WO2009087046A1

WO2009087046A1 - Hand washing assessment method

Info

Publication number: WO2009087046A1
Application number: PCT/EP2008/068012
Authority: WO
Inventors: Michael John Hoptroff; Katherine Mary Thompson
Original assignee: Unilever Plc; Unilever N.V.; Hindustan Unilever Limited
Priority date: 2008-01-04
Filing date: 2008-12-19
Publication date: 2009-07-16
Also published as: MY179556A

Abstract

The invention provides a method for assessing the effectiveness of skin washing in a subject, comprising the steps of : (a) applying a patch on or over a target area of the skin of a subject before washing of the target area, followed by (b) assessing the appearance of the patch after washing of the target area; in which the patch comprises a pH sensitive indicator which is designed to undergo a visually perceptible change in colour when contacted with an alkaline composition, such as soap or other anionic surfactant, during washing of the target area, and in which assessment of the appearance of the patch after washing provides an indication to the assessor whether an alkaline composition, such as soap or other anionic surfactant, has or has not been used to wash the target area.

Description

HAND WASHING ASSESSMENT METHOD

Field of the Invention

The present invention relates to a method for assessing the effectiveness of hand washing in a subject.

Background and Prior Art

The World Health Organisation (WHO) estimates that diarrhoea and respiratory infections are responsible for two-thirds of child deaths. The vast majority of child mortality occurs among the world' s poorest populations in low- and middle- income countries.

Hands play a key role in the spread of diarrhoeal pathogens and some respiratory infections. Hand washing interrupts the transmission of disease agents and so can significantly reduce diarrhoea and respiratory infections, as well as skin infections and trachoma. Recent research has shown that washing hands with soap at critical moments (particularly after contact with human excreta and before handling food) can cut diarrhoea risk by almost half. Another recent study indicates that hand washing with soap has the potential to reduce respiratory infections by about a third.

While soap is found in most households, it is often reserved for washing clothes, dishes and bathing. Hand washing with soap is not common, though practice varies from country to country. The main reason given why rates of hand washing with soap are so low is that it is simply not a habit. The widely held belief is that if hands look clean, they are clean .

The food services and healthcare industries have developed various systems intended to address the problem of insufficient hand washing.

US 6,029,600 describes a non-electrical device for reminding restroom users to wash and cleanse their hands. An aerosol spray can or a pump spray activated by the opening of the restroom door releases a visible dye stain onto the hand of the user. The stain is removed by the cleanser supplied in the restroom. Suitable stains are described as easily washable, non-toxic materials such as a diluted food colouring.

US2005/0231373 describes a similar marking mechanism in the form of a standalone device. A removable dye is applied to the hands of a user pressing down on an absorbent pad containing the dye. The device also incorporates an audible signal which is triggered when marking takes place and serves as an alert to a person monitoring hand washing that marking has occurred.

GB-A-2, 408, 330 describes a method of assessing a wound to thereby facilitate the selection of subsequent treatment using a film, preferably comprised of a hydrogel, which may be incorporated into a dressing, and which incorporates a pH colour indicator. As stated above, one of the key elements of effective hand washing is the use of a cleansing composition such as soap or other anionic surfactant, rather than water alone.

A problem associated with the above devices is that they may not necessarily indicate whether or not soap has been used in the hand washing process. For example, the dyes used in US2005/0023373 are described as removable, albeit more slowly, with water only.

Another problem associated with the above devices is that hand marking is user-actuated and so may not necessarily be carried out properly or optimally when users are young children .

The incorporation of pH-sensitive indicators into cleansing products has been described in the prior art.

WO 06/105193 discloses compositions for disinfecting hard surfaces with a colour change indicator comprising mixtures of a biocide, a surfactant and a pH indicator. The indicator colour is lost on drying the surface reinforcing the idea that the surface is now hygienically clean.

WO 06/10526 discloses the incorporation of a pH indicator into a soap product such that the product changes colour after a period of time to cue that adequate washing has occurred. The trigger for the colour change is provided by absorption of carbon dioxide from the air. WO 06/023736 describes the use of suspended microencapsulated coloured particles which rupture after a given period of time by mechanical shear causing a change in the colour of the soap lather. This indicates when a sufficiently long washing process has taken place to adequately clean the hands.

US 2005/0090414 describes delivering a pH indicator from a liquid soap and mixing via a dual-chamber dosing device with encapsulated bicarbonate. Physical abrasion releases the base, increasing the pH of the soap solution and causing a colour change after a defined period of time.

A problem associated with the above proposals is that they do not allow a third party such as a parent or primary caregiver to monitor the use of soap in the washing process, unless that parent or primary caregiver actually supervises the washing process and observes any colour change directly.

The present invention provides a low-cost and effective method to promote the use of soap, and in particular hand washing with soap.

The invention also provides a solution to the problem of distinguishing between proper hand washing with soap and less effective hand washing using water alone.

The invention also enables a parent or caregiver to monitor the washing process (especially hand washing) in subjects such as young children, without the need for direct supervision .

Summary of the Invention

The invention provides a method for assessing the effectiveness of skin washing in a subject, comprising the steps of:

(a) applying a patch on or over a target area of the skin of a subject before washing of the target area, followed by

(b) assessing the appearance of the patch after washing of the target area;

in which the patch comprises a pH sensitive indicator which is designed to undergo a visually perceptible change in colour when contacted with an alkaline composition, such as soap or other anionic surfactant, during washing of the target area,

and in which assessment of the appearance of the patch after washing provides an indication to the assessor whether an alkaline composition, such as soap or other anionic surfactant, has or has not been used to wash the target area .

Detailed Description of the Invention

Studies have shown that the duration of washing is also important in ensuring effectiveness of the process. Accordingly it is preferred that the patch for use in the method of the invention is designed to undergo a visually perceptible change after a suitable period of washing.

By "suitable period" in the context of this invention is generally meant a period of washing up to about 5 minutes, preferably up to about 45 seconds, more preferably from 5 to 45 seconds, most preferably from 10 to 40 seconds, ideally from 15 to 35 seconds.

By "visually perceptible" in the context of this invention is meant visible to the naked eye.

The patch may suitably be provided in the form of a device or scaffold incorporating the pH sensitive indicator, and designed to facilitate contact between the pH sensitive indicator and the washing medium during the skin washing process. Examples of suitable forms of device or scaffold include transfers or stickers, bracelets, rings and necklaces.

In a preferred embodiment, the patch may comprise a device or scaffold, conveniently a ring, which may comprise (or consist of) a textile element which may be coated or impregnated with a pH indicator. The pH indicator may have been coated onto or impregnated into the patch in a polymer, for example a film-forming polymer as herein described, by techniques well known to those skilled in the art. In some instances the patch may comprise a ring comprising a sleeve of textile, for example from 2 mm to 15 mm long, preferably 2 mm to 10 mm long, e.g. 2 to 5 mm long, and of a diameter appropriate to fit snugly around a user's finger, e.g. 4 cm to 9 cm circumference. The patch may be coated or impregnated with indicator as herein described.

Conveniently, the pH indicator in the patch and thereby the patch itself may be reusable. This can be achieved for example by treating the patch with an acid, for example a weak acid, such as can be found in citrus fruit juice, for example lemon, orange or lime juice, or vinegar. This embodiment may be useful where the patch is in the form of device or scaffold such as a transfer or sticker, bracelet, ring or necklace, especially a ring.

In a further embodiment, the device or scaffold may comprise a separate textile element. The textile element is preferably hydrophilic, and may be woven or non-woven. The textile element is preferably absorbent. Conveniently the textile element comprises cotton. Textile elements which are hydrophilic and/or absorbent have been found to be particularly suitable as they facilitate penetration of a polymer solution containing a pH indicator within the yarn and fabric structure, increasing the resistance of the film to mechanical abrasion. The hydrophilic nature of the textile aids wetting, allowing even uptake of the polymer solution providing a uniform thickness and colour intensity. The high wetting also aids uptake of the alkaline solution and consequent rapid colour changing of the indicator in use. Such embodiments may be particularly suited to being reusable. In such instances the textile element is preferably coated or impregnated with a polymer, conveniently a film-forming polymer as described herein. The textile is preferably white or dyed with another colour to a light pastel shade.

Conveniently the textile element is capable of wicking and also being wetted. Wetting and wicking are discussed in Textile Research Journal, E. Kissa, 1996, 66 (10), pp 660- 668. Wicking test methods and apparatuses are described Textile Research Journal, P. Harnett and P.N. Mehta, July 1984, pp 471-478, and US 2002/0120410. Wettability can be described as the displacement of fibre-air interface with a fibre-liquid interface. It can be measured by British Standard BS 4554:1970; preferably the wetting time is less that 200 second, preferably less than 20 seconds, preferably less than 5 seconds, preferably less than 3 seconds, preferably less than 1 second. The contents of all of these documents are incorporated herein by reference.

In a preferred embodiment the device or scaffold is conveniently a ring which may be made of any material, for example a metal or plastics material, having an area for retention of a textile element. The textile element may be permanently or releasably retained in the device or scaffold (e.g. ring) . The textile element may be coated or impregnated with a polymer containing the pH indicator.

Alternatively, a patch of pH sensitive indicator could be applied directly to the target area by means of a marker pen, spray applicator, stamp pad, brush applicator, sponge applicator or similar device. In a particularly preferred embodiment, the patch is designed to be applied to the fingertips, where cleansing is most important for disease prevention.

Accordingly, the patch is ideally of a size and shape which is small enough to be applied to a fingertip of a subject (usually a young child) , yet large enough to be easily visualised by the naked eye during use.

A suitable example would be a small disc having a diameter ranging from 1 to 10 mm, preferably from 2 to 5 mm.

Alternatively, if the patch is designed to be applied to a device or scaffold such as a ring, then it could suitably take the form of a thin strip or band having a width ranging from 1 to 10 mm, preferably from 2 to 5 mm.

A range of different patch sizes could be provided in the form of a set, so that a parent or caregiver can select the most appropriate size for their child. Alternatively, if the patch of pH sensitive indicator is applied directly to the target area by means of pen-type devices such as those described above, then the size and shape of patch applied can be conveniently user-determined.

Patches suitable for use in the invention may suitably be prepared from a mixture containing the pH sensitive indicator, at least one polymer, preferably a film-forming polymer, and a solvent. By "film-forming polymer" means a polymer capable of forming, by itself or in the presence of an auxiliary film- forming agent, a macroscopically continuous film, preferably a cohesive film and more preferably a film whose cohesion and mechanical properties are such that the film can be isolated and handled in isolation, for example when the film is made by pouring or casting the mixture onto a surface such as a non-stick backing paper. The film could alternatively be obtained by casting, blow-moulding, extrusion or blown extrusion of the film-forming polymer.

Advantageously, the film-forming polymer serves to entrap or immobilise the pH sensitive indicator, which helps to reduce the risk of smearing or staining of the pH sensitive indicator onto areas of the skin or other articles which it is not desired to mark. It also enables a wider variety of pH sensitive indicators to be used in the method of the invention .

A patch suitable for use in the invention may suitably be made by a method comprising the following steps:

(a) preparing a mixture containing at least one pH sensitive indicator, at least one film-forming polymer and a solvent;

(b) pouring or casting the mixture onto a surface such as a non-stick backing paper, and

(c) drying the mixture so as to form a solid or semisolid film on the surface, the solid or semisolid film bearing the pH sensitive indicator. Such a solid or semisolid film may be applied directly to the skin as a transfer or sticker by placing the solid or semisolid film on the skin with the non-stick backing paper uppermost, moistening the backing paper, and applying gentle pressure before peeling off the non-stick backing paper, leaving the film adhered to the skin.

Alternatively the solid or semisolid film could be incorporated into a piece of jewellery designed to contact the skin surface, such as a bracelet or necklace or preferably a ring.

Alternatively, a mixture containing at least one pH sensitive indicator, at least one film-forming polymer and a solvent could be applied directly to the skin (by a brush or pen-type device or similar) , so that a solid or semisolid film bearing the pH sensitive indicator is formed on the skin surface as the mixture dries.

Film-forming polymers suitable for use in the invention can be of synthetic origin or of natural origin, and can be of nonionic, cationic, anionic or amphoteric character.

Examples of suitable film-forming polymers include:

synthetic, nonionic film-forming polymers such as homopolymers of vinyl pyrrolidone, homopolymers of N-vinyl formamide, copolymers of vinyl pyrrolidone and vinyl alcohol, copolymers of vinyl pyrrolidone and vinyl acetate, copolymers of vinyl pyrrolidone and dimethylaminoalkylmethacrylates, in which alkyl means methyl, ethyl or propyl, terpolymers of vinyl pyrrolidone, vinyl acetate and vinyl propionate, polyacrylamides, homopolymers of vinylacetate (PVAc) , polyvinylacetate phthalate, homopolymers of vinyl alcohol (PVA), copolymers of vinyl alcohol and ethylene, copolymers of vinyl alcohol and vinyl acetate, polyethylene glycols, polyethylene glycol/polyvinylalcohol copolymers and polyethylene glycol/polypropylene glycol copolymers;

synthetic, anionic, film-forming polymers such as branched or non-branched vinyl acetate/crotonic acid copolymers, terpolymers of acrylic acid, alkyl acrylates and N- alkylacrylamides, (such as acrylic acid/ethyl acrylate/N-t- butylacrylamide terpolymers), vinyl pyrrolidone/vinyl acetate copolymers, terpolymers of vinyl acetate, crotonate and vinyl alkanoate (such as vinyl acetate/crotonate/vinyl neodecanoate copolymers) , copolymers of vinyl alcohol and acrylic acid, and methyl vinyl ether/maleic acid anhydride copolymers and their monoesters;

synthetic, cationic, film-forming polymers such as poly (allylamine) , copolymers of vinylpyrrolidone and quaternized derivatives of dialkylaminoacrylate and dialkylaminomethacrylate, copolymers of vinylpyrrolidone and methylvinylimidazolium salts (e.g. chloride), terpolymers of dimethyl diallyl ammonium chloride, sodium acrylate and acrylamide, terpolymers of vinylpyrrolidone, dimethylamino ethyl methacrylate and vinyl caprolactam, polymeric quaternary ammonium salts of hydroxyethylcellulose reacted with a trimethyl ammonium substituted epoxide, copolymers of vinylpyrrolidone and methacrylamidopropyltrimethylammonium chloride, diquaternary polydimethylsiloxanes, cationic diallyl quaternary ammonium- containing polymers (such as dimethyldiallylammonium chloride homopolymer and copolymers of acrylamide and dimethyldiallylammonium chloride and cationic polyacrylamides ;

natural or naturally-derived film-forming polymers such as chitosan, hydroxyalkylchitosan, hydroxyalkylchitin, shellac, polyaminoacids, peptides, starch, modified starch, gelatin, sodium alginate, xanthan gum, tragacanth gum, guar gum, acacia gum, arabic gum, pectin, carrageenan, dextrin, maltodextrin, polydextrose, pullulan, cellulose and cellulose derived film-forming polymers (such as methylcellulose, ethylcellulose, propylcellulose, carboxymethy1cellulose, hydroxyethy1cellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose) ;

synthetic, amphoteric, film-forming polymers such as dimethyldiallylammonium chloride/acrylamide/acrylic acid terpolymers, copolymers of acrylic acid and dialkyldiallylammonium salts (such as dimethyldiallylammonium chloride) , (meth) acrylethylbetaine/alkyl (meth) acrylate copolymers (such as N-methacryloyloxyethyl-N, N-dimethylammonium-α-N- methylcarboxybetaine/alkyl methacrylate copolymers) , sodium acrylate/acrylamidopropyltrimethylammonium chloride copolymers and octylacrylamide/acrylates/butylaminoethyl methacrylate copolymers. A preferred class of suitable film-forming polymers for use in the invention are homopolymers and copolymers of vinyl alcohol, most preferably homopolymers of vinyl alcohol (PVA) . These materials provide physically robust films and good colour change kinetics.

Mixtures of any of the above described materials could also be used, in order to achieve preferred film forming capabilities, mechanical properties, and kinetics of colour change.

Suitable pH sensitive indicators for use in the invention may be naturally occurring or synthetic.

Preferred pH sensitive indicators for use in the invention give a clear visually perceptible colour change when the pH of their environment is raised from near neutrality such as the pH of water or the slightly acidic conditions typical of skin (pH 5.5-6.5) to a slightly alkaline value (pH 8.5 or above) . The pH range over which a colour change from the acidic (HIn) form to the (In^") form is observed may be slightly shifted for an indicator borne on a solid or semisolid film when compared to the same indicator in aqueous solution. When preparing solid or semisolid films according to the method described above, it may be necessary to adjust pH of the indicator in solution to ensure that the indicator is present in its acidic (HIn) form before incorporation into the solid or semisolid film.

It may also be desirable in some circumstances for the colour change provided by the pH sensitive indicator to be reversible, for example when the patch is provided in the form of a durable device such as a piece of jewellery as described above. For example, depending on the choice of pH sensitive indicator, the device could be immersed in water for a period of time to reverse the colour change. More preferably the device could be treated with a slightly acidic solution (ideally an easily available household commodity such as vinegar or fruit juice) , so as to reverse the colour change and thus render the device suitable for several repeat uses.

Examples of suitable pH sensitive indicators are listed below. The Colour Index numbers (CI.) are taken from the Rowe Colour Index, 3rd edition, Society of Dyers and Colourists , Bradford, England, 1971.

Alizarin (1, 2-dihydroxyanthraquinone, Mordant Red 11, CI 58000), an organic pigment in madder which turns from yellow at neutral pH to pinkish-purple under alkaline conditions

Quinizarin (1, 4-dihydroxyanthraquinone) , a dye found in plants including madder and senna, which turns from yellow at neutral pH to reddish under alkaline conditions - Juglone (5-hydroxy-l, 4-naphthoquinone, Natural Brown 7, CI 75500), a colorant extracted from walnuts which turns from yellow at neutral pH to purple under alkaline conditions Curcumin ( [ (IE, 6E) -1, 7-bis (4-hydroxy-3-methoxyphenyl) - 1,6- heptadiene-3, 5-dione) ] , Natural Yellow 3, C.I. 75300) the principal colorant in turmeric (food colorant ElOO) which turns from yellow at neutral pH to reddish-purple under alkaline conditions Phenolphthalein (3, 3-bis (p-hydroxyphenyl) phthalide; 3, 3-bis (4-hydroxyphenyl) -1 (3H) -isobenzofuranone) , a synthetic indicator which turns from colourless at neutral pH to pink under alkaline conditions Meta-cresolsulfonephthalein (Metacresol purple) , a synthetic indicator which turns from yellow at neutral pH to purple under alkaline conditions - Ort.ho-cresolsulfonephthalein (Cresol red) , a synthetic indicator which turns from yellow at neutral pH to red under alkaline conditions

Thymolsulfonephthalein (Thymol blue) , a synthetic indicator which turns from yellow at neutral pH to blue under alkaline conditions α-Naphtholphthalein, a synthetic indicator which turns from pale pink at neutral pH to greenish-blue under alkaline conditions Phenolsulfonephthalein (Phenol Red) , a synthetic indicator which turns from yellow at neutral pH to red under alkaline conditions

Hessian Bordeaux, (4, 4-bis (4-amino-l-naphthylazo) -2, 2- stilbenesulphonic acid) , a synthetic indicator which turns from blue at neutral pH to red under alkaline conditions

- Brilliant Yellow (2, 2 ' (1, 2-Ethenediyl) -bis [5- [ (4- hydroxyphenyl) -azo] benzenesulfonic acid] disodium salt, Direct Yellow 4 , CI 24890) which turns from yellow at neutral pH to orange-red under alkaline conditions Mixtures of any of the above described pH sensitive indicators could also be used in order to achieve the desired colour changing characteristics.

Specific examples of preferred pH sensitive indicators include Alizarin (C. I.58000), Curcumin (C. I.75300), and to a slightly lesser extent Phenolsulfonephthalein (Phenol Red) .

Curcumin is particularly preferred since this gives a fast colour change which is also reversible.

The invention is further illustrated with reference to the following, non-limiting examples, in which all percentages are by weight based on total weight unless otherwise specified.

EXAMPLES

Example 1

A 10% solution of hydrolysed PVA (molecular weight 85,000 to 124,000) was prepared in water. The pH of the solution was adjusted to a value of 6 to 6.5 by dropwise addition of dilute acid. The solution was mixed in a 1:1 volume ratio with an aqueous solution containing 0.05 g/L of curcumin. A small spot of the resulting solution was dropped onto a glass backing slide and allowed to dry to produce a film of approximately 1 cm diameter.

An aqueous soap slurry was prepared by dissolving 8 g of bar soap in 100 g water. A fingertip was dipped in the soap - I i

slurry and used to transfer a small quantity of the soap solution onto the film. The soap solution was gently rubbed across the surface of the film for 5 seconds. Over this time the film changed colour from yellow-brown to purplish-red. The colour darkened still further when the film was left to stand for an additional 20 to 30 seconds. The reddish colour was retained when the soap solution was rinsed from the film by holding the film under a running cold water tap for 10 seconds and when the film was allowed to dry in the air. The polymer film did not become sticky and the dye colour did not leach onto the skin or onto the surrounding glass substrate .

The soap treated surface was then gently rubbed by fingertip with household vinegar for 5 seconds. The film returned to its original yellow-brown colour and is allowed to dry once more .

The soap treatment and vinegar regeneration process can be repeated up to 5 times before the surface of the film starts to become eroded.

Example 2

A curcumin-PVA film was prepared as described in Example 1. The pH sensitive film was rubbed with water in the absence of any soap product. No colour change is observed. The film can be fully immersed in water for periods of minutes or hours without a colour change occurring. Accordingly, the curcumin-PVA film can be used as an indicator of whether soap has (or has not) been used in the handwashing process.

Example 3

A 10% solution of hydrolysed PVA (molecular weight 85,000 to 124,000) was prepared in water. The pH of the solution was adjusted to a value of 6 to 6.5 by dropwise addition of dilute acid. The solution was mixed in a 9:1 volume ratio with an ethanolic solution containing 0.1 g/L of curcumin. A small spot of the resulting solution was dropped onto a glass backing slide and allowed to dry to produce a film of approximately 1 cm diameter. The film was treated with a soap solution as detailed in Example 1 for 5 seconds. Over this time the film changed colour from yellow-brown to purplish-red. The colour change was more uniform than that from Example 1.

Example 4

A PVA-curcumin solution was prepared as in Example 3. A piece of woven cotton fabric was placed horizontally on a flat glass petri dish. The PVA-curcumin solution was dosed at the centre of the piece of cloth at a level of Ig per cm² of exposed fabric surface. The PVA-curcumin mixture was gently applied across the fabric surface using a paint brush and allowed to soak evenly into the fabric. The petri dish and contents was heated in a 40 deg C oven for 3 hours to evaporate the solvents. The polymer-dye impregnated fabric was then gently removed from the glass surface. A sample of the fabric was treated with a soap solution prepared as in Example 1. The colour changed from bright yellow to pink within 5 seconds.

The impregnated textile could be cut into any desired shape, for example to produce a sleeve suitable for use as a ring.

Claims

1. A method for assessing the effectiveness of skin washing in a subject, comprising the steps of:

(b) assessing the appearance of the patch after washing of the target area;

2. A method according to claim 1, in which the patch is prepared from a mixture containing the pH sensitive indicator, at least one film-forming polymer and a solvent.

3. A method according to claim 2, in which the patch is made by a method comprising the following steps: (a) preparing a mixture containing at least one pH sensitive indicator, at least one film-forming polymer and a solvent;

(c) drying the mixture so as to form a solid or semisolid film on the surface, the solid or semisolid film bearing the pH sensitive indicator.

4. A method according to claim 2 or claim 3, in which the film-forming polymer is a homopolymer or copolymer of vinyl alcohol, preferably a homopolymer of vinyl alcohol (PVA) .

5. A method according to any one of claims 2 to 4, in which the pH sensitive indicator is alizarin (C. I.58000), curcumin (C. I.75300) or phenolsulfonephthalein (Phenol Red), preferably curcumin.

6. A method according to any one of the preceding claims wherein the patch comprises a device or scaffold incorporating a pH sensitive indicator.

7. A method according to claim 6 wherein the device or scaffold is in the form of a transfer, sticker, bracelet, necklace or ring, preferably a ring.

8. A method according to claim 7 wherein the device or scaffold includes a textile element.

9. A method according to claim 8 wherein the textile element is hydrophilic.

10. A method according to claim 8 or claim 9 wherein the textile element is absorbent.

11. A method according to any of claims 6 to 10 wherein the pH indicator is coated or impregnated into the patch in a polymer, conveniently a film- forming polymer.

12. A method according to any one of the preceding claims wherein the patch is reusable.

13. A method according to any one of the claims 8 to 11 wherein the textile element is permanently or releasably retained.

14. A method according to any of the preceding claims wherein the pH indicator is Curcumin or Alizarin.

RECTIFIED SHEET (RULE91) ISA/EP