6%, 7%, 10%.
Advantageously the chelating agent is chosen from nitrilotriacetic acid
(NTA), C6H9NO 6 , its salts or esters thereof. NTA is a chemical compound
which is generally used as a chelating agent and which forms coordination
compounds with metal ions (chelates) such as Ca2+ , Cu2+ Or Fe3+ . NTA is
easily biodegradable and is almost completely removed during wastewater
treatment. Most preferred is the NTA in the form of its salts, of which the
trisodic form of NTA orTrisodium Nitrilotriacetate is most preferred. Other
variants of NTA such as nitrilotriacetic acid sodium salt, nitrilotriacetic acid
monosodium salt, nitrilotriacetic acid disodium salt, nitrilotriacetic acid
disodium salt monohydrate, nitrilotriacetic acid trisodium salt monohydrate
could be used as suitable alternatives to NTA.
According to the present invention it was found that such a composition
combining a chelating compound, such as NTA (or one of its derivatives
such as salts, esters, phosphates etc), at least one biocide and a
metasilicate is surprisingly effective and has a wide biocidal properties
spectrum that extends to bacteria, viruses, spores and fungi.
Advantageously the composition of the invention is a high level or "Ievel2"
disinfectant according to European standard.
The biocide itself can be chosen form a range of compounds.
According to the Biocidal Products Directive 98/8/EC (BPD), the
classification of biocides can be broken down into 23 product types (i.e.
application categories), with several comprising different subgroups.
Whilst the present invention is not limited to any particular type of biocide,
it is particularly preferred that the composition of the invention to belong to
disinfectants and general biocidal products of the classification and more
particularly to Product-type one of this main group, that is human hygiene
biocidal products.
However, it is envisaged that other product-types are encompassed by the
invention including the following sub-groups:
*
Product-type 2 : Private area and public health area disinfectants and
other biocidal products
*
Product-type 3 : Veterinary hygiene biocidal products
*
Product-type 4 : Food and feed area disinfectants
*
Product-type 5 : Drinking water disinfectants
Of course the present invention is not limited to the use of only one biocide
but can in fact include one of more together with the metasilicate and
chelating agent in the composition.
A preferred biocide(s) is to be found amongst the bactericides and
comprises certain types of quaternary ammonium compounds (quats)
which are well known for their biocidal activity. For example,
Benzalkonium chloride (alkyl dimethyl benzyl ammonium chloride) is a
mixture of alkylbenzyl dimethylammonium chlorides of various alkyl chain
lengths. It is commonly used as an antiseptic. The greatest bactericidal
activity is associated with the Ci 2-Ci 4 alkyl derivatives. It has been
considered one of the safest synthetic biocides known, and has a long
history of efficacious use. Benzalkonium chloride is readily soluble in
water, alcohol, and acetone. However, formulation requires great care as
benzalkonium can be inactivated by certain organic compounds, including
soap, and must not be mixed with anionic surfactants.
Standard concentrates are manufactured as 50% and 80% w/w solutions,
and sold under trade names such as BC50, BC80, BAC50, BAC80, etc.
The 50% solution is purely aqueous, while more concentrated solutions
require incorporation of rheology modifiers (alcohols, polyethylene glycols,
etc.) to prevent increases in viscosity or gel formation under low
temperature conditions. Because of ease of handling and manufacture,
BAC50 formulations are generally preferred in the composition of the
invention.
Benzalkonium chloride solutions are rapidly acting anti-infectious agents
with a moderately long duration of action. They are in fact active against
bacteria and also some viruses, fungi, and spores. However, on the
whole, bacterial spores have been shown to be resistant to such
compounds. Solutions are bacteriostatic or bactericidal according to their
concentration. Gram-positive bacteria are generally more susceptible than
gram-negative. Activity is not greatly affected by pH, but increases
substantially at higher temperatures and prolonged exposure times.
Metasilicates are used as degreasing agents and as buffering agents.
Also they are known as having some anti-viral properties. According to a
preferred embodiment of the invention, water soluble sodium metasilicates
Na2SiO 3 , and especially sodium metasilicate pentahydrate, are preferably
used in the composition.
In a further embodiment a surfactant may be included in the aqueous
composition but preferably at a low concentration such as less than 10%.
Preferably the ratio of chelating agent to biocide in the composition
remains unchanged. Use of such a surfactant can increase the efficacy of
the aqueous biocidal composition. The usual concentration of surfactant
in biocidal compositions for human hygiene is 15-20%. As such the
composition of the present invention provides a biocidal composition
having no or a comparatively reduced concentration in surfactant for a
comparably high efficacy thereby presenting advantages for the
environment and in terms of efficiency and costs.
The biocidal composition of the present invention has shown biocidal
activity without the presence of a surfactant. However, some surfactants
can be added in order to improve the efficacy of the biocidal composition
of the invention. It should be noted that the concentration of surfactant is
distinctly lower than the one used in prior art biocidal compositions.
A surfactant which has been used is available under the trade name
Surfac.UN90/90 is C9-H alcohol ethoxylate with 9 MEO at 90.000%. Other
surfactants are Surfac CDG, Surfac B4, Surfac UN65/95, Surfac TKPP.
Depending upon the particular application of the composition, the
concentration of the various components of the invention may vary
depending upon the particular ingredient used.
However, a concentrated composition can advantageously be formulated
for ease of handling and storage. The concentrate composition can then
be diluted with water to obtain the following preferred concentration (% by
weight) of compounds:
BAC50 35%, UN90 11%, Metso 5%, NTA 5%,
BAC50 27%, UN90 11%, Metso 5%, NTA 5%
BAC50 23%, UN90 12%, Metso 5%, NTA 8%,
BAC50 11%, UN90 18%, Metso 5%, NTA 5%, or
BAC50 5%, UN90 7.5%, Metso 5%, NTA 5%.
Wherein "BAC50" is a Benzalkonium Chloride 50.000% C12-C14 Alkyl
Benzyl Dimethyl Ammonium Chloride from the company Univar in 50%
water, "UN90" is the UN90/90 surfactant above mentioned, "Metso" is
sodium metasilicate pentahydrate and "NTA" refers to the trisodic form of
NTA. The same notations are used herein below:
The composition of the invention, and in particular the specific formulations
above listed, can be used as a liquid disinfectant which can be used
externally for the washing of human skin (for example as a spray or wipes
or as a hand wash).
For such uses the composition of the invention advantageously includes
wash ingredients such as: Versene (tetrasodium EDTA), Surfac CDE/G
(coconut diethanolamide >90% and diθthanolamide 1-5%), Empipearl XA
200/X ( Ci2-Ci 8 alkyl amido propyl dimethylamine betaines sodium, Ci 0-Ci 6
alkyl ethoxy sulphate), Nipagen M (methyl-4-hydroxybenzoate),
Nipasol N
(propyl-4-hydroxybenzoate).
The composition of the invention, and in particular the specific formulations
above listed, can also be used in a wide range of applications such as a
surface cleaner in wipes, sprays, foggers (automatic room sprayer),
sanitizer etc, or as a fabric conditioner. It can used in hospitals, medical or
dental practices, in particular to sanitize medical instruments, in nursing
and care homes and in schools.
For fabric conditioner an additional surfactant such as Sepantex VK90
(alkylester ammonium isopropanol) can be advantageously used.
As a sanitizer the following additional compounds can be used: water,
glycerin, Nipaguard PDU (a mixture of propylene glycol, diazolidinyl urea,
methyl- 4-hydroxybenzoate and propyl-4-hydroxybenzoate) and
Polysorbate 20 (polyoxyethylene (20) sorbiton monolaureate.)
The composition of the invention can be formulated to have a safe and
alkaline pH of around 10-1 1. The pH of the invention is maintained by
controlling the NTA and the Metso. A pH of 10-1 1 is advantageous for safe
handling and storage of the product. The alkaline pH is also effective as a
boost to the cleaning properties of the product.
In order that those skilled in the art can more fully appreciate the aspects
of the invention, the following examples are set forth, which examples are
given solely for the purposes of illustration, and should not be considered
as expressing limitations unless so set forth in the appended claims.
Example 1 : Test Formu lation I
A biocidal composition according to the invention has been tested against
various infectious organisms and it has been demonstrated that this
composition is a high level, or "level 2" disinfectant. The composition
tested in the following experiments has the following formulation which will
be referred to hereinafter as Formulation I
- BAC 50
5 wt%,
- Un 90
7.5 wt%,
- Metso
5 wt%, and
- NTA
5 wt%.
The solvent being water.
This formulation was kept at 4°C and in darkness before being diluted with
hard water for the tests to take place.
The effectiveness of the formulation as described above was tested by
carrying out standard suspension tests. Such tests follow a recognised
approach to disinfectant validation which is the CEN (European
Committees for normalisation) 216 work programme and provides a
systematic approach to the testing of disinfectants. From this a number of
BS EN methods have been derived for which there are clearly defined
pass criteria. In particular the BS EN methods are standard suspension
tests that are phase 2 /step 1 tests. This means that the test is
appropriate to the intended use of the biocidal product. Suspension tests
are set up with 1 part microorganism suspension, the count of
microorganisms to be within defined limits, 1 part interfering substance
and 8 parts biocidal agent. Contact between the biocide and micro¬
organism/interfering substance mixture is maintained for a defined time at
a defined temperature (20 QC). The biocide is then neutralised
appropriately. Controls ensure correct neutralisation of the product has
occurred and internally validate the test. Tests require that the biocidal
agent reduces the microbiological viability by 5 Logi 0 (bacteria) or 4 Logi 0
(Fungi) or 3 Logi 0 (spores) or 3 or 4 Logi 0 (viruses).
Experiment 1 - Clostridium Difficile under BS EN 1276-1997
Chemical disinfectants and antiseptics - Quantitative suspension test for
the evaluation of bactericidal activity of chemical disinfectants and
antiseptics used in food, industrial, domestic and institutional areas (phase
2 , step 1)
Test Method and its validation
The method used is filtration-neutralization.
Neutralizer - Lecithin 3g/l,
Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l, phosphate
buffer 0.0025 mol/l, sterilized by autoclave.
Experimental conditions
Product diluent used: sterile synthetic hard water.
Product test concentrations: 10% v/v; 20% v/v 80%v/v
Appearance product dilutions: clear
Contact time: t = 5 min + 10 s
Test temperature: 20°C ± 1°C
Interfering substance: 0.3 g/l bovine albumin
Stability of mixture: No precipitation
Temperature of incubation: 37°C ± 1°C
Identification of strains: Clostridium difficile NCTC 1 1209.
The results are shown in Table 1
Conclusion
According to testing carried out under conditions specified in EN 1276,
Formulation I possesses bactericidal activity at a concentration of 10.00%
V/V after five minutes at 20°C under clean conditions (0.3 g/L bovine
serum albumin) for referenced strains Clostridium difficile NCTC 11209.
Table 1
Experiment 2 - Community Acquired - Methicillin Resistant
Staphylococcus Aureus under BS EN 1276-1997
Chemical disinfectants and antiseptics - Quantitative suspension test for
the evaluation of bactericidal activity of chemical disinfectants and
antiseptics used in food, industrial, domestic and institutional areas (phase
2 , step 1)
Test Method and its validation
The method used is filtration-neutralization.
Neutralizer - Lecithin 3g/l,
Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l, phosphate
buffer 0.0025 mol/l, sterilized by autoclave.
Experimental conditions
Product diluent used: sterile synthetic hard water.
Product test concentrations: 5% v/v; 10% v/v 80%v/v
Appearance product dilutions: clear
Contact time: t = 5 min ± 10 s
Test temperature: 2 O0C ± 1°C
Interfering substance: 0.3 g/l bovine albumin
Stability of mixture: No precipitation
Temperature of incubation: 37°C ± 1°C
Identification of strains: CA-MRSA SMRSARL 02.6225.E
The test results are shown in Table 2
Conditions
According to testing carried out under conditions specified in EN 1276
Formulation I possesses bactericidal activity at a concentration of 5.00%
V/V after five minutes at 20°C under clean conditions (0.3 g/L bovine
serum albumin) for referenced strains community acquired methicillin
resistant staphylococcus aureus strain SMRSARL 02.6225. E .
Table 2
en
Experiment 3 - Test Report under BS EN 1040-1 997
Chemical disinfectants and antiseptics - Suspension test for the
evaluation of bactericidal activity of chemical disinfectants and antiseptics
used in food, industrial, domestic and institutional areas (phase 1)
Test Method and its validation
The method used is filtration-neutralization.
Neutralizer - Lecithin 3g/l,
Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l, phosphate
buffer 0.0025 mol/l, sterilized by autoclave.
Experimental conditions
Product diluent used: sterile synthetic hard water.
Product test concentrations: 20% WV; 50% WV 80%WV
Appearance product dilutions: clear
Contact time: t = 5 min ± 10 s
Test temperature: 20 0C ± 1°C
Interfering substance: 0.3 g/l bovine albumin
Stability of mixture: No precipitation
Temperature of incubation: 37°C ± 1°C
Identification of strains: Pse υdomonas aeruginosa ATCC 15442
Staphylococcus aureus ATCC 6538
The results are shown in Table 3
Conclusion
According to EN 1040 (1997), Formulation I possesses bactericidal activity
for the referenced strains Pseudomonas aeruginosa ATCC 15442 and
Staphylococcus aureus ATCC 6538 at the working concentration (20%
WV as tested). In order to qualify the product as an antiseptic and/or
chemical disinfectant for a defined purpose, it will be evaluated using
additional standard tests which are appropriate to its intended use.
Table 3
Viable1 Count
Test Organism
Bacterial
Bacterial
Neutralizer
Dilution-
Test
Suspension
toxicity
neutralization
Suspension
Nv (see
control (Nx)
control (Ny)
(See 5.4.1 .4)*
A.2)*
(see
(see A.4.1.2)*
A.4.1.2)*
(N)
Pseudomonas
3.3 x 108
1. 1 x 10 3
9.0 x 101
8.9 x 10 1
4.0 x 108
1.7 x 10 3
8.9 x 10 1
1.3 x 10 2
Aeruginosa
ATCC 15442
Staphylococcus
aureus
ATCC 6538
For the two strains tested:
N is between 1.5 x 10 8 and 5.0 x 10 8 cfu/ml;
Nv is between 6.0 )< 102 and 3 x 10 cfu/ml;
Nx is equal or greater than 0.05 Nv
Ny is equal or greater than 0.05 Nv
The neutralization is validated with Ithe neutralizer tested for the test
concentrations of 80% V/V of product as received and for the two Jstrains tested.
* The
references above refer to British standard EN 1040-1997
Experiment 4 - Test Report under BS EN 1275-1997
Chemical disinfectants and antiseptics - Basic fungicidal activity (phase 1)
Test Method and its validation
The method used is filtration-neutralization. Neutralizer - Lecithin 3g/l,
Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l, phosphate
buffer 0.0025 mol/l, sterilized by autoclave.
Experimental conditions
Product diluent used: sterile synthetic hard water.
Product test concentrations: 20% WV; 50% WV 80%WV
Appearance product dilutions: clear
Contact time: t = 5 min ± 10 s
Test temperature: 20°C ± 1°C
Interfering substance: 0.3 g/l bovine albumin
Stability of mixture: No precipitation
Temperature of incubation: 37°C ± 1°C
Identification of strains:
Candida albicans ATCC 10231
Aspergillus niger ATCC 16404
The results are shown in Table 4 .
Conclusion
According to EN 1275, Formulation I possesses fungicidal activity for the
referenced strains Candida albicans ATCC 10231 and Aspergillus niger
ATCC 16404 at the working concentration (20% WV as tested). In order
to qualify the product as an antiseptic and/or chemical disinfectant for a
defined purpose, it will be evaluated using additional standard tests which
are appropriate to its intended use.
Table 4
*The references above refer to British standard EN 1275-1997
Experiment 5 - Test Report under BS EN 1276-1997
Chemical disinfectants and antiseptics - Quantitative suspension test for
the evaluation of bactericidal activity of chemical disinfectants and
antiseptics used in food, industrial, domestic and institutional areas (phase
2 , step 1)
Test Method and its validation
The method used is filtration-neutralization.
Neutralizer - Lecithin 3g/l,
Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l, phosphate
buffer 0.0025 mol/l, sterilized by autoclave.
Experimental conditions
Product diluent used: sterile synthetic hard water.
Product test concentrations: 20% W/V; 50% VWV 80%WV
Appearance product dilutions: clear
Contact time: t = 5 min ± 10 s
Test temperature: 2 O0C ± 10C
Interfering substance: 0.3 g/l bovine albumin
Stability of mixture: No precipitation
Temperature of incubation: 37°C ± 1°C
Identification of strains:
Pseudomonas aeruginosa ATCC 15442
Escherichia coli ATCC 10536
Staphylococcus aureus ATCC 6538
Enterococcus hirae ATCC 8043
The results are shown in Table 5
Conclusion
According to testing carried out under conditions specified in EN 1276,
Formulation I possesses bactericidal activity at a concentration of 20.00%
V/V after five minutes at 20°C under clean conditions (0.3 g/L bovine
serum albumin) for referenced strains Pseudomonas aeruginosa ATCC
15442, Escherichia coli ATCC 10536, Staphylococcus aureus ATCC 6538
and Enterococcus hirae ATCC 8043.
Table 5
CO
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Experiment 6 - Test Report under BS EN 1650 -1998
Chemical disinfectants and antiseptics - Quantitative suspension test for
the evaluation of fungicidal activity of chemical disinfectants and
antiseptics used in food, industrial, domestic and institutional areas (phase
2 , step 1)
Test Method and its validation
The method used is filtration-neutralization.
Neutralizer - Lecithin 3g/l,
Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l, phosphate
buffer 0.0025 mol/l, sterilized by autoclave.
Experimental conditions
Product diluent used: sterile synthetic hard water.
Product test concentrations: 20% v/v; 50% v/v 80%v/v
Appearance product dilutions: clear
Contact time: t = 5 min ± 10 s
Test temperature: 20°C ± 1°C
Interfering substance: 0.3 g/l bovine albumin
Stability of mixture: No precipitation
Temperature of incubation: 37°C ± 1°C
Identification of strains:
Candida albuminv ATCC 10231 .
Aspergillus niger ATCC 16404
The results are shown in Table 6
Conclusion
According to EN 1650, Formulation I possesses fungicidal activity for the
referenced strains Candida albicans ATCC 10231 and Aspergilus niger
ATCC 16404 at the working concentration (20% WV as tested).
Table 6
Experiment 7 - Test Report under BS EN 13704:2002
Chemical disinfectants - Quantitative suspension test for the evaluation of
sporicidal activity of chemical disinfectants and antiseptics used in food,
industrial, domestic and institutional areas (phase 2 , step 1)
This test measures the efficacy of the biocidal agent against bacterial
endospores. These are generally considered to be the most resistant
microbial structures to biocides. Passing this test enables the biocide to be
considered to be a "high level" disinfectant. Eight parts biocide are
contacted with 1 part interfering substance and 1 part spore suspension of
Bacillus subtilis spores at a defined concentration. The contact time is 60
minutes at 20 eC.
Test Method and its validation
The method used is filtration-neutralization. Neutralizer - Lecithin 3g/l,
Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l, phosphate
buffer 0.0025 mol/l, sterilized by autoclave.
Experimental conditions
Product diluent used: sterile synthetic hard water.
Product test concentrations: 20% v/v; 50% v/v; 80%v/v
Appearance product dilutions: clear
Contact time: t = 5 min ± 10 s
Test temperature: 20°C ± 1°C
Interfering substance: 0.3 g/l bovine albumin
Stability of mixture: No precipitation
Temperature of incubation: 37°C ± 1°C
Identification of strains: Bacillus subtilis ATCC 6633.
The results are shown in Table 7
Conclusion
According to EN 13704, Formulation I possesses sporicidal activity for the
referenced strains Bacillus subtilis ATCC 6633 at the concentration (20%
VA/ as tested).
Table 7
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Experiment 8 - Test Report Standard Test method for Efficacy of
Antimicrobial Agents against Viruses in Suspension
Bovine Viral Diarrhoea Virus (Hepatitis C Virus surrogate)
Test Method and its validation
The method used is filtration-neutralization. Neutralizer - Dulbecco's
modified Eagles medium + 5% v/v Foetal bovine serum.
Experimental conditions
Product diluent used: sterile hard water.
Product test concentrations: 2% V/V; 10% V/V; 80%V/V
Appearance product dilutions: colourless, clear product solution
Contact time: t = 5 min ± 10 s
Test temperature: 2 O0C ± 1°C
Interfering substance: 0.6 g/l foetal bovine serum
Stability of mixture: Precipitate absent throughout the test
Temperature of incubation: 37°C ± 1°C
Identification of virus: Bovine Viral Diarrhoea Virus 1 (ATCC VR1422). BT cells.
The results are shown in Table 8 .
Conclusion
Formulation I1 when diluted at 2.00% (V/V) in hard water, possesses
virucidal activity in five minutes at 20°C under clean conditions (06 g/l
protein as foetal bovine serum) for Bovine viral diarrhoea virus-
(Hepatitis C virus surrogate)
Table 8
Suspension test results for the efficacy of Formulation I
disinfectant against Bovine Viral Diarrhoea
*
This result is due to residual cytotoxicity of the disinfectant at this
concentration of disinfectant.
10
Comments:
Virucidal activity is based on a reduction in virus viability of a
minimum of 4 LOGi 0. This therefore represents a minimum value.
Stock BVDV: 1.48 x 108
15
Residual efficacy control: minimum positive cpe for BVDV at 10 4
dilution of Formulation 1 disinfectant after neutralisation.
Experiment 9 - Test Report Standard Test method for Efficacy of
Antimicrobial Agents against Viruses in Suspension
Feline Calicivirus (Human Norovirus surrogate)
Test Method and its validation
The method used is dilution-neutralization. Neutralizer - Dulbecco's
modified Eagles medium + 5% v/v Foetal bovine serum.
Experimental conditions
Product diluent used: sterile hard water.
Product test concentrations: 2% V/V; 10% V/V; 80%V/V
Appearance product dilutions: colourless, clear product solution
Contact time: t = 5 min ± 10 s
Test temperature: 20°C ± 1°C
Interfering substance: 0.6 g/l foetal bovine serum
Stability of mixture: Precipitate absent throughout the test
Temperature of incubation: 37°C ± 1°C
Identification of virus: Feline calicivirus (FCV)/FEA cells (human
norovirus surrogate).
The results are shown in Table 9 .
Conclusion
Formulation
I,
when diluted at 2.00% (V/V) in hard water, possesses
virucidal activity in five minutes at 20°C under clean conditions (0,6
g/l protein as foetal bovine serum) for feline calicivirus (human
norovirus surrogate)
Table 9
Suspension test results for the efficacy of Formulation 1
Disinfectant against Feline Calicivirus
*
This result is due to residual cytotoxicity of the disinfectant at this
concentration of disinfectant.
10
Comments:
Activity based on a reduction in virus viability of a minimum of 4
LOG 10-
This therefore represents a minimum value.
15
Stock FCV: 2.14 x 10 9.
Residual efficacy control: minimum positive cytopathic effect (cpe) for
FCV at 10-4 dilution of Formulation I disinfectant after neutralisation.
Experiment 10 - Vancomycin Resistant Enterococcus under BS EN
0
1276-1 997
Chemical disinfectants and antiseptics - Quantitative suspension test
for the evaluation of bactericidal activity of chemical disinfectants and
antiseptics used in food, industrial, domestic and institutional areas
(phase 2 , step 1)
Test Method and its validation
The method used is filtration-neutralization.
Neutralizer - Lecithin
3g/l, Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l,
phosphate buffer 0.0025 mol/l, sterilized by autoclave.
Experimental conditions
Product diluent used - sterile synthetic hard water.
Product test concentrations: 5% VWV; 10% VWV; 80% WV
Appearance product dilutions: clear
Contact time: t = 5 min ± 10 s
Test temperature: 20°C ± 10C
Interfering substance: 0.3 g/l bovine albumin
Stability of mixture: No precipitation
Temperature of incubation: 37°C ± 1°C
Identification of strains: Vancomycin Resistant Enterococcus NCTC
12201
Conclusion
According to testing carried out under conditions specified in EN
1276-1997, Formulation I possesses bactericidal activity at a
concentration of 5.00% WV after five minutes at 20°C under clean
conditions (0.3 g/L bovine serum albumin) for referenced strains
Vancomycin Resistant Enterococcus NCTC 12201 .
Table 10
CO
Experiment 11 - Methicicillin Resistant Staphylococcus Aureus 15 and 16
under BSEN 1276 (1997)
Test Method and its validation
The method used is filtration-neutralization.
Neturalizer: Lecithin 3g/l,
Polysorbate 80 30g/l, sodium thiosulphate 5g/l, L-histidine 1g/l, phosphate
buffer 0.0025mol/l, sterilized by autoclave
Experimental conditions
Product diluent used: sterile synthetic hard water.
Product test concentrations: 10% VWV; 20% VWV; 80%V/V
Appearance product dilutions: clear
Contact time: t = 5 min ± 10 s
Test temperature: 20°C ± 1°C
Interfering substance: 0.3 g/l bovine albumin
Stability of mixture: No Precipitation
Temperature of incubation: 37°C ± 1°C
Identification of MRSA Strain:
EMRSA 15:CC22 (SMRL No. 00.9521 .M)
EMRSA 16 : CC30 (SMRL No. 00.9523. R)
The results are shown in Table 11.
Although each country have their own names for MRSA, clones MLST
Typing data which groups MRSA into Clonal Complexes (CC) made up of
related Sequence Types (ST) is the widely adopted international
nomenclature.
The majority of MRSA infections worldwide are caused by
MRSA clones which fall within five Clonal Complexes. A representative of
each of these Clonal Complexes was used in this test. EMRSA 15 is the
dominant MRSA in the UK, followed by EMRSA 16. In Scotland these two
clones account for 72% and 20%, respectively, of all MRSA referred to the
Scottish MRSA reference laboratory. EMRSA-16 and other clones with
Clonal Complex 30 are found worldwide. They are the second
commonest MRSA in the USA (9%) and are a common cause of
community acquired MRSA both the USA and Australia.
According to testing carried out under condition specified in EN 1276
(1997) Formulation I possesses bactericidal activity at a concentration of
10.00% V/V after 5 minutes at 20 0C under clean conditions (0,3 g/L bovine
serum albumin) for referenced strains EMRSA 15:CC22 (SMRL No.
00.9521 .M), EMRSA 16:CC30 (SMRL No. 00.9523.R).
Table 1 1
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Example 2 - Efficacy of Test Formulation 2 against Acinetobacter
Baumannii under clean and dirty conditions
Test Formulation 2 (by wt %) is as follows:
BAC 50
27%
Surfactant UN90
11%
Metasilicate Pentahydrate 4.5%
NTA
4.5%
The test suspension is prepared and an organic load (Bovine Serum
Albumin) or interfering substance is added to the suspension at different
concentrations to simulate both clean and dirty conditions. This is
performed to investigate how well the agent will perform under both
conditions.
Test Organisms A cinetobacter baumannii ATCC 19606
Media and Reagents
Minimal Salts Medium - MSM
Buffered Peptone Water containing 5% Polysorbate 80-BUP5
Buffered peptone water -BUP
Tryptone soya broth - TSB
Tryptone soya agar - TSA
Sterile 0.45 µm syringe filters
Bovine Serum Albumin
Purified Water
Formulation 2 was made up fresh on day of testing and diluted 10OmIs
concentrated solution and 1OmIs tap water.
Preparation of Inoculum
Acinetobacter baumannii was sub cultured from a stock culture into 10ml
TSB and incubated at 36°C ± 1°C for 18-24 hours. After incubation, the
culture was centrifuged at 4500rpm for 3 minutes, the supernatant
discarded and the pellet resuspended in MSM.
Preparation of Bovine Serum Albumin (Interfering Substance)
Two solutions of BSA were prepared to simulate the clean and dirty
conditions. For clean conditions, dissolve 0.3g BSA into 100ml purified
water, and filter sterilized using a 0.45 µm syringe filter. This gives a final
concentration of BSA in the test procedure of 0.3g/litre. For dirty
conditions dissolve 3g BSA into 100ml purified water, and filter sterilized
using a 0.45 µm syringe filter. This gives a final concentration of BSA in
the test procedure of 3g/litre.
Procedure
Suspension Test in the Presence of Bovine Serum Albumin (BSA)
Dispense 9ml of the diluted formulation 2 into a sterile vial, and allow to
equilibrate in a water bat set at 20 0C ± 10C. Add 1ml of the filtered BSA
for clean conditions. Introduce 0.1 ml of the inoculum into the test
suspension and mix thoroughly. Withdraw 1ml aliquots at 5 , 15, 30 and 60
minutes and transfer to 200ml volume of BUP5 to give a 1/200 dilution.
Maintain at 20 0C ± 10C for 5 minutes. After 5 minutes, remove a 1ml
aliquot from the 1/200 dilution, and use to prepare 4 further tenfold
dilutions in 9ml volumes of BUP5. 1ml aliquots from each dilution are
dispensed into Petri dishes and 15-2OmI TSA added. All plates were
incubated at 36°C ± 10C
Control Count
To determine the number of viable organisms inoculated into the test
material, a control count was carried out for the test organism. A 10ml
volume of BUP was challenged with 0.1ml of the inoculum and 1ml was
transferred to a 200ml volume of BUP. Maintain at 20 0C ± 10C for 5
minutes. After 5 minutes, remove a 1ml aliquot from the 1/200 dilution and
use to prepare 4 further tenfold dilutions in 9ml volumes of BUP. Remove
1ml from each dilution and use to prepare pour plates with TSA.
Validation
Validation is performed to ensure adequate neutralization of the active
occurs at the first product dilution.
The inoculum is diluted in MSM to give a count of 1000-10000 cfu ml 1,
allow to equilibrate in a water bat set at 20 0C ± 10C. 2mls of the
equilibrated inoculum is added, to give approximately 10-100 organisms
per ml, to 200ml volume of BUP5 plus 1ml of the interfering substance for
clean conditions and mix thoroughly. 1ml of the diluted cleaning solution
is added to the inoculated diluent and the resulting solution is shaken to
mix. The temperature is maintained at 20 0C ± 10C and 1ml is removed
after 5 minutes and again after 30 minutes and used to prepare pour
plates using TSA. The procedure is repeated with a 200 ml volume of
both BUP and BUP5 without product for the validation control counts.
The above test and validation method was repeated using the interfering
substance for dirty conditions. Each run was then repeated a further two
times for each condition.
All plates were incubated at 36°± 1°C for 48 hours and the number of
colony forming units were counted.
The above test and validation method was repeated using the interfering
substance for dirty conditions. Each run was then repeated a further two
times for each condition.
Results are shown in Tables A and B. The following acceptance criterion
was a log reduction of 5 log.
Conclusion
From the results it can be seen that for the cleaning agent good biocidal
activity was seen within 5 minutes against Acinetobacter baumanni or
both clean and dirty conditions, meeting the acceptance criteria as
recommended in the British and European Standards BS EN 1276:1997.
Table A - Results for Cleaning Solution Testing - Clean Conditions
Table B - Results for Cleaning Solution Testing - Dirty Conditions
Example 3 - Comparative test of the combined and individual effects
of the components of Formulation I
Table 12 shows the efficacy of thirteen solutions of a varied mixture of
each of the chemicals used in the composition against E.Coli and
Poliovirus. The aim of this experiment was to obtain a better
understanding of the contribution of each of the individual raw materials to
the composition and to investigate the possibility of a synergetic effect.
Table 12 shows the results of this experiment.
Table 12
- No Biocidal activity
+ Intermediate activity
++ Biocidai activity
The following conclusions can be taken from Table 11.
BAC 50
No effect
BAC 50 + Surfactant
Bactericidal Effect
Metas (i.e. sodium metasilicate pentahydrate) Viral Effect
NTA
No Effect
NTA + BAC 50
Intermediate viral activity
Results for sample 2 shows Metas to have a high level of viral activity.
Results for the surfactant alone (sample 3) showed a bactericidal effect.
Surprisingly, with no Metas present, (sample 7), the formulation has an
intermediate viral activity with NTA + BAC 50.
As expected sample 10 showed BAC 50 + Surfactant to have a
bactericidal effect.
The combination of ingredients exhibits an unexpected efficacy against
bacteria, viruses and spores. Even though certain individual components
contained in the composition of this invention are capable of bactericidal
action by themselves, the combination of these ingredients, results in a
synergetic effect which is active against spores and viruses as well.
The particular advantage of compositions according to the invention such
as Formulation I (Sample 13) lies in the fact that as it is water based,
biodegradable, non corrosive and safe to work with in any environment,
having no adverse human impact, unlike a lot of competitor products
which being alcohol based are flammable. At worst they are only a minor
irritant to the skin. More importantly whilst being biodegradable and water
based they do show substantial biocidal activity over a broad specificity as
evidenced under the tests outlined in the examples above.
Claims
1.
A biocidal composition comprising a chelating agent, metasilicate and
at least one anti-bacterial biocide.
2.
The composition of claim 1 wherein said metasilicate is a disodium
trioxosilicate.
3.
The composition of claim 2 wherein said disodium trioxosilicate is
disodium trioxosilicate pentahydrate.
4.
The composition of any one of claims 1 to 3 , wherein the chelating
agent is chosen from nitrilotriacetic acid (NTA) or its derivatives.
5.
The composition of any one of claims 1 to 3 , wherein the chelating
agent is chosen from the group consisting of ethylene glycol
tetraacetic acid, diethylene triamine pentaacetic acid and their salts
and esters thereof.
6.
The composition as claimed in any one of the preceding claims
wherein the concentration of said metasilicate ranges from 3 to 6 wt%.
7.
The aqueous composition as claimed in any one of claims 1 to 5,
wherein the concentration of said biocide agent ranges from 5 to 35
wt%.
8.
The composition as claimed in any one of the preceding claims
wherein the concentration of said chelating agent ranges from 4 to 8
wt%.
9.
The composition as claimed in any one of the preceding claims
wherein said biocide is chosen in the group consisting of quaternary
ammonium compounds and alkyl dimethyl betaines and mixtures
thereof.
10. The composition as claimed in claim 9 , wherein said biocide is chosen
from the group consisting of quaternary ammonium compounds.
11. The composition as claimed in any one of the preceding claims
wherein said composition further comprises a surfactant.
12. The composition as claimed in claim 11, wherein the concentration of
said surfactant in the composition is less than 10 wt%.
13. The composition as claimed in claim 12, wherein said surfactant is
chosen from the group consisting of Synperonic 9 1 10, Surfactant
UN90, Synperonic LF/RA 280 and Synperonic LF/RA 30.
14. The composition as claimed in any one of claims 11 to 13, wherein
said surfactant is Synperonic UN90/90.
15. The composition as claimed in any one of the preceding claims,
wherein said composition is an aqueous composition.
16. Use of the composition as claimed in any one of the preceding claims
as a biocidal composition.
17. Method of disinfecting which comprises the step of applying a
composition as claimed in any one of claims 1 to 15 to a surface
and/or a body part to disinfect.
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