Note: Descriptions are shown in the official language in which they were submitted.
~~ 65945
WO 95/00613 PCT/EP94/02002
Cleaning and disinfecting formulations
FIELD OF THE INVENTION
This invention relates to a cleaning and disinfect-
ing formulation for disinfecting floors and walls,
especially in hospitals, or for the antiseptic treatment
of healthy skin.
BACKGROUND OF THE INVENTION
Nosocomial infections represent a major public
health problem on account of their frequency, their
potential seriousness and their economic and social
consequences. They can be controlled by the application
of efficient measures, for example by observing strict
hygiene and disinfection procedures in various places,
for example in the treatment of floors, walls and sur-
faces of all kinds and in the treatment of surgical
instruments and equipment.
The French Standards Committee AFNOR defines disin-
fection as a process with a short-term outcome which
enables microorganisms to be eliminated or destroyed
and/or unwanted viruses present in contaminated neutral
environments to be inactivated.
At the present time, the active substances most
commonly used for disinfection in hospitals are alde-
hydes, quaternary ammonium compounds and phenols. The
aldehydes typically used for disinfection are formalde-
hyde and glutaraldehyde. Although they have a particu-
larly broad action spectrum, they are toxic both to users
and to patients.
The inhalation of formaldehyde by human beings has
a lachrymatory effect at concentrations above 0.1 ppm and
causes irritation of the upper respiral=ory tracts with
inflammation of the nasal, throat and :Larynx mucosa at
concentrations of 1 to 5 ppm and lung oedema at concen-
trations of 50 ppm. In hospitals, the use of relatively
large quantities of formaldehyde in anatomy/pathology
~t.
..- .R.
CA 02165945 2004-07-22
2
laboratories and in haemodialysis units has caused acute
poisoning. In addition, irritation of the eyes can occur
in certain people exposed to formaldehyde in a concentra-
tion above 0.4 ppm. In the event of local exposure of
the mucosa, contact with formaldehyde can cause irrita-
tion, changes in the cornea or eczema-like lesions..
After prolonged inhalation, glutaraldehyde can cause
irritation of the respiratory organs in conjunction w:Lth
coughing attacks in human beings. Where it acts locall;r
on the mucosa, glutaraldehyde can cause irritation,
erythema or changes in the cornea.
By virtue of their bactericidal and cleaning proper-
ties, quaternary ammonium compounds are often used as
disinfectants in hospitals. In human beings, however,,
numerous cases of skin disease with contact allergies
have been observed. Users who had used products based on
quaternary ammonium either repeatedly or over prolonged
periods showed oversensitivity reactions. The inhalation
of mists or aerosols containing quaternary ammonium salt;
can result in lung oedema. Accordingly, there is a need
to find a non-toxic cleaning and disinfecting formulation
which could be used in hospitals instead of aldehydes and
quaternary ammonium compounds.
The problem addressed by the present invention is to
provide a new disinfecting and cleaning formulation which
will not have any toxic effects on the user and which can
readily be used for disinfecting surfaces.
THE SUMMARY OF THE INVENTION
The present invention relates to a cleaning and
disinfecting formulation containing a tertiary alkylamine
and an amine oxide in a ratio by weight of 0.2:1 to 2:1.
According to the invention, the active substance is
an alkylamine with a bactericidal, fungicidal and~viruci
dal effect. This substance is inhibited to only a very
slight extent by the presence of interference substances.
In addition, it is completely safe both in toxicological
CA 02165945 2004-07-22
3
terms and in ecological terms. It has been found to b~e
of advantage to combine a synergistic quantity of an
amine oxide with the above-mentioned alkylamine in order
to obtain a significant increase in the antimicrobial
effect in conjunction with a simultaneous cleaning
effect. In one preferred embodiment of the invention
designed to achieve maximum activity, the tertiary
alkyl amine makes up from 2 to 15% by weight and preferab~-
ly from 2 to 9% by weight of the composition as a whole.
The tertiary alkyl amine is preferably a compound corre-
sponding to the following general formula:
/ (CH2)s-NH2
R~-N\
(CH2)s-NH2
in which R1 is an alkyl radical containing 8 to 16 and -
preferably 10 to 14 carbon atoms. The amine oxide i:~
preferably a compound corresponding to the following
general formula:
Hs
R2-N.__-~O
CH3
in which R2 is an alkyl radical containing 8 to 16 and
preferably 10 to 14 carbon atoms.
It has been found that the best results can be
obtained when the radicals R1 and RZ are identical and, in
particular, when Rl or RZ is a lauryl radical. In this
case, the alkylamine is diaminopropyl laurylamine while
the amine oxide is dimethyl laurylamine oxide.
In addition, the composition according to the
invention may contain a low-foaming nonionic surfactant
CA 02165945 2004-07-22
4
in a quantity of 2 to 20o by weight and preferably 2 to
13% by weight, based on the composition as a whole, and
a fragrance in a quantity of 0 . 005 to 1 o by weight anct
preferably 0.05 to 0.6% by weight, based on the compo:~i-
tion as a whole. The nonionic surfactant may be an
aliphatic polyalkoxyl derivative, for example a fatty
alcohol ethylene oxide propylene oxide. It is preferably
used in a ratio by weight of 0.2:1 to 4:1 to the alkylamine
or the amine oxide.
15
DETAILED DESCRIPTION OF THE INVENTION
The present invention may best be described in ter_m:~
of the following preferred composition according to r_he
invention which was found to show high activity:
Diaminopropyl laurylamine 5.40
Dimethyl laurylamine oxide 6%
Polyalkoxyl derivative 8%
Fragrance 0.1%
Aqueous auxiliaries qsf 100
The composition mentioned above is prepared in a
TM
mixer into which purified water (Aqua purificata) , the
dimethyl laurylamine oxide, the diaminopropyl laurylamine
and the polyalkoxyl derivative are introduced while
shaking in the order shown.
For the purposes of end use, the composition is
diluted with water in a ratio of 20 ml of solution to 8
liters of water which corresponds to an in-use concentra-
tion of 0.25%. The product according to the invention is
a clear, amber-colored solution with a pH value of 1.1.2
~ 0.5. It may be packed, for example, either in 20 m.l
bags or in 5 liter canisters. By virtue of its anti~sep-
tic effect and its non-toxicity according to the AFNOR
Standards, which will be discussed hereinafter, the
composition according to the invention may be used fc>r
the antiseptic treatment of healthy skin.
The antimicrobial effect of the preferred composi-
CA 02165945 2004-07-22
tion according to the invention (above-cited example) was
demonstrated in accordance with the AFNOR Standards. The
results attached as appendixes relate to the variouas
tests carried out by the membrane filter method. These
5 tests involved the following determinations:
- determination of the bactericidal effect in accord-
ance with AFNOR NF T 72-151 (Appendix 1),
- determination of the bactericidal effect in the
presence of an interference substance (hard water)
in accordance with AfNOR NF T 72-171 (Appendix 2),
- determination of the bactericidal effect in the
presence of an interference substance (albumin) in
accordance with AfNOR NF T 72-171 (Appendix 3),
- determination of the fungicidal effect in accordance
with AfNOR NF T 72-201 (Appendix 4).
The bactericidal effect of the formulation was also
TM
tested against Mycobacterium Tuberculosis by the Bactec
TM
TB method. The Bactec TB Medium (12B) is a nutrient
TM
medium based on enriched Middelbrook 7H9. The mycobac-
teria use a carbon-14-labeled fatty acid present in the
medium and give off COZ to the air.
If mycobacterial growth is present in the 12B
TM
bottles, the Bactec 460 measures the radioactivity caused
by the 14 C02 released and displays it numerically on a
scale of 0 to 999. These values are known as the growth
index (GI). The increase in the GI is proportional to
the bacterial count. After the Mycobacterium Tuber-
culosis suspension has been exposed to the disinfectant,
the mycobacteria remaining are counted by injecting each
diluted germ suspension into two 12B bottles for several
dilutions. The increase in the GI corresponds to the
presence of mycobacteria.
TM
The method in question comprises using a McFarland
3 suspension in sterile distilled water which is prepared
from a pure culture of Mycobacterium Tuberculosis taken
CA 02165945 2004-07-22
6
TM
from a Lowenstein nutrient medium. Dilutions are pre-
pared from this "mother" solution progressively by a
factor of 10 up to a factor 109. 100 ~1 of each dilution
are inoculated into a 12B medium containing 3 ml of
culture medium (dilution 1/40 in each 12B bottle). 200
~.l of pure or dilute disinfectant are then contacted with
TM
200 ~,1 of Mycobacterium Tuberculosis with McFarland 3
over a certain period in a shaken 35 ml centrifuge tube.
After the contact time, the tube is filled with sterile
distilled water and then centrifuged for 30 minutes. at
3000 r.p.m.. The supernatant phase is siphoned off and
the residue is taken up in 2 ml of distilled water (dilu-
tion 1/10 up to 10-5). The substance is then poured into
two 12B bottles containing 4 ml of nutrient solution»
100 ~1 of the l0 1 dilution (dilution 1/40)
100 ~1 of the 10-2 dilution
100 ~tl of the 10 3 dilution
100 ~1 of the 10-4 dilution
100 ~1 of the 10-5 dilution
TM
The count and the test are read off from the Bactec
TB reader. The results expressed as the GI are set out
in the Tables appearing in Appendix 5 for the individual
contact times, being read off after double inoculation of
the 12B nutrient media at the times T1, T2, T3, T6, T7,
T8, T10, T13, T15, T16, T17 and T20. The toxicological
studies carried out with the preferred composition
according to the invention as defined above produced the
following results:
a) Acute toxicity in male mice after oral administra-
tion
- LD50 > 2.5 ml/kg
b) Index of acute eye irritation
- 9.3 to 24 hours (product with mild irritant
effect)
c) Index of primary skin irritation
2165945
WO 95/00613 7 PCT/EP94/02002
- 0.71 (product with mild irritant effect)
d) Index of primary skin irritation of the product in
the in-use concentration (dilution to 0.25%)
- 0.25 (product with no irritant effect)
CA 02165945 2004-07-22
8
E x a m p 1 a s
Appendix 1
Determination of bactericidal activity:
Standard AFNOR T 72-151, November 1987
I. Sample data:
Product designation and ,
batch number: Cleaning and disinfecting formulation
for floors and surfaces
Manufacturer: PARAGERM
Analysis period: May 1993
Active substances and
concentrations: Confidentially disclosed
II. Test conditions:
Test temperature: 20°C
Diluent used in the tests: Sterile distilled water
III. Procedure determined after the preliminary test:
Type of membranes and data: Millipore filter Type HA, pore
size 0.45
Rinsing liquid Composition
- polysorbate 3%
- lecithin 0.3%
- histidine 0.1%
- sodium thiosulfate 0.5%
Number of rinses with this liquid: 3
Volume used for each rinse: 50 ml
Neutralizing agent added to the counting medium and
concentration:
Neutralizing nutrient medium for counting
Composition:
beef extract 3 g
tryptic casein peptone 5 g
216545
WO 95/00613 9
PCT/E P94/02002
glucose 1 g
lecithin 1
g
polysorbate 7 g
agar agar in powder form 15 g
distilled water 1000 ml
Determination of product:
of the bactericidal the
activity
SPECTRUM 4 0 SPECTRUM 5 X
Results of the preliminary tests under the descr ibed
conditions
Tested Strains, Collection Colonies Counted
Concentrations and Number in the
of the Product Collection N N~
n
(V/V)
0.125% Pseudomonas aeruginosa 163 153 121
CIP A 22
0.25% Escherichia coli 125 84 8
CIP 54 127
0.25% Staphylococcus aureus 132 124 130
CIP 53 154
0.25% Enterococcus hirae 85 94 65
CIP 5 855
SPECTRUM 4
0.25% Mycobacterium smegmatis 127 75 70
CIP 7 326
SPECTRUM 5
X165945
WO 95/00613 10 PCT/EP94/02002
Results of the actual tests under the described conditions
Colonies counted
(+ = more than 150
colonies)
Concentration of
S T R A I N S N N' n the product in
50 contact with the
to bacteria
150 pH (% V/V)
Min. Max. 0.06% 0.125% 0.25%
*Pseudomonas
aeruginosa 163 151 121 6.10 6.40 2 0
*Escherichia _
Coll 125 84 80 6.10 6.40 12 1
*Staphylococcus
aureus 132 124 30 6.10 6.40 0 0 0
*Enterococcus
hirae 85 94 65 6.10 6.40 0 0 0
SPECTRUM 4
*Mycobacterium
smegmatis 127 75 50 6.10 6.40 0 0
SPECTRUM 5
Bactericidal concentration of the tested product in relation to:
Pseudomonas aeruginosa S 0.06%
Escherichia coli 5 0.06%
Staphylococcus aureus <_ 0.06%
Enterococcus hirae 5 0.06%
Mycobacterium smegmatis 5 0.125%
Based on all the strains tested, the product corresponds to
the Standard AFNOR T 72-151 in a concentration of 0.125%.
265945
WO 95/00613 11 PCT/EP94/02002
Appendix 2
Determination of bactericidal activity in the presence of
interference substances
Standard AFNOR T 72-171
I. Salmple da
Product designation and
batch number: Cleaning and disinfecting formulation
for floors and surfaces
Manufacturer: PARAGERM
Analysis period: May 1993
Active substances and
concentrations: Confidentially disclosed
II. Test conditions:
Test temperature: 20°C
Diluent used in the tests: Sterile distilled water
Interference substances: Hard water
III. Procedure determined after the preliminarv test-
Type of membranes and data: Millipore filter Type HA, pore
size 0.45 a
Rinsing liquid Composition
- polysorbate 3%
- lecithin 0.3%
- histidine 0.1%
- sodium thiosulfate 0.5%
Number of rinses with this liquid: 3
Volume used for each rinse: 50 ml
Neutralizing agent added to the counting medium and
concentration:
Neutralizing nutrient medium for counting
Composition:
beef extract 3 g
tryptic casein peptone 5 g
2165945
WO 95/00613 12 PCT/EP94/02002
glucose
1 g
lecithin 1 g
polysorbate 7 g
agar agar in powder form 15 g
distilled water 1000 ml
Determina tion of the bactericidal ivity of product:
act the
SPECTRUM 4 X SPECTRUM 5 O
Results of preliminary tests under the described
the conditions
Tested Strains, Collection Colonies Counted
Concentrationsand Number in the
of the ProductCollection N N' n
(V/V)
0.5% Pseudomonas aeruginosa 62 61 58
CIP A 22
0.25% Escherichia coli 147 126 108
CIP 54 127
0.25% Staphylococcus aureus 97 104 100
CIP 53 154
0.5% Enterococcus hirae 72 92 89
CIP 5 855
SPECTRUM 4
Mycobacterium smegmatis
CIP 7 326
SPECTRUM 5
X165945
WO 95/00613 13 PCT/EP94/02002
Results of the actual tests under the described conditions
Colonies counted
(+ = more than 150
colonies)
Concentration of
S T R A I N S N N' n the product in
50 contact with the
to bacteria
150 pH (% V/V)
Min. Max. 0.125% 0.25% 0.5%
*Pseudomonas
aeruginosa 62 61 58 6.20 8.10 + 53
*Escherichia
coli 147 126 108 6.40 6.20 0 0
*Staphylococcus
aureus 97 104 100 6.40 6.70 0 0
*Enterococcus
hirae 72 92 89 6.70 8.10 0 0 0
SPECTRUM 4
*Mycobacterium
smegmatis
SPECTRUM 5
Bactericidal concentration of the tested product in relation to:
Pseudomonas aeruginosa <_ 0.5%
Escherichia coli 5 0.125%
Staphylococcus aureus S 0.125%
Enterococcus hirae 5 0.125%
Mycobacterium smegmatis <_
Based on all the strains tested, the product corresponds to
the Standard AFNOR T 72-171 in a concentration of 0.5% in the
presence of hard water.
2165945
WO 95/00613 14 PCT/EP94/02002
appendix 3
Determination of bactericidal activity in the presence of
interference substances
Standard AFNOR T 72-171
I. Sample da
Product designation and
batch number: Cleaning and disinfecting formulation
for floors and surfaces
Manufacturer: PARAGERM
Analysis period: May 1993
Active substances and
concentrations: Confidentially disclosed
II. Test conditions:
Test temperature: 20°C
Diluent used in the tests: Sterile distilled water
Interference substances: Albumin - yeast extract
III. Procedure determined after the preliminary test~
Type of membranes and data: Millipore filter Type HA, pore
size 0.45 ~,
Rinsing liquid Composition
- polysorbate 3%
- lecithin 0.3%
- histidine 0.1%
- sodium thiosulfate 0.5%
Number of rinses with this liquid: 3
Volume used for each rinse: 50 ml
Neutralizing agent added to the counting medium and
concentration:
Neutralizing nutrient medium for counting
Composition:
beef extract 3 g
tryptic casein peptone 5 g
2165945
WO 95/00613 15 PCT/EP94/02002
glucose 1 g
lecithin 1 g
polysorbate 7 g
agar agar in powder form 15 g
distilled water 1000 ml
Determina tion of the bactericidal ivity of the product:
act
SPECTRUM 4 X SPECTRUM 5 O
Results of the preliminary tests under the descr ibed conditions
Tested Strains, Collection Colonies
Counted
Concentrations and Number in the
of the Product Collection N N' n
(V/V)
0.5% Pseudomonas aeruginosa 172 186 163
CIP A 22
0.5% Escherichia coli 90 64 68
CIP 54 127
0.5% Staphylococcus aureus 53 56 53
CIP 53 154
0.5% Enterococcus hirae 102 101 109
CIP 5 855
SPECTRUM 4
Mycobacterium smegmatis
CIP 7 326
SPECTRUM 5
2165945
'WO 95/00613 16 PCT/EP94/02002
Results of the actual tests under the described conditions
Colonies counted
(+ = more than 150
colonies)
Concentration of
S T R A I N S N N' n the product in
50 contact with the
to bacteria
150 pH (% V/V)
Min. Max. 0.25% 0.5%
*Pseudomonas
aeruginosa 172 186 163 6.70 8
*Escherichia
Coli 90 64 68 6.70 0
*Staphylococcus
aureus 53 56 53 6.70 26
*Enterococcus
hirae 102 101 109 6.70 95
SPECTRUM 4
*Mycobacterium
smegmatis
SPECTRUM 5
Bactericidal concentration of the tested product in relation to:
Pseudomonas aeruginosa <_ 0.5%
Escherichia coli _< 0.5%
Staphylococcus aureus 5 0.5%
Mycobacterium smegmatis <_
Based on all the strains tested, the product corresponds to
the Standard AFNOR T 72-171 in a concentration of 0.5%.
2165945
WO 95/00613 17 PCT/EP94/02002
Appendix 4
Determination of fungicidal activity:
Standard AFNOR T 72-201, September 1987
I. Sample data:
Product designation and
batch number: Cleaning and disinfecting formulation
for floors and surfaces, filtration
Manufacturer: PARAGERM
Analysis period: May 1993
Active substances and
concentrations: Confidentially disclosed
II. Test conditions:
Test temperature: 20°C
Diluent used in the tests: Sterile distilled water
III. Procedure determined after the preliminary test'
Type of membranes and data: Millipore filter Type HA, pore
size 0.45
Rinsing liquid Composition
- polysorbate 3%
- lecithin 0.3%
- histidine 0.1%
- sodium thiosulfate 0.5%
Number of rinses with this liquid: 3
Volume used for each rinse: 50 ml
Neutralizing agent added to the counting medium and
concentration: not applicable
2165945
WO 95/00613 18 PCT/EP9~/02002
Results of the preliminary tests under the described conditions
Tested Strains, Collection Colonies Counted
Concentrations and Number in the
of the Product Collection N N' n
(v/v)
Penicillinum verrucosum var.
cyclopium IP 1231-80 = LCP 282
Cladosporium cladosporioides
IP 1232-80 = LCP 484
Absidia corymbifera
IP 1129-75 = LCP 1942
0.5% Candida albicans
IP 1180-79 = LCP 409 =
ATCC 2091 111 108 95
Results of the actual tests under the described conditions
Colonies counted
(+ = more than 150
colonies)
Concentration of
S T R A I N S N N' n the product in
50 contact with the
to bacteria
150 pH (% V/V)
Min. Max. 0.06% 0.125%
*Penicillinum
verrucosum
*Cladosporium
cladosporioides
* Absidia
corymbifera
* Candida
albicans 111 108 95 6.10 6.40 0 0
X165945
WO 95/00613 19 PCT/EP94/02002
Fungicidal concentration of the tested product in relation to:
Penicillinum verrucosum: %
Cladosporium cladosporioides: %
Absidia corymbifera: %
Candida albicans: <_ 0.6%
Appendix 5
Strain: M. Tuberculosis tested without disinfectant
From a McFarland 3 suspension of the strain, 100 ~1 of each
dilution (10-1 to 10-5) are inoculated into a 12B bottle
Tl T2 T3 T6 T7 T8 T10 T13 T15 T16 T17 T21
12th 13th 14th 17th 18th 19th 21st 24th 26th 27th 28th 1st
May May May May May May May May May May May July
1 14 64 109 999 Pur. - - - - - - -
2 1 8 14 175 204 314 999 Pur. - - - -
3 0 0 0 4 8 19 148 584 750 810 890 999
4 0 0 0 0 0 1 18 84 154 170 261 595
0 0 0 0 0 0 0 1 4 4 8 29
Pur. - Purified
Disinfectant for floors and surfaces diluted to 0 25% tested
at 50% with M. Tuberculosis
Contact time : 5 minutes with shakinct AK
T1 T2 T3 T6 T7 T8 T10 T13 T15 T16 T17 T21
12th 13th 14th 17th 18th 19th 21st 24th 26th 27th 28th 1st
May May May May May May May May May May May July
1 0 0 0 68 88 124 398 999 Pur. - - -
2 0 0 0 0 4 4 29 120 188 250 298 815
3 4 0 0 0 0 0 8 50 135 175 324 885
4 0 0 0 0 0 0 0 4 4 4 5 18
5 0 0 0 0 0 0 0 0 0 0 0 0
Pur. - Purified
2165945
'O 95/00613 20 PCT/EP94/02002
The M. Tuberculosis strain tested without disinfectant grew to 10-5
after culture for 15 days.
In the presence of the 0.25% disinfectant, growth is observed at 10-4
after culture for 13 days.
No growth for the first few days with the 10-1 dilution.
Disinfectant for floors and surfaces diluted to 0.550 tested
at 50% with M. Tuberculosis
Contact time 5 minutes with ~~hakina AM
T1 T2 T3 T6 T7 T8 T10 T13 T15 T16 T17 T21
12th 13th 14th 17th 18th 19th 21st 24th 26th 27th 28th 1st
May May May May May May May May May May May July
- 0 0 0 74 94 135 474 999 Pur. - - -
1
2 0 0 0 8 10 14 54 288 398 540 621 999
3 0 0 0 0 0 0 1 18 24 28 34 84
4 0 0 0 0 0 0 0 11 18 21 24 68
0 0 0 0 0 0 0 0 0 0 0 0
~ ~
Pur. - Purified
The development is similar to that indicated i.n the previous Table.
It should be understood that the detailed description and
specific examples which indicate the presently preferred embodiments
of the invention are given by way of illustration only since various
changes and modifications within the spirit and scope of the
appended claims will become apparent to those of ordinary skill in
the art upon review of the above description.
