STABLE PERFUMING COMPOSITIONS HAVING GOOD SPRAY PROPERTIES

Abstract

Disclosed is a composition in the form of an oil-in-water emulsion and including: a) at least one oil; b) at least one cross-linked anionic polyelectrolyte; c) at least one water-in-oil emulsifying surfactant including an alkylpolyxyloside having a non-linear branched alkyl radical and having 10 to 36 carbon atoms; d) at least one oil-in-water emulsifying surfactant; e) at least one hydrophobic perfuming substance; and f) water.

Claims

1. A perfuming composition provided in the form of an emulsion of oil-in-water type and comprising: a) at least one oil, b) at least one crosslinked anionic polyelectrolyte, c) at least one emulsifying surfactant of water-in-oil type comprising an alkyl polyxyloside possessing a nonlinear branched alkyl radical and comprising from 10 to 36 carbon atoms, d) at least one emulsifying surfactant of oil-in-water type, e) at least one hydrophobic perfuming substance, and f) water.

2. The composition as claimed in claim 1, comprising, per 100% of its weight: a) from 1% to 30% by weight of oil; b) from 0.05% to 2.5% by weight of the crosslinked anionic polyelectrolyte; c) from 0.05% to 2.5% by weight of the surfactant of water-in-oil type; d) from 0.5% to 5% by weight of the surfactant of oil-in-water type; e) from 0.5% to 10% by weight of the hydrophobic perfuming substance; f) from 50% to 97.9% by weight of water.

3. The composition as claimed in claim 1, wherein the emulsifying surfactant of water-in-oil type comprises at least one composition of alkyl polyglycosides represented by the formula (I):
R.sub.2—O—(X).sub.x—H  (I) in which x represents a decimal number of between 1.05 and 2.5, X represents the xylosyl or α,β-D-xylopyranosyl radical, obtained from the deletion of the hemiacetal hydroxyl group of α,β-D-xylopyranose, and R.sub.2 represents a radical of formula (II):
CH(C.sub.tH.sub.2t+1)(C.sub.vH.sub.2v+1)CH.sub.2—  (II) in which t is an integer of between 6 and 18, v is an integer of between 4 and 18 and the sum t+v is greater than or equal to 10 and less than or equal to 22.

4. The composition as claimed in claim 3, wherein the composition consists of a mixture of compounds represented by the formulae (I.sub.1), (I.sub.2), (I.sub.3), (I.sub.4) and (I.sub.5):
R.sub.2—O-(G).sub.1-H  (I.sub.1)
R.sub.2—O-(G).sub.2-H  (I.sub.2)
R.sub.2—O-(G).sub.3-H  (I.sub.3)
R.sub.2—O-(G).sub.4-H  (I.sub.4)
R.sub.2—O-(G).sub.5-H  (I.sub.5) in the respective molar proportions a1, a2, a3, a4 and a5, such that: the sum a1+a2+a3+a4+a5 is equal to 1, and that the sum a1+2a2+3a3+4a4+5a5 is equal to x.

5. The composition as claimed in claim 3, wherein the emulsifying surfactant of water-in-oil type comprises a fatty alcohol of formula (III):
CH(C.sub.tH.sub.2t+1)(C.sub.vH.sub.2v+1)CH.sub.2—OH  (III), in which t is an integer of between 6 and 18, v is an integer of between 4 and 18 and the sum t+v is greater than or equal to 14.

6. The composition as claimed in claim 1, wherein the emulsifying surfactant of oil-in-water type comprises: a compound of formula (IV):
R.sub.1—(C═O)[O—CH.sub.2—CH(OH)—CH.sub.2].sub.p—OH  (IV), in which p represents an integer greater than or equal to 1 and less than or equal to 15 and in which the group R.sub.1—(C═O)— represents a saturated or unsaturated and linear or branched acyl radical comprising from 6 to 22 carbon atoms, and/or a composition of alkyl polyglycosides represented by the formula (VI):
R.sub.3—O-(G).sub.x′-H  (VI) in which x′ represents a decimal number of between 1.05 and 2.5, G represents the glucosyl or α,β-D-glucopyranosyl radical, obtained from the deletion of the hemiacetal hydroxyl group of α,β-D-glucopyranose, and R.sub.3 represents a radical chosen from the elements of the group consisting of the n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl and n-behenyl radicals.

7. The composition as claimed in claim 6, wherein the composition consists of a mixture of compounds represented by the formulae (VI.sub.1), (VI.sub.2), (VI.sub.3), (VI.sub.4) and (VI.sub.5):
R.sub.3—O-(G).sub.1-H  (VI.sub.1)
R.sub.3—O-(G).sub.2-H  (VI.sub.2)
R.sub.3—O-(G).sub.3-H  (VI.sub.3)
R.sub.3—O-(G).sub.4-H  (VI.sub.4)
R.sub.3—O-(G).sub.5-H  (VI.sub.5) in the respective molar proportions a1, a2, a3, a4 and a5, such that: the sum a1+a2+a3+a4+a5 is equal to 1, and that the sum a1+2a2+3a3+4a4+5a5 is equal to x.

8. The composition as claimed in claim 6, wherein the emulsifying surfactant of oil-in-water type comprises a compound of formula (IV) and at least one compound of formula (V):
HO—[CH.sub.2—CH(OH)—CH.sub.2—O].sub.n—H  (V), in which n, which is identical to or different from p, represents an integer of greater than or equal to 1 and of less than or equal to 15.

9. The composition as claimed in claim 6, wherein the emulsifying surfactant of oil-in-water type comprises a composition of alkyl polyglycosides and a fatty alcohol of formula (VII):
R′.sub.3—OH  (VII), in which R′.sub.3 represents a radical chosen from the elements of the group consisting of the n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl and n-behenyl radicals, and with R′.sub.3 identical to or different from R.sub.3.

10. The composition as claimed in claim 3, wherein the R.sub.2 radical represents the 2-octyl-1-dodecyl radical in the formula (II) obtained from the deletion of the hydroxyl group of 2-octyl-1-dodecanol, and for which t is equal to 10 and v is equal to 8 in the formula (II).

11. The composition as claimed in claim 1, wherein the crosslinked anionic polyelectrolyte comprises a proportion of greater than or equal to 25 mol % of monomer units resulting from 2-methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulfonic acid in free acid or partially or completely salified form.

12. The composition as claimed in claim 1, wherein the hydrophobic perfuming substance is chosen from the elements of the group consisting of the following olfactory families: Citrus, Floral, Fougère, Chypre, Woody.

13. A process for perfuming the skin, hair, scalp, lips, clothes or household linen, the process comprising at least one stage of applying to said skin, hair, scalp, lips, clothes or household linen of the composition as defined in claim 1.

14. A container comprising a spraying means and a packaging means, and containing a composition as defined in claim 1.

15. The composition as claimed in claim 2, wherein the emulsifying surfactant of water-in-oil type comprises at least one composition of alkyl polyglycosides represented by the formula (I):
R.sub.2—O—(X).sub.x—H  (I) in which x represents a decimal number of between 1.05 and 2.5, X represents the xylosyl or α,β-D-xylopyranosyl radical, obtained from the deletion of the hemiacetal hydroxyl group of α,β-D-xylopyranose, and R.sub.2 represents a radical of formula (II):
CH(C.sub.tH.sub.2t+1)(C.sub.vH.sub.2v+1)CH.sub.2—  (II) in which t is an integer of between 6 and 18, v is an integer of between 4 and 18 and the sum t+v is greater than or equal to 10 and less than or equal to 22.

16. The composition as claimed in claim 4, wherein the emulsifying surfactant of water-in-oil type comprises a fatty alcohol of formula (III):
CH(C.sub.tH.sub.2t+1)(C.sub.vH.sub.2v+1)CH.sub.2—OH  (III), in which t is an integer of between 6 and 18, v is an integer of between 4 and 18 and the sum t+v is greater than or equal to 14.

17. The composition as claimed in claim 2, wherein the emulsifying surfactant of oil-in-water type comprises: a compound of formula (IV):
R.sub.1—(C═O)[O—CH.sub.2—CH(OH)—CH.sub.2].sub.p—OH  (IV), in which p represents an integer greater than or equal to 1 and less than or equal to 15 and in which the group R.sub.1—(C═O)— represents a saturated or unsaturated and linear or branched acyl radical comprising from 6 to 22 carbon atoms, and/or a composition of alkyl polyglycosides represented by the formula (VI):
R.sub.3—O-(G).sub.x′-H  (VI) in which x′ represents a decimal number of between 1.05 and 2.5, G represents the glucosyl or α,β-D-glucopyranosyl radical, obtained from the deletion of the hemiacetal hydroxyl group of α,β-D-glucopyranose, and R.sub.3 represents a radical chosen from the elements of the group consisting of the n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl and n-behenyl radicals.

18. The composition as claimed in claim 3, wherein the emulsifying surfactant of oil-in-water type comprises: a compound of formula (IV):
R.sub.1—(C═O)[O—CH.sub.2—CH(OH)—CH.sub.2].sub.p—OH  (IV), in which p represents an integer greater than or equal to 1 and less than or equal to 15 and in which the group R.sub.1—(C═O)— represents a saturated or unsaturated and linear or branched acyl radical comprising from 6 to 22 carbon atoms, and/or a composition of alkyl polyglycosides represented by the formula (VI):
R.sub.3—O-(G).sub.x′-H  (VI) in which x′ represents a decimal number of between 1.05 and 2.5, G represents the glucosyl or α,β-D-glucopyranosyl radical, obtained from the deletion of the hemiacetal hydroxyl group of α,β-D-glucopyranose, and R.sub.3 represents a radical chosen from the elements of the group consisting of the n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl and n-behenyl radicals.

19. The composition as claimed in claim 4, wherein the emulsifying surfactant of oil-in-water type comprises: a compound of formula (IV):
R.sub.1—(C═O)[O—CH.sub.2—CH(OH)—CH.sub.2].sub.p—OH  (IV), in which p represents an integer greater than or equal to 1 and less than or equal to 15 and in which the group R.sub.1—(C═O)— represents a saturated or unsaturated and linear or branched acyl radical comprising from 6 to 22 carbon atoms, and/or a composition of alkyl polyglycosides represented by the formula (VI):
R.sub.3—O-(G).sub.x′-H  (VI) in which x′ represents a decimal number of between 1.05 and 2.5, G represents the glucosyl or α,β-D-glucopyranosyl radical, obtained from the deletion of the hemiacetal hydroxyl group of α,β-D-glucopyranose, and R.sub.3 represents a radical chosen from the elements of the group consisting of the n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl and n-behenyl radicals.

20. The composition as claimed in claim 5, wherein the emulsifying surfactant of oil-in-water type comprises: a compound of formula (IV):
R.sub.1—(C═O)[O—CH.sub.2—CH(OH)—CH.sub.2].sub.p—OH  (IV), in which p represents an integer greater than or equal to 1 and less than or equal to 15 and in which the group R.sub.1—(C═O)— represents a saturated or unsaturated and linear or branched acyl radical comprising from 6 to 22 carbon atoms, and/or a composition of alkyl polyglycosides represented by the formula (VI):
R.sub.3—O-(G).sub.x′-H  (VI) in which x′ represents a decimal number of between 1.05 and 2.5, G represents the glucosyl or α,β-D-glucopyranosyl radical, obtained from the deletion of the hemiacetal hydroxyl group of α,β-D-glucopyranose, and R.sub.3 represents a radical chosen from the elements of the group consisting of the n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl and n-behenyl radicals.

Description

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Preparation and Evaluation of Oil-In-Water Emulsions According to the Invention and of Comparative Oil-In-Water Emulsions.

1) Preparation of the Emulsions According to the Invention and of the Comparative Emulsions

[0232] Six emulsions according to the invention, denoted (F.sub.1) to (F.sub.6), and eight comparative emulsions, denoted (E.sub.1) to (E.sub.8), the proportions by weight of the constituents of which are recorded in table 1 below, are prepared, the contents by weight of the polyelectrolytes being indicated as percentage of polymeric dry matter, by employing the process below.

[0233] The constituents of the fatty phase are introduced successively into a beaker, mixed and brought to a temperature of 20° C. after a stage of heating at 40° C.; the mixing is carried out using a mechanical stirrer equipped with a propeller-type stirring rotor, at a speed of 100 revolutions/minute.

[0234] The constituents of the dispersed phase are mixed at ambient temperature in a beaker using a mechanical stirrer at a speed of 2000 revolutions/minute and the thickening agent is then gradually added.

[0235] The aqueous phase is added in one go to the fatty phase at ambient temperature and at a stirring speed of 4000 revolutions/minute with a stirrer equipped with a shearing rotor. This stirring is then maintained for five minutes. The preserving agent is subsequently added and the resulting mixture is mixed at ambient temperature and at a stirring speed of 4000 revolutions/minute with a stirrer equipped with a shearing rotor. The emulsion obtained is then drained out.

TABLE-US-00001 TABLE 1 Compositions (F.sub.1), (F.sub.2), (F.sub.3), (F.sub.4), (F.sub.5) and (F.sub.6) according to the invention (F.sub.1) (F.sub.2) (F.sub.3) (F.sub.4) (F.sub.5) (F.sub.6) Phase A Water  60%  60%  60%  60%  60%  60% Fluidifeel Easy.sup.(1)  1.2%  1.2%  1.2%  1.2%  1.2%  1% Emogreen L 15.sup.(2)  2%  0%  2%  0%  2%  2% C.sub.8—C.sub.10 triglyceride.sup.(3)  0%  2%  0%  2%  0%  0% “Raspberry” perfume.sup.(4)  2.5%  0%  2.5%  0%  2.5%  2.5% “Citrus tonic” perfume.sup.(5)  0%  2.5%  0%  2.5%  0%  0% Sepinov ™ EMT 10 .sup.(6)  0.4%  0.4%  0%  0%  0%  0% Sepimax ™ Zen.sup.(8)  0%  0%  0.4%  0.4%  0.2%  0.2% Fluidanov ™ 20X.sup.(7)  0.5%  0.5%  0.5%  0.5%  0.5%  0.5% Fluidifeel Easy.sup.(1)/ 2.4/1 2.4/1 2.4/1 2.4/1 2.4/1 2/1 Fluidanov ™ 20X.sup.(7) ratio Phase B Water q.s. q.s. q.s. q.s. q.s. q.s. 100% 100% 100% 100% 100% 100% Phase C Euxyl ™ PE 9010.sup.(9)  1%  1%  1%  1%  1%  1%

TABLE-US-00002 TABLE 2 Comparative compositions (E.sub.1), (E.sub.2), (E.sub.3), (E.sub.4), (E.sub.5), (E.sub.6), (E.sub.7) and (E.sub.8) (E.sub.1) (E.sub.2) (E.sub.3) (E.sub.4) (E.sub.5) (E.sub.6) (E.sub.7) (E.sub.8) Phase A Water  60%  60%  60%  60%  60%  60%  60%  60% Fluidifeel  1.2%  1.2%  1.2%  1.2%  1.5%  1.5%  1.5%  1.5% Easy.sup.(1) Emogreen  2%  0%  2%  0%  2%  0%  2%  0% L 15.sup.(2) C.sub.8—C.sub.10  0%  2%  0%  2%  0%  2%  0%  2% triglyceride.sup.(3) “Raspberry”  2.5%  0%  2.5%  0%  2.5%  0%  2.5%  0% perfume.sup.(4) “Citrus tonic”  0%  2.5%  0%  2.5%  0%  2.5%  0%  2.5% perfume.sup.(5) Sepinov ™  0.4%  0.4%  0%  0%  0.4%  0.4%  0.4%  0.4% EMT 10 .sup.(6) Sepimax ™  0%  0%  0.4%  0.4%  0%  0%  0%  0% Zen Fluidanov ™  0%  0%  0%  0.5%  0.5%  0.5%  1%  1% 20X.sup.(7) Fluidifeel — — — 2.4/1 3/1 3/1 3/2 3/2 Easy.sup.(1)/ Fluidanov ™ 20X.sup.(7) ratio Phase B Water q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. 100% 100% 100% 100% 100% 100% 100% 100% Phase C Euxyl ™  1%  1%  1%  1%  1%  1%  1%  1% PE 9010.sup.(9)
(1): Fluidifeel™ Easy is a mixture of lauryl polyglucoside with a degree of polymerization of 1.20, of myristyl polyglucoside with a degree of polymerization of 1.20 and of polyglyceryl-6 laurate, used as emulsifying agent of oil-in-water type to prepare cosmetic emulsions.
(2): Emogreen L15 is a mixture of linear alkanes, isoalkanes and cycloalkanes comprising from 15 to 19 carbon atoms used as an emollient agent for the preparation of cosmetic formulations.
(3): Caprylic/capric triglycerides is a mixture of equal weights of glyceryl trioctanoate and glyceryl tridecanoate.
(4) “Raspberry” perfume (or “raspberry in blue” by Luzi No. 650074) is a fruity fragrance with a floral and woody tendency.
(5) “Citrus tonic” perfume (or Tonic E) supplied by Mane is a fresh, floral and musky fragrance.
(6) Sepinov™ EMT 10: Polymeric thickening agent (INCI name: Hydroxyethyl Acrylate/Acryloyldimethyltaurate Acrylate Copolymer), used as rheology modifier for cosmetic formulations.
(7) Fluidanov™ 20X is a mixture comprising, per 100% of its weight, from 70% to 90% by weight of 2-octyl-1-dodecanol and from 10% to 30% by weight of 2-octyl-1-dodecyl polyxyloside.
(8) Sepimax™ Zen (INCI name: Polyacrylate Crosspolymer-6) is a polymeric thickening agent used as rheology modifier for cosmetic formulations.
(9): Euxyl™ PE9010 (INCI name: Phenoxyethanol & Ethylhexylglycerin): composition used as preserving agent.

2) Evaluation of the Properties of the Emulsions According to the Invention and of the Comparative Emulsions

[0236] 2.1 Evaluation of the Stability of the Compositions (F.sub.1) to (F.sub.6) According to the Invention and of the Comparative Compositions (E.sub.1) to (E.sub.8)
a) An amount of 100 ml of the composition to be tested and contained in a 250 ml flask is introduced into a climate-controlled chamber regulated at 25° C., for a period of time of one month.

[0237] The visual appearance of the composition tested is evaluated before stabilizing in the chamber and after a period of time of one month in said climate-controlled chamber, which appearance will be described according to observation either as “Homogeneous”, meaning a stability of the composition tested, or as “Heterogeneous”, meaning an instability of the composition tested.

b) An amount of 100 ml of the composition to be tested and contained in a 250 ml flask is introduced into a climate-controlled chamber regulated at 45° C., for a period of time of one month.

[0238] The visual appearance of the composition tested is evaluated before stabilizing in the chamber and after a period of time of one month in said climate-controlled chamber, which appearance will be described according to observation either as “Homogeneous”, meaning a stability of the composition tested, or as “Heterogeneous”, meaning an instability of the composition tested.

c) The results obtained are recorded in table 3 below.
2.2 Evaluation of the Spray Factor of the Compositions (F.sub.1) to (F.sub.6) According to the Invention and of the Comparative Compositions (E.sub.1) to (E.sub.8)
Principle: the objective of this test is to assess the quality of emission of fluid compositions through a spray nozzle.
This involves measuring the emission surface area of the fluid on a cardboard support positioned at a fixed distance from the spray packaging, so as to qualitatively assess the spraying of the fluid tested.
Thus, the fluids which are emitted in the form of a jet, with a very small spray surface area, do not offer a good spray, whereas the fluids emitted in the form of a high surface area provide a high spray quality.

Protocol:

[0239] The products to be tested are packaged in test bottles 24 hours before carrying out the evaluation and stored in a climatic-controlled chamber, the temperature of which is regulated at 20° C.
The 100 ml glass bottles are equipped with a spray nozzle of Pompa AVLP 18/415 type sold by Eurovetrocap SPA.
Cardboard folders are cut into strips 16 centimeters high and fixed to a support at a distance of 7 centimeters from the body of the spray bottle.
The spray devices are primed once and then a second priming pressure is applied before taking the measurements.
The spray devices are placed on the support 7 centimeters from the strip of cardboard folder, the nozzle facing said strip of cardboard.
Two consecutive pressures are then applied and the appearance of a damp stain on the strip of cardboard, in the form of a circular surface area, is observed.
The following are then measured, for each composition, without taking into account the scattered specks: [0240] the distance L.sub.1i corresponding to the width of the circular stain [0241] the distance L.sub.2i corresponding to the width of the circular stain Four measurements are carried out for each composition tested.
For each series of compositions for which it is desired to evaluate the spray factor, these measurements are also carried out with a suitable control fluid, in the case in point here with demineralized water and namely: [0242] the distance L.sub.1w corresponding to the width of the circular stain of the sprayed water [0243] the distance L.sub.2w corresponding to the width of the circular stain of the sprayed water.
The surface area is subsequently calculated for each composition (i) tested and for the water, followed by the “spray factor”, as follows: [0244] surface area composition tested (Si): Si=L.sub.1i×L.sub.2i×π/4 [0245] surface area water (Sw): Sw=L.sub.1w×L.sub.2w×π/4 [0246] spray factor (in %): SF=(Si/Sw)×100
In the context of the present invention relating to perfuming compositions, a spray factor (SF) will be deemed satisfactory and meeting the needs of the technical problem if it is greater than or equal to 50%, more particularly greater than or equal to 60%.

2.3 Results.

[0247] The results of the evaluations obtained for the compositions (F.sub.1) to (F.sub.6) according to the invention are recorded in table 3 below and those obtained for the comparative compositions (E.sub.1) to (E.sub.8) are recorded in table 4 below.

TABLE-US-00003 TABLE 3 Evaluation of the stability and of the spray factor of the compositions (F.sub.1), (F.sub.2), (F.sub.3), (F.sub.4), (F.sub.5) and (F.sub.6) according to the invention. (F.sub.1) (F.sub.2) (F.sub.3) (F.sub.4) (F.sub.5) (F.sub.6) Stability Homogeneous Homogeneous Homogeneous Homogeneous Homogeneous Homogeneous one liquid liquid liquid liquid liquid liquid month 45° C. Stability Homogeneous Homogeneous Homogeneous Homogeneous Homogeneous Homogeneous one liquid liquid liquid liquid liquid liquid month 25° C. Spray 80% 100% 68% 79% 90% 100% factor (SF) in %

TABLE-US-00004 TABLE 4 Evaluation of the stability and of the spray factor of the comparative compositions (E.sub.1), (E.sub.2), (E.sub.3), (E.sub.4), (E.sub.5), (E.sub.6), (E.sub.7) and (E.sub.8) (E.sub.1) (E.sub.2) (E.sub.3) (E.sub.4) (E.sub.5) (E.sub.6) (E.sub.7) (E.sub.8) Stability Hetero- Hetero- Hetero- Hetero- Hetero- Hetero- Hetero- Hetero- one month geneous geneous geneous geneous geneous geneous geneous geneous 45°C Stability Hetero- Stable Homo- Homo- Hetero- Hetero- Hetero- Hetero- one month geneous geneous geneous geneous geneous geneous geneous 25°C Spray factor n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. (SF) in %

2.4 Comments.

[0248] The results obtained show the need to include an emulsifying surfactant of water-in-oil type (S.sub.1), in this instance a composition of 2-octyldodecyl polyxyloside and of 2-octyl-1-dodecanol, in the perfuming composition in order to achieve the stability of the perfuming composition at 25° C. and 45° C., as shown by comparing (F.sub.1) and (E.sub.1), (F.sub.2) and (E.sub.2), (F.sub.3) and (E.sub.3), and (F.sub.4) and (E.sub.4). These results show the presence of this stabilizing technical effect for different perfumed substances and for different anionic polyelectrolytes (Sepimax™ Zen comprising a crosslinked anionic polyelectrolyte, the polymeric backbone of which comprises in particular a monomeric unit of formula (M1) and of hydrophobic nature, whereas the crosslinked anionic polyelectrolyte contained in Sepinov™ EMT10 is a crosslinked copolymer comprising only hydrophilic units).

[0249] The compositions according to the invention are also characterized by high spray factors, some of them being as effective as a spray of demineralized water used as positive control.