Claims:CLAIMS

We claim:
1. An insecticide incorporated fabric comprising of:

a) a pre-defined amount of active ingredient(s) and optionally a synergist converted into plurality of master batches in form of granules by using a polymer base matrix along with a pre-defined amount of adsorbent; and

b) a pre-determined amount of polyethylene and a coloring pigment mixed with said granules and extruded in forms of yarns;
wherein,
said active ingredient(s) with or without a synergist are incorporated into plurality of filaments during extrusion that yield in an insecticide incorporated fabric;
said adsorbent mixes uniformly with said polymer base matrix and active ingredient(s) with or without a synergist at a temperature below 250°C;
said insecticide incorporated fabric maintains bio-efficacy after more than 20 washes, thereby ensuring elongated wash durability and shelf life;
said insecticide incorporated fabric prevents insect bites thereby reducing the transmission of diseases;
said adsorbent is nano-particles of silica of a predefined size ranging from 10 to 100 nm with a particle surface area ranging between 50 to 1000 m2/g to impart a balance between release and retention of said active ingredient(s) and synergist; and
said adsorbent is added in an amount ranging from 0.1% to 1.5% based on said active ingredient(s) and synergist (if present) loading and their chemical nature.

2. The insecticide incorporated fabric as claimed in claim 1, wherein said shelf life is from 60 to 66 months.

3. The insecticide incorporated fabric as claimed in claim 1, wherein said adsorbent prevents bleeding or blooming of active ingredient(s) with or without a synergist at surface of said filament after washing.

4. A method for manufacturing insecticide incorporated fabric, comprising steps of:

a) converting an active ingredient(s) and optionally a synergist into plurality of master batches in form of granules by using a polymer as a base matrix along with an adsorbent;

b) preparing a mixture by mixing said granules obtained in step a) with polyethylene and a coloring pigment; and

c) extruding said mixture obtained in step b) in form of yarns and incorporating said active ingredient(s) with or without a synergist into plurality of filaments that provide insecticide incorporated fabric;
wherein,
said filaments includes but not limited to monofilaments, multi-filaments;
said active ingredient(s) with or without a synergist are infused or incorporated into said yarns or filaments or by combining two or more incorporated said filaments or yarns using a process of extrusion in order to make said insecticide incorporated fabric having a wash durability beyond 20 washes with a balanced release and retention properties and a prolonged shelf-life ranging from 60 to 66 months;
said adsorbent is nano particles of silica of a predefined size ranging from 10 to 100 nm with a particle surface area ranging between 50 to 1000 m2/g to imparts said balanced release and retention of said active ingredient(s); and
said adsorbent has adsorption and desorption abilities for each active ingredient(s) and synergist to controls release of each active ingredient(s) and synergist and prevent bleeding or blooming of active ingredient(s) and synergist (if present) at surface of said filament after washing.
5. The method as claimed in claim 4, wherein said polymer is preferably high density polyethylene (HDPE).

6. The method as claimed in claim 4, wherein said polyethylene is selected from including but not limited to high density polyethylene (HDPE), linear low-density polyethylene (LLDPE), low-density polyethylene (LDPE) and polypropylene.

7. The method as claimed in claim 4, wherein concentration of said active ingredient(s) with or without a synergist in said master batch depends on the nature of active ingredient(s) and synergist, thermal stability to withstand extrusion temperature, and the target concentration of said active ingredient(s) with or without a synergist in said fabric.

8. The method as claimed in claim 4, wherein said insecticidal fabric is designed to have at least one active ingredient, more than one active ingredient(s) or active ingredient(s) and a synergist.

9. The method as claimed in claim 4, wherein said active ingredient(s) and synergist includes but is not limited to alpha-cypermethrin and piperonyl butoxide (PBO), respectively.

10. The method as claimed in claim 4, wherein said adsorbent is added in an amount ranging from 0.1% to 1.5% based on said active ingredient(s) and synergist.
, Description:FIELD OF THE INVENTION
The present invention relates to an insecticide incorporated fabric and method of manufacturing thereof. More particularly, the present invention relates to an insecticide incorporated fabric with an improved polymer composition that maintains a balance between release and retention to control the release of insecticides with or without a synergist incorporated into the fabric and method of manufacturing thereof.

BACKGROUND OF THE INVENTION
Vector borne diseases such as malaria, dengue, chikungunya fever and alike are generally caused by mosquitoes. But the mosquitoes are not the causative for the diseases as they transmit the diseases from an infected human being to several healthy human beings when they bite to take a blood meal. For protection and cure from these diseases numerous vaccines are available which work by targeting the causative agent but as a prevention several methods are adopted to reduce the transmission of theses disease from an infected person to a healthy person such as insect repellents, nets, insecticides and alike.
According to research and surveys, among all the methods, insecticide treated mosquito net was observed as an extremely effective method for reducing mosquito bites and transmission of diseases. The number of deaths from malaria in the year 2018 is 405,000, compared to 585,000 in 2010 and the malaria deaths were over a million in 2000. The mosquito net is a generally a meshed curtain that is draped circumferentially over a frame of bed or any furniture, this net acts as protection barrier against bites from mosquitoes as well as other flies. The mosquito net is made to be an effective mesh without obscuring visibility or ventilation levels.
The traditional mosquito nets are made of polyester or high density polyethylene (HDPE) but the conventional nets were not effective enough so to upsurge efficacy of the nets, they are pre-treated with a suitable insecticide that are commonly known as insecticide treated nets (ITNs) that provide not only a physical barrier but also a chemical barrier that helps to prevent human beings sleeping under them from mosquito bites. As insecticide treated nets (ITNs) knockdown and kill mosquitoes that land on them the disease spreading vector population is reduced.
The bio-efficacy related to currently available insecticide treated nets (ITNs) tends to lose bio-efficacy on washing the nets as there is no balance between retention and release of insecticide. Also, these nets start losing the bio-efficacy after about 10 washes, and retention is not alone a sufficient condition for sustained bio-efficacy. Mosquitoes landing on an insecticide treated net are killed because of the insecticide present on the surface of the yarn (or filament). Hence, satisfactory amount of active ingredient should be released from the core of the polyolefin yarn, netting material, to populate the yarn surface after washing. It is important that the bio-availability of the active ingredient on the yarn surface is critical for the performance of the net. There should be delicate balance between the two conflicting factors, retention of the active ingredient (wash resistance) and the release of the active ingredient to surface to make it bio-available to kill the mosquitoes landing on the net.
JP2011529926A discloses the use of a polymer mix containing LDPE (low density polyethylene) and HDPE (high density polyethylene) for manufacturing yarns incorporated with deltamethrin and a synergist namely piperonyl butoxide (PBO). But there is no balance between retention and release of textile product in this net, which further leads to wastage of textile product in the net. Moreover, higher aqueous solubility of piperonyl butoxide (PBO) has impact not only on its release but also on the release of deltamethrin.
WO2010046348A1 describes an improved textile product comprising an insecticide and at least one more different active ingredient with the usage of two different yarns that is one for each active ingredient or one for active ingredient and the other for the synergist. If both the chemicals are in the same yarn the release or retention property one will influence the other. Moreover, the use of an additive, an oil or a wax, that changes the solubility of the chemical in the polyolefin and thus effect the release property of the polymer matrix with respect to that chemical which is invariably an insecticide or a synergist.
KR20170007108A discloses a process for producing pesticide insecticide netting for pine tree propagation and to its net fabric. The net fabric is woven based from a synthetic fiber yarn in which the insecticidal composition is mixed and fixed to the surface. But, bio-efficacy of this invention decreases with the number of washes and also there is no balance between retention and release of textile product which further leads to wastage of textile product in the net.
Therefore, there is a need of an improved insecticide incorporated fabric with a polymer composition having an adsorbent such as silica in the nano-size and consequently large surface area that is wash durable and maintains a balance between release and retention to control the release of insecticides and synergist in the fabric.

OBJECT OF THE INVENTION
The main object of the present invention is to provide an insecticide incorporated fabric with an improved polymer composition to reduce the transmission of vector borne diseases.
Another object of the present invention is to provide a method for manufacturing an insecticide incorporated fabric that substantiate proper release of chemicals to achieve the delicate balance between release versus retention of the active ingredient(s) with or without a synergist.
Yet another object of the present invention is to provide an insecticide incorporated fabric that has the ability to regain the bio-efficacy within 24 hours after washing.
Yet another object of the present invention is to provide an insecticide incorporated fabric that controls the release of insecticides and synergist in the fabric.
Still another object of the present invention is to provide an insecticide incorporated fabric that uses an adsorbent such as silica in the nano-size and consequently large surface area to control the retention versus release properties.

SUMMARY OF THE INVENTION
The present invention relates to an insecticide incorporated fabric and a method of manufacturing thereof that is wash durable and substantiates proper release of chemicals to achieve the delicate balance between release versus retention of the active ingredients and/or synergist by using an adsorbent such as silica in nano-size and consequently large surface area to control the retention versus release properties.
In an embodiment, the present invention provides an insecticide incorporated fabric comprising of a pre-defined amount of active ingredient(s) with or without a synergist converted in form of granules by using a polymer base matrix along with a pre-defined amount of adsorbent; and a pre-determined amount of polyethylene and a coloring pigment mixed with the granules and extruded in forms of yarns and the active ingredient(s) alone or with a synergist are incorporated into plurality of filaments that results in an insecticide incorporated fabric, wherein, the extrusion is performed at temperature ranging from 90°C to 250°C, the adsorbent mixes uniformly with the polymer and active ingredient at a temperature below 250°C; the fabric maintain bio-efficacy beyond 20 washes, thereby ensuring elongated wash durability and shelf life, the adsorbent is nano-particles of silica of a predefined size to impart a balance between release and retention of the active ingredient(s) and synergist; and the adsorbent is added in an amount ranging from 0.1% to 1.5% based on the active ingredient(s) and synergist (if present) loading and their chemical nature.
In another embodiment, the present invention provides a method for manufacturing insecticide incorporated fabric, comprising steps of: a) converting at least one active ingredient alone or with a synergist into plurality of master batches by using a polymer as a base matrix; b) preparing a mixture by mixing the granules obtained in step a) with polyethylene and a coloring pigment; and c) extruding the mixture obtained in step b) in form of yarns and incorporating the active ingredient(s) with or without a synergist into plurality of filaments that results in insecticide incorporated fabric; wherein, the active ingredient(s) with or without a synergist are infused or incorporated into the yarns or filaments or by combining two or more incorporated in the filaments or yarns using a process of extrusion in order to make the insecticide incorporated fabric wash durable with a balance of release and retention properties and a prolonged shelf-life; the adsorbent is nano-particles of silica of a predefined size, the adsorbent has adsorption and desorption abilities for active ingredient(s) to control release of each active ingredient(s) and synergist and prevents bleeding or blooming of active ingredient(s) and synergist (if present) at surface of the filament after washing.
The present invention relates to an insecticide incorporated fabric and manufacturing thereof with a wash durability of beyond 20 washes and prolonged shelf life by maintaining a balance between retention and release of active ingredient(s) i.e. insecticides for preventing mosquito bites and reducing the transmission of diseases in an economical way.
The above objects and advantages of the present invention will become apparent from the hereinafter set forth brief description of the drawings, detailed description of the invention, and claims appended herewith.

BRIEF DESCRIPTION OF THE DRAWING
An understanding of the method for manufacturing insecticide incorporated fabric of the present invention may be obtained by reference to the following drawings:
Figure 1 is a graphical representation of results obtained when different amounts of adsorbate (piperonyl butoxide (PBO) or alpha-cypermethrin) adsorbed on the adsorbent silica.

DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described hereinafter with reference to the accompanying drawings in which a preferred embodiment of the invention is shown. This invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those skilled in the art.
The present invention now will be described hereinafter with reference to the detailed description, in which some, but not all embodiments of the invention are indicated. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout. The present invention is described fully herein with non-limiting embodiments and exemplary experimentation.
The present invention provides an insecticide incorporated fabric and manufacturing thereof for preventing mosquito bites and reducing the transmission of diseases in an economical way that substantiate proper release of chemicals to achieve the delicate balance between release versus retention of the active ingredients and synergist to enhance the wash durability and prolong the shelf life.
In an embodiment, the present invention provides an insecticide incorporated fabric comprising of a pre-defined amount of active ingredient(s) with or without a synergist converted in form of granules by using a polymer base matrix along with a pre-defined amount of adsorbent; and a pre-determined amount of polyethylene and a coloring pigment mixed with the granules and extruded in forms of yarns and the active ingredient(s) with or without a synergist are incorporated into plurality of filaments that results in an insecticide incorporated fabric, wherein, the extrusion is performed at temperature ranging from 90°C to 250°C, the adsorbent mixes uniformly with the polymer, active ingredient(s) with or without a synergist at a temperature below 250°C; the fabric maintains bio-efficacy beyond 20 washes, thereby ensuring elongated wash durability and shelf life, the adsorbent is nano-particles of silica of a predefined size to impart a balanced between release and retention of the active ingredient(s); and the adsorbent is added in an amount ranging from 0.1% to 1.5% based on the active ingredient(s) and synergist (if present) loading and their chemical nature
In another embodiment, the present invention provides a method for manufacturing insecticide incorporated fabric, comprising steps of: a) converting at least one ingredient(s) with or without a synergist into plurality of master batches by using a polymer as a base matrix; b) preparing a mixture by mixing the granules obtained in step a) with polyethylene and a coloring pigment; c) extruding the mixture obtained in step b) in form of yarns and incorporating the active ingredient(s) with or without a synergist into plurality of filaments that results in insecticide incorporated fabric; wherein, the active ingredient(s) with or without a synergist are infused or incorporated into the yarns or filaments or by combining two or more incorporated in the filaments or yarns using a process of extrusion in order to make the insecticide incorporated fabric wash durable with a balance of release and retention properties and a prolonged shelf-life; the adsorbent is nano-particles of silica of a predefined size to impart the balance between release and retention of the active ingredient(s) with or without a synergist; the adsorbent has adsorption and desorption abilities for active ingredient(s) and synergist to control release of them and prevent bleeding or blooming of active ingredient(s) and synergist (if present) at surface of the filament after washing.
The active ingredient(s) alone or with a synergist are converted into plurality of master batches by using a polymer as a base matrix along with an adsorbent in which the active ingredient(s) and synergist include but are not limited to alpha-cypermethrin and piperonyl butoxide, respectively. The adsorbent is nano particles of silica of the predefined size ranging from 10 to 100 nm with a particle surface area ranging between 50 to 1000 m2/g to impart balance between release and retention of the active ingredient(s). The adsorbent is added in an amount ranging from 0.1% to 1.5% based on the active ingredient(s) and the synergist (if present) loading and their chemical nature during the manufacture of the active ingredient with or without a synergist master batch in a twin-screw extruder wherein the adsorbent mixes uniformly with the molten polymer, active ingredient and synergist (if present) at a temperature below 250°C. The temperature range is adjusted depending on the thermal stability of the synergist and active ingredient(s).
A mixture is prepared by mixing the granules with polyethylene and a coloring pigment. The adsorbent has adsorption and desorption abilities for each active ingredient(s) to control release of each active ingredient(s) and prevent bleeding or blooming of active ingredient(s) and synergist (if present) at surface of the filament after washing. The polyethylene is selected from a group including but not limited to high density polyethylene (HDPE), linear low-density polyethylene (LLDPE), low-density polyethylene (LDPE) and polypropylene. Further, the mixture is extruded in form of yarns and incorporating the active ingredient(s) with or without a synergist into plurality of filaments that results in insecticide incorporated fabric. The extrusion is performed at a temperature ranging from 90°C to 250°C. The filaments include but are not limited to monofilaments, multi-filaments and the active ingredient(s) with or without a synergist are infused or incorporated into the yarns or filaments or by combining two or more incorporated the filaments or yarns using a process of extrusion in order to make the insecticide incorporated fabric wash durable with combination of release and retention properties and a prolonged shelf-life.
The yarns are then knitted to form the insecticide incorporated fabric a wash durable insecticidal fabric that have sustained bio-efficacy due to the balance between release and retention property of the said fabric that are used for different applications such as the long-lasting insecticidal nets.
The basic principle of working of the silica adsorbent is close to the chromatographic separation of organic compounds. Different insecticides and synergist vary in their affinity for silica and thus differ in their adsorption and desorption behaviour. The chemical compounds that are strongly adsorbed, released slowly during washing and thus the release is controlled and the bleeding or blooming of chemical is avoided during and after washing. For instance, adsorption density of alpha-cypermethrin on nano silica used is much lower than that of piperonyl butoxide.

EXAMPLE 1
Manufacture of insecticide incorporated fabric with alpha-cypermethrin
A product i.e. Product-1 is made which is alpha-cypermethrin incorporated fabric. Alpha-cypermethrin content in the fabric is 4.35 g/kg to 7.25 g/kg. In the manufacture of the insecticide incorporated fabric, the technical grade alpha-cypermethrin is converted into master-batch granules in a twin-screw extruder. This step is necessary to scale down concentration of alpha-cypermethrin from about 95% and convert it into granules that are convenient to handle in the down-stream process. A mater-batch containing 15% alpha-cypermethrin was extruded in a twin- screw extruder. During the manufacture of the master batch, 15% alpha-cypermethrin, 0.12 to 0.18% silica adsorbent and a 0.1 to 0.2% antioxidant such as Irgafos® taken are mixed with linear low-density polyethylene and fed into a twin-screw extruder. The antioxidant does not limit to Irgafos®, but several other antioxidants are commercially available for polymer processing. Extrusion temperature was maintained below 240°C. The master-batch granules thus obtained are mixed with high density polyethylene (HDPE) and fed into a single-screw extruder to manufacture the yarns incorporated with alpha-cypermethrin. The amount of master-batch mixed with high density polyethylene (HDPE) granules is based on the target concentration of alpha-cypermethrin and the temperature of the extruder was maintained below 240°C. The high density polyethylene (HDPE), yarns thus extruded are incorporated with alpha-cypermethrin and are directly warped onto knitting beams. The knitting beams are loaded onto knitting machines and the yarns are warp knitted into polyethylene fabrics incorporated with alpha-cypermethrin at the target dosage.

EXAMPLE 2
Manufacture of insecticide incorporated fabric with alpha-cypermethrin and piperonyl butoxide
A product i.e. Product-2 is manufactured which is incorporated with alpha-cypermethrin and piperonyl butoxide (PBO). Product-2 therefore, contains an active ingredient and a synergist and the combination is not limited to alpha-cypermethrin and piperonyl butoxide (PBO). A skilled person may come up with combination of a different insecticide and a synergist. The product is aimed to increase the bio-efficacy to knockdown and kill mosquito populations that have developed resistance towards pyrethroid such as alpha-cypermethrin. Product-2 is incorporated with alpha-cypermethrin ranging from 4.50 g/kg to 7.50 g/kg along with piperonyl butoxide (PBO) 1.65 g/kg to 2.75 g/kg. The master-batch was manufactured in a twin-screw extruder. Technical grade alpha-cypermethrin 15%, technical grade piperonyl butoxide 5%, the silica adsorbent 0.15 to 0.2% and (antioxidant) 0.1 to 0.2% were mixed thoroughly with linear low-density polyethylene (LLDPE) and fed into a twin-screw extruder and the granules were extruded at a temperature below 230°C. The chemical master-batch granules contain about 15% alpha-cypermethrin and 5% piperonyl butoxide (PBO) and is used to extrude yarns incorporated with both alpha-cypermethrin and the synergist piperonyl butoxide (PBO) for knitting fabrics of Product-2. As the target dosage of product-2 is alpha-cypermethrin 4.50 g/kg to 7.50 g/kg along with piperonyl butoxide (PBO) 1.65 g/kg to 2.75 g/kg about 4.5% of the master-batch is mixed thoroughly with high density polyethylene (HDPE) granules and fed into single screw extruder. The let-down-ratio (LDR) of the mater-batch is fixed based on its chemical assay. High density polyethylene (HDPE) yarns incorporated with the insecticide alpha-cypermethrin and the synergist PBO at the target dosage were extruded maintaining the temperature below 230°C. The yarns were directly warped onto beams and then knitted into fabrics containing alpha-cypermethrin and piperonyl butoxide (PBO). The fabrics are then cut and stitched into the form of bed nets (Product-2).

EXAMPLE 3
Manufacture of insecticide incorporated fabric with alpha-cypermethrin and higher concentration of piperonyl butoxide
A product i.e. Product-3 is manufactured that is a polyethylene fabric similar to Product-2 but with a higher concentration of piperonyl butoxide (PBO). Product-3 contains alpha-cypermethrin 5.8 g/kg ± 25% and PBO 10 g/kg ± 25%. Technical grade alpha-cypermethrin 8 %, technical grade piperonyl butoxide 12.5%, the silica adsorbent ranging from 0.20 – 0.31% and Irgafos® (antioxidant) 0.1 to 0.2% were mixed thoroughly with linear low-density polyethylene (LLDPE) and fed into a twin-screw extruder and the granules were extruded at a temperature below 230°C. The chemical master-batch granules contain about 8% alpha-cypermethrin and 12.5% piperonyl butoxide (PBO) and is used to extrude yarns incorporated with both alpha-cypermethrin and the synergist piperonyl butoxide (PBO). As the target dosage of product-3 is alpha-cypermethrin 4.35 g/kg to 7.25 g/kg along with PBO 7.5 g/kg to 12.5 g/kg about 8% of the master-batch is mixed thoroughly with high density polyethylene (HDPE), granules and fed into single screw extruder. The let-down-ratio (LDR) of the mater-batch is fixed based on its chemical assay. high density polyethylene (HDPE) yarns incorporated with the insecticide alpha-cypermethrin and the synergist piperonyl butoxide (PBO) at the target dosage were extruded maintaining the temperature below 230°C. The yarns were directly warped onto beams and then knitted into fabrics containing alpha-cypermethrin and piperonyl butoxide (PBO). The fabrics may then be cut and stitched into the form of bed nets (Product-3).

EXAMPLE 4
Adsorption of piperonyl butoxide (PBO) and alpha-cypermethrin on nano grade silica
Piperonyl butoxide or alpha-cypermethrin (adsorbate) solutions of different concentrations varying from 50 mg/L to 1000 mg/L were prepared. Exact amount of nano grade silica was weighed (to 1 mg) into a 100 mL conical flask. Adsorbate solution 20 mL of known concentration was pipette out into the flask and the solution was agitated in an equilibrium shaker and left overnight to ensure completion of adsorption of the adsorbate to attain equilibrium. The solution was then filtered off and analyzed for concentration of the adsorbate left behind in the solution. From the initial concentration and the concentration left behind after adsorption, the amount of adsorbate adsorbed was calculated. Each trial was run with a duplicate and if the difference between the duplicates is > 5% the trail was repeated. Average of the duplicate runs was taken for all experimental determinations. Determination of alpha-cypermethrin was carried out in a gas chromatography with fame ionization detector (GC-FID) using dioctyl phthalate as internal standard. The method is similar to the Collaborative International Pesticides Analytical Council (CIPAC) method with reference no. (454/LN/M/3.2) however, no extraction using xylene is needed. Similarly, piperonyl butoxide (PBO) concentration were determined in GC-FID using octadecane as the internal standard, a method similar to CIPAC method 33/LN/(M)/3.
Figure 1 shows a graphical representation of results obtained when the amounts of adsorbate (piperonyl butoxide (PBO) or alpha-cypermethrin) adsorbed on the adsorbent (silica). The maximum adsorption density of piperonyl butoxide (PBO) on the nano grade silica is 325 mg/g and that of alpha-cypermethrin is only 55 mg/g. Adsorption density of piperonyl butoxide (PBO) is almost six times that of alpha-cypermethrin on the nano silica particles that offer a large surface area for adsorption. This differential adsorption behavior is the underlying principle for improving the retention of the more soluble piperonyl butoxide (PBO) incorporated into the polymer matrix. In the absence of silica in the polymer composition piperonyl butoxide (PBO) bleeds in an uncontrolled fashion during washing. The use of nano grade silica in the polymer composition controls the release and maintains a balance to sustain the bio-availability of the chemicals on the fiber surface.

EXAMPLE 5
Chemical analysis of the insecticide incorporated fabrics
Determination of alpha-cypermethrin incorporated into fabric:
Alpha-cypermethrin in the product was extracted by completely dissolving the net by refluxing in xylene and the solution was then analyzed by gas chromatography with flame ionization detector (GC-FID) using dioctyl phthalate as internal standard. This is based on the Collaborative International Pesticides Analytical Council (CIPAC) method for the determination of alpha-cypermethrin incorporated into polyethylene long-lasting insecticidal nets (LLINs).
Determination of piperonyl butoxide incorporated into fabric:
The analytical method is based on Collaborative International Pesticides Analytical Council (CIPAC) method (33/LN/(M)/3) for the determination of piperonyl butoxide incorporated into polyethylene net along with alpha-cypermethrin. Piperonyl butoxide (PBO) present in the net is extracted by refluxing with xylene and then analyzed by gas chromatography with flame ionization detector (GC-FID). Octadecane was used as the internal standard.
Sampling:
From an entire net, 5 pieces of 25 cm × 25 cm were cut with scissors and put into a 1 L screw capped glass bottle for determination of alpha-cypermethrin content. Similarly, samples are collected for the determination of piperonyl butoxide (PBO) content. Further, Table 1 represents the results of chemical analysis.

Table 1
Results of chemical analysis of the insecticidal fabrics
Sample number Product-1 Product-2 Product-3
Alpha-cypermethrin content (g/kg) Alpha-cypermethrin content (g/kg) PBO content (g/kg) Alpha-cypermethrin content (g/kg) PBO content (g/kg)
1 5.81 6.03 2.18 5.90 10.18
2 5.80 5.94 2.19 5.84 10.27
3 5.78 6.07 2.21 5.96 10.29
4 5.82 6.10 2.28 5.89 10.37
5 5.85 6.01 2.22 5.84 10.25
Mean 5.81 6.03 2.22 5.89 10.27
Std Dev. 0.026 0.061 0.039 0.050 0.069
RSD% 0.445 1.016 1.765 0.846 0.669

EXAMPLE 6
Determination of retention/release index (wash resistance)
Sampling:
From one entire net, 8 times 3 pieces of 25 cm × 25 cm were cut with scissors (1 piece on the roof and 2 pieces on each large side) and put into 1 L screw capped glass bottles for determination of retention index after 0, 1, 3, 5, 10, 15, 20 and 25 washes (3 pieces for each wash cycle) (= 8 samples of 3 pieces; 24 pieces). Further, Table 2 represents the retention results of active ingredient or synergist in the products.

Table 2
Retention of active ingredient or synergist in the products
Wash number Chemical content (g/kg) Retention (%)
w.r.t. Wash-0
Sample 1 Sample 2 Sample 3 Sample 4 Mean
Alpha-cypermethrin content (g/kg) of Product-1
0 5.82 5.85 5.78 5.80 5.81
1 5.78 5.80 5.75 5.76 5.77 99.31
3 5.74 5.72 5.67 5.73 5.72 98.32
5 5.62 5.64 5.62 5.60 5.62 96.69
10 5.43 5.44 5.39 5.40 5.42 93.16
15 5.37 5.34 5.32 5.34 5.34 91.91
20 5.25 5.22 5.26 5.27 5.25 90.32
25 5.15 5.09 5.13 5.10 5.12 88.04
Alpha-cypermethrin content (g/kg) for Product-2
0 6.02 5.95 5.99 6.04 6.00
1 5.94 5.90 5.95 5.97 5.94 99.00
3 5.89 5.87 5.91 5.93 5.90 98.33
5 5.82 5.79 5.80 5.78 5.80 96.63
10 5.56 5.62 5.60 5.57 5.59 93.13
15 5.47 5.53 5.45 5.51 5.49 91.50
20 5.37 5.39 5.40 5.42 5.40 89.92
25 5.29 5.32 5.27 5.25 5.28 88.04
PBO content for Product-2 (g/kg)
0 2.24 2.19 2.26 2.20 2.22
1 2.22 2.18 2.22 2.18 2.20 98.99
3 2.19 2.16 2.17 2.15 2.17 97.53
5 2.10 2.08 2.12 2.09 2.10 94.38
10 2.03 2.05 2.02 2.01 2.03 91.23
15 1.93 1.91 1.96 1.90 1.93 86.61
20 1.82 1.80 1.79 1.85 1.82 81.66
25 1.72 1.73 1.68 1.70 1.71 76.83
Alpha-cypermethrin content (g/kg) of Product-3
0 5.86 5.76 5.78 5.82 5.81
1 5.80 5.73 5.75 5.78 5.77 99.31
3 5.73 5.69 5.71 5.70 5.71 98.32
5 5.64 5.60 5.58 5.63 5.61 96.68
10 5.41 5.37 5.35 5.39 5.38 92.68
15 5.29 5.31 5.30 5.27 5.29 91.17
20 5.22 5.24 5.20 5.18 5.21 89.75
25 5.12 5.11 5.08 5.09 5.10 87.86
PBO content (g/kg) of Product-3
0 10.24 10.29 10.27 10.22 10.26
1 10.13 10.15 10.12 10.11 10.13 98.76
3 9.92 9.98 10.01 9.95 9.97 97.17
5 9.50 9.45 9.58 9.60 9.53 92.95
10 9.22 9.17 9.26 9.19 9.21 89.81
15 8.89 8.85 8.82 8.86 8.86 86.35
20 8.39 8.42 8.32 8.33 8.37 81.57
25 8.12 8.09 8.14 8.16 8.13 79.25
Washing procedure:
CIPAC procedure MT 195 was followed for determination of wash resistance index.
Preparation of stock solution of the CIPAC washing agent as per MT 195
The stock solution of the washing agent was prepared as follows: The bottle of polyoxyethylene glycol (25) monostearate (CAS number 9004-99-3 or 37231-60-0) was heated to about 50°C to melt and reduce its viscosity. The bottle was turned through 180 degrees a few times to ensure homogeneity. The approximate weight of 12.0 ±0.2 g sodium oleate (CAS number 143-19-1) and 8.0 ±0.2 g of polyoxyethylene glycol (25) monostearate was added into the 80 ml deionised water in a 100 mL glass flask. The mixture was heated about 50°C turning through 180 degrees frequently or stirring by a magnetic stirrer until the mixture becomes clear and homogeneous.
b) Washing
A 2.5 ml of the stock solution of the washing agent was added into a 500 ml of deionised water at 30 ± 2°C in a 1 L glass bottle. A piece of net sample was inserted with deionised water at 30 ± 2°C in a 1 L glass bottle and it is capped. The bottle was inverted for 10 times at an angle 180 degree. The bottle is placed in the water bath in an upright position free from vibration (30 ± 2°C) and not under direct sunlight. After 10 minutes, the net sample was removed using tweezers, and any remaining adherent drops of wash fluid are removed by gentle shaking.
c) Rinsing
The washed net sample was inserted into a 1 L glass bottle containing 500 ml of deionised water at 30 ± 2°C, capped and inverted 10 times (180°). The bottle was placed in the water bath in an upright position free from vibration and not under direct sunlight. After 10 minutes, the net sample was removed using tweezers, the deionised water was replaced, the sample was inserted into the 1 L bottle and the rinsing step was repeated once more.
d) Heating
Using tweezers, the net sample was pulled out from rinsing fluid and carefully any remaining adherent water drops were removed by gentle shaking. The sample was dried for 30 minutes at room temperature on a line and protected from direct sunlight. The sample was folded carefully twice in each direction, the rolled samples were placed in a glass bottle, capped and stored in an oven at 40 ± 2°C for 22 hours ± 2 hours before starting the next washing cycle.
e) Analysis
The washed net samples were analysed individually for alpha-cypermethrin and piperonyl butoxide following the respective methods. Co-determination of piperonyl butoxide (PBO) and alpha-cypermethrin is not possible because of the interference of citric acid used in the determination of alpha-cypermethrin. Citric acid was added to the solution for the determination alpha-cypermethrin to prevent epimerisation at the injection port.

Example 7
Regeneration time
Regeneration time is the period of time required for the regain of bio-efficacy after washing the insecticide incorporated fabric. During washing, the active ingredient on the surface of the filaments are washed away and the bio-efficacy decreases. The active ingredient has to diffuse from the core of the fibre, the reservoir to accumulate on the surface to compensate for the depleted amount. The time required to rebuild and attain equilibrium, the regeneration time depends on the release characteristics of the fabric incorporated with the insecticide. This is essential to regain the bio-efficacy before washing. In order to determine the time period required for regeneration of the LN after standard washing and holding at 30 °C, bioassays were carried out at constant intervals of time (+1, +2, +3, +5, +7 days) on 4 net samples (made for each of the products) washed and dried three times consecutively (nets washed three times are expected to deplete surface insecticide on the net). Insecticide bioavailability curves (24 hours’ mortality and 60 min KD), as measured by 3 minutes exposure in cone bioassays, were established for 4 unwashed nettings and 4 nettings washed three times consecutively. The time required (in days) to reach the plateau of efficacy were the period required for full regeneration of the net. Further, Table 3 illustrates the results of regeneration time study in cone bioassay.

Table 3
Results of knockdown and mortality (Anopheles gambiae; susceptible) for nets washed three times for regeneration time
Bio-efficacy endpoint Product tested 0 Day-1 Day-2 Day-3 Day-5 Day-7
Knockdown (KD) Product-1 100% 100% 100% 100% 100% 100%
Product-2 100% 100% 100% 100% 100% 100%
Product-3 100% 100% 100% 100% 100% 100%
Mortality Product-1 100% 100% 98% 100% 96% 95%
Product-2 100% 100% 100% 100% 100% 100%
Product-3 100% 100% 100% 100% 100% 100%

According to these data, the regeneration time based on mortality is one day (24 hours). This data indicates the restoration of bio-efficacy in 24 hours after washing the product to remove the surface concentration of the active ingredient and/or the synergist. This data clearly indicates the release property of the insecticide incorporated products manufactured.

EXAMPLE 8
Effect of washing on bio-efficacy: laboratory cone bio-assay
Washing:
The resistance of the insecticide incorporated fabrics (nets) to washing was determined through standard bioassays carried out on nets washed at intervals required for regeneration (as determined above), using the standard wash procedure, and dried and held at 30°C. Bioassays were done after 1, 3, 5, 10, 15, 20 and 25 washes. Each bioassay was done after the regeneration time and just before the next wash. For practical reasons, nets were not washed during the weekend but stored at 30°C until the next washes. Curves were drawn using respectively percentage mortality and knockdown (KD) versus number of washes.
Cone bioassay procedure:
The standard cone test measures knockdown and mortality of mosquitoes exposed in a small chamber to a piece of treated netting for a 3-minute exposure time. Five, non-blood fed, 2-5 days old Anopheles gambiae mosquitoes were exposed to netting materials (25x25 cm) for 3 minutes, after which they were held for 24 hours with access to sugar solution. Four cones were used on the same net sample. 50 mosquitoes on each net (10 replicates) and nettings samples were tested and results pooled for analysis (5 × 10 × 4 = 200 mosquitoes for each product). Mosquitoes exposed to untreated nets as well as deltamethrin treated nets (25 mg/m2) were used as negative and positive control respectively. Bioassays were carried out at 27 + 2°C and 75 + 10% RH. Knockdown was measured after 60 minutes’ post-exposure and mortality after 24 hours.
Results of knockdown (KD) and mortality for Products 1, 2 and 3 against susceptible and pyrethroid resistant An. stephenci are presented Table 4 and Table 5, respectively. Both knockdown and mortality are above 85% until 25 washes for all the three products. Though the alpha-cypermethrin only product performs poorly against resistant species the alpha-cypermethrin + PBO incorporated nets (Product-2 and Product-3) perform better against resistant mosquitoes. Product-3 with 10 g/kg piperonyl butoxide (PBO) outperforms the other two products and sustains the bio-efficacy even after washing several times. Usually the high concentration of piperonyl butoxide (PBO) makes the chemical bleed faster and the depletion in concentration leads to drop in bio-efficacy on washing. In the present example, piperonyl butoxide (PBO) is retained and at the same time released to make it bioavailable to effect knockdown and mortality. This is achieved by the use of silica adsorbent in the polymer composition. The retention of PBO and alpha-cypermethrin are presented in Table 2. The sustained bio-efficacy of Product-2 and Product-3 and their superior performance over a marked available net (Comparator net) are presented in the experimental hut study discussed in Example 9.

Table 4
Knockdown (KD) effect and mortality for An. stephenci (Susceptible) exposed to Product-1, Product-2 and Product-3 after several cycles of washes (W)
Endpoint W-0 W-1 W-3 W-5 W-10 W-15 W-20 W-25
KD Product-1 100% 100% 100% 100% 95.5% 98.0 87.5% 86.5%
Product-2 100% 100% 100% 100% 100% 100% 100% 100%
Product-3 100% 100% 100% 100% 100% 100% 100% 100%
Mortality Product-1 100% 100% 100% 98.0% 90.0% 83.5% 88.0% 85.5%
Product-2 100% 100% 100% 98.0% 91.5% 86.5% 83.5% 88.0%
Product-3 100% 100% 100% 100% 92.0% 95.0% 92.0% 90.5%

Table 5
Knockdown (KD) effect and mortality for An. stephenci (Resistant) exposed to Product-1, Product-2 and Product-3 after several cycles of washes (W)
Endpoint W-0 W-1 W-3 W-5 W-10 W-15 W-20 W-25
KD Product-1 96.0% 37.5% 44..0% 20.0% 17.5% 5.0% 0% 0%
Product-2 100% 87.0% 69.0% 72.0% 29.0% 15.0% 5.0% 7.0%
Product-3 100% 100% 100% 100% 100% 98.0% 89.0% 69.5%
Mortality Product-1 52.0% 27.0% 11.0% 4.5% 0% 0% 0% 0%
Product-2 92.0% 45.0% 22.0% 20.0% 15.0% 7.0% 2.0% 5.0%
Product-3 100% 100% 85.0% 74.5% 62.0% 46.5% 44.5% 30.5%

EXAMPLE 9
Experimental huts study
Standard washing procedure:
Net samples (25 cm x 25 cm) were individually introduced into 1 litre beakers containing 0.5 l deionized water, with 2 g/l of “savon de Marseille” (pH 10–11) added just before and fully dissolved. Beakers were immediately introduced into a water bath at 30°C and shaken for 10 minutes at 155 movements per minute. The samples were then removed and rinsed twice for 10 minutes in clean deionized water in the same shaking conditions as stated above. Nets were dried at room temperature for 2 hours then stored in aluminium foil at 30°C in the dark (climatic chamber) between washes
Experimental hut trails:
The trials were carried out in three different locations in Africa. Experimental huts at all three sites are of the West African design. Seven experimental huts were used for the study at each site. To prevent any contamination from previous trials, hut walls were refurbished by plastering and the cement allowed to cure for 1 month prior to the evaluation.
Test items and experimental hut treatments:
Thirteen (13) replicate nets were used per LN type at each study site – 12 were used for the hut trial (6 for unwashed arm and 6 for washed arm) and 1 was used for chemical analysis and supplementary cone bioassays. Nine (9) treatments were thus tested in the experimental huts at each study site.
Net type Insecticide treatment washing status are given below:
Untreated Net None unwashed
Product-1 Alphacypermethrin (5.8 g/kg) only unwashed
Product-1 Alphacypermethrin (5.8 g/kg) only washed 20 times
Product-2 Alphacypermethrin (6.0 g/kg) + piperonyl butoxide (PBO) (2.2 g/kg) unwashed
Product-2 Alphacypermethrin (6.0 g/kg) + piperonyl butoxide (PBO) (2.2 g/kg) washed 20 times
Product-3 Alphacypermethrin (5.8 g/kg) + piperonyl butoxide (PBO) (10 g/kg) unwashed
Product-3 Alphacypermethrin (5.8 g/kg) + piperonyl butoxide (PBO) (10 g/kg) washed 20 times
Commercial LN Permethrin (20 g/kg) + piperonyl butoxide (PBO) (10 g/kg) unwashed
Commercial LN (20 g/kg) + piperonyl butoxide (PBO) (10 g/kg) washed 20 times
The nets were placed in an aluminium bowl containing 10 L of water and 2 g/L of vegetable soap. The nets were washed for a total of 10 minutes and then the nets are agitated with a pole at 20 rotations per minute for 3 minutes and allowed to soak for 4 minutes and agitated again for 3 minutes. The nets are rinsed twice with clean water following the same procedure, dried horizontally in the shade and stored at ambient temperature between washes. Total of seven replicate nets of each candidate net were washed and 6 were used for the study and the 7th for the chemical analysis.
Hut trial design:
Treatments were initially allocated randomly to the experimental huts. To avoid any potential bias due to hut position, the treatments were rotated through the 9 experimental huts every week of the trial using a randomized Latin Square Design. The randomized Latin square design (LSD) was chosen to minimize carry over effects between treatments. The hut trial run through a complete Latin square design (7 weeks). Based on power analysis, the study was considered to have enough power to demonstrate a 25% difference in the impact between the main treatment arms of the study if the average number of mosquitoes collected per treatment arm per day is greater than 3. Data collection was done for 6 days within each week. On the 7th day of the week, the huts were thoroughly cleaned in preparation for the next rotation cycle. Six replicate nets were tested per hut treatment and the nets were swapped daily within each week of the trial.
Volunteer sleepers:
At each study site, seven (7) consenting human volunteer sleepers slept in the huts from dusk to dawn daily throughout the trial. To account for individual attractiveness to mosquitoes, sleepers were rotated daily between the 9 experimental huts using a Latin square design. Mosquitoes were collected by volunteer sleepers daily from the room, veranda trap and inside the net for each experimental hut and brought to the laboratory for identification and scoring.
Outcome measures:
The following outcome measures were used to assess the efficacy of the treatments in the experimental huts:
Primary end points
Mortality - the proportion of dead mosquitoes collected in each experimental hut treatment after a 24 h holding period.
Blood-feeding rate – the proportion of blood-fed mosquitoes collected in each experimental hut treatment.
Secondary end points
Deterrence – the proportional reduction in number of mosquitoes entering huts with treated nets
Insecticide induced exiting rates estimated from the proportions of mosquitoes collected from the verandas of treatment and control huts
Blood-feeding inhibition - the proportional reduction in blood feeding in huts with insecticide treated nets relative to controls with untreated nets
Personal protection - the reduction in mosquito biting by insecticide treated nets relative to untreated nets, as derived from the formula
% personal protection=100×((Bu-Bt))/Bu
where Bu is the total number blood-fed mosquitoes in the huts with untreated nets, and Bt is the total number blood-fed in the huts with treated nets.

Table 6
Results of experimental hut studies in three sites
Site -1 Site -2 Site -3
Mortality
0 wash 20 washes 0 wash 20 washes 0 wash 20 washes
Product-1 20% 21% 10% 8% 12% 7%
Product-2 31% 28% 30% 19% 24% 14%
Product-3 33% 27% 40% 36% 31% 22%
Comparator net 16% 12% 26% 11% 15% 9%
Blood feeding inhibition
Product-1 56% 46% 38% 35% 33% 31%
Product-2 79% 71% 48% 47% 74% 72%
Product-3 91% 71% 54% 47% 85% 54%
Comparator net 49% 14% 34% 32% 65% 0%

Example 10
Shelf-life study
Shelf-life study on Product-1 containing alpha-cypermethrin alone was carried out for 66 months as per Gaitonde Committee Protocol. Product-1 in the form of finished rectangular nets were packed in LDPE pouches and stored at three different agro-climatic locations under ambient temperature conditions in India. Time point of analysis were 0, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60 and 66 months after storage. Duplicate samples were drawn at each time point and sent to laboratory for analysis. Alpha-cypermethrin content of the samples were determined following CIPAC method 454/LN/M/3.2 and bursting strength of the samples were determined as per ISO 13938-2:1999. The results of alpha-cypermethrin content and bursting strength of the samples are presented in Table 7.

Table 7
Results of alpha-cypermethrin content and bursting strength values of the samples collected at specific time points during the 66-months shelf-life study
Months Site Bursting strength (kPa) Alpha-cypermethrin content (g/kg)
Sample1 Sample 2 Sample1 Sample 2
0 1 542.5 557.1 5.86 5.91
2 552.5 561.3 5.80 5.93
3 555.2 556.2 5.93 5.82
6 1 554.1 554.9 5.83 5.83
2 551.7 556.4 5.84 5.81
3 558.6 565.4 5.80 5.86
12 1 538.6 554.1 5.76 5.82
2 554.2 559.5 5.76 5.75
3 549.9 556.3 5.79 5.77
18 1 549.1 551.7 5.77 5.71
2 549.6 548.0 5.74 5.75
3 551.0 554.1 5.72 5.75
24 1 548.3 551.2 5.71 5.71
2 548.6 547.7 5.70 5.70
3 549.7 553.4 5.70 5.69
30 1 548.2 550.8 5.67 5.67
2 548.7 546.3 5.65 5.66
3 550.5 553.4 5.64 5.63
36 1 547.7 550.1 5.63 5.63
2 548.6 546.5 5.61 5.62
3 550.0 553.1 5.60 5.60
42 1 547.0 550.1 5.60 5.60
2 547.9 546.5 5.58 5.57
3 549.3 552.9 5.59 5.60
48 1 546.7 549.4 5.58 5.58
2 548.1 546.6 5.52 5.52
3 547.5 549.5 5.50 5.51
54 1 547.0 550.4 5.51 5.50
2 548.1 549.6 5.49 5.48
3 550.1 551.5 5.47 5.48
60 1 546.9 549.1 5.44 5.45
2 547.5 547.2 5.45 5.44
3 548.5 549.2 5.44 5.45
66 1 547.7 550.5 5.42 5.43
2 547.7 546.8 5.43 5.42
3 549.5 548.0 5.41 5.41

The present invention relates to an insecticide incorporated fabric and manufacturing thereof with a wash durability beyond 20 washes and prolonged shelf life i.e. 66 months by maintaining a balance between retention and release of active ingredient(s) i.e. insecticides for preventing mosquito bites and reducing the transmission of diseases in an economical way.
Many modifications and other embodiments of the invention set forth herein will readily occur to one skilled in the art to which the invention pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.