Background to the Invention Many biostatic agents currently used in plastics, paints or other coatings are organic in nature. A problem with biostatic agents of this type is that they tend to diffuse to the surface of the plastic or coating, and can then be leached out by weathering, or photochemically decayed by light. Furthermore, many such organic biostatic agents have limited biological acceptability, and therefore cannot be used for applications such as food or pharmaceutical packaging, where they may leak into and be dissolved by the contents of the package.

The biostatic activity of zinc oxide is well known, and it is of low toxicity. Accordingly, it has been used for many years for wound dressings, calamine lotion, baby products etc., where its mild soothing properties assist in healing skin infections, and injuries.

Zinc oxides for use in medical and pharmaceutical applications are normally prepared by the French process in which pure zinc metal is boiled from crucibles and oxidised by mixing the vapour with air. The purity of the product obtained from this process depends on the purity of the zinc metal used and selecting an appropriately pure metal allows pharmaceutical quality to be produced. Zinc oxides prepared by this process typically have a particle size in the range between 0. 1 and 1. 0 microns and a surface area of around 4 to 6 m2/g.

The biostatic activity of zinc oxide on paint moulds was reported by Salvin many years ago ; see Industrial and Engineering Chemistry (1944) 36 (4) : 336-340. More specifically, Salvin reported that the efficacy of zinc

oxide as a biostat was related to its particle size, with zinc oxides having small particle size and a concomitant large surface area giving the best results.

Summary of the Invention Surprisingly, and in contrast to the teachings of Salvin, it has now been found that a porous form of zinc oxide having an average particle size in the range 1 to 30 Am and having a surface area in the range 20 to 150 m2/g, is an effective biostatic agent, which out-performs the fine particle zinc oxide preferred by Salvin.

This novel biostatic agent finds particular use in the fields bf plastics, paints and other coating applications, due to the fact that it does not migrate and it is stable to light., Furthermore, as zinc oxide is non-toxic, it can be used. for applications such as food/beverage or pharmaceutical packaging.

Description of the Invention The zinc oxide of the present invention has a porous "sponge like"morphology, resulting in a large internal surface area. The zinc oxide has an average particle size in the range 1 to 30 Am and a surface area in the range 20 to 150 m/g, and preferably has an average particle size in the range 3 to 10 Am and a surface area in the range 40 to 100 m/g, most preferably 70 to 100 m2/g.

In the context of this Application, particle size is intended to mean particle diameter. In the case of the zinc oxide of the present invention average, or mean, particle size was measured by scanning electron microscopy and laser diffraction techniques. Measurement of specific surface area was conducted by gas adsorption and the BET method : Comparison with the specific surface area obtained through calculation based upon a spherical particle shape, indicates that at least 90% of the surface area, and typically more, for instance 99% of the specific surface area, can be attributed to internal porosity.

A further gas adsorption technique, the BJH method for determination of pore area distribution, has also revealed

that at least 50% of the surface area is attributed to pores of a maximum size of 30 nm, preferably with around 60% of the surface area being attributed to pores having a maximum size of 30 nm, more preferably 20 nm.

The zinc oxide of the present invention is manufactured by careful thermal decomposition of crystalline zinc compounds, such as the hydroxide, carbonate, basic carbonate, nitrate or carboxylate salts.

Typically it will be prepared by thermal decomposition of zinc carbonate under conditions of temperature and time selected to give the requisite high surface area. A preferred, commercially-available, form of the zinc oxide of the present invention is sold under the trade name Decelox Bio, by Elementis Pigments Limited, UK ; this material has a surface area of around 80 m2/g.

When required, the zinc oxide can be manufactured to pharmaceutical purity specifications for trace metals, by selecting a suitably pure starting material for the preparation process.

The zinc oxide of the present invention is particularly effective as a biostatic agent in plastic materials, paints and other coatings. It can be used in packaging materials, for instance containers and films, particularly for food or pharmaceuticals. The zinc oxide is heat stable at the temperatures typically encountered under plastic processing and use conditions, and is unaffected by light. The FDA and other regulatory bodies approve zinc oxide with a pharmaceutical purity for food packaging. Indeed, zinc oxide is essential for animals and human nutrition, and there is a recommended daily allowance (RDA) of 15 mg for the human diet. Furthermore, its enhanced W absorbing properties make the zinc oxide of the present invention useful in paints and coatings, particularly for exterior surfaces, enhancing the maximum period for which those surfaces can be exposed to the environment, before re-painting or re-coating is required.

The zinc oxide may be used alone to confer biostatic properties, or it may, advantageously, be used with another biostatic agent, depending upon its intended application.

For instance, the zinc oxide may be used in combination with an approved organic biostatic agent when its intended ,.... application is that of food/beverage or pharmaceutical packaging, or an approved inorganic biostatic agent.

Surprisingly good results, and indeed synergistic results, have been observed when the zinc oxide is used in combination with Triclosan, i. e. 2, 4, 4'-trichloro-2'- hydroxyphenyl ether (Irgasan DP 300, as supplied by Ciba).

Similar results may be obtained through the use of zinc oxide according to the present invention in combination with other, compounds of the same class as Triclosan, i. e. other substituted phenyl ethers, and in particular chloro- substituted phenyl and hydroxyphenyl ethers.

Other suitable organic biocides for use in combination with the zinc oxide of the present invention include phenolic'biocides other than Triclosan, for instance Bayer's Preventol range of products, and generally any biocide specifically sold for use in paint, plastics and coatings.

Suitable inorganic biocides for use in combination with the zinc oxide of the present invention include cuprous oxide, for instance for antifouling marine coatings, and other inorganic biocides sold for use in paints, plastics and coatings in general.

The amount of zinc oxide used will depend upon its intended application. Suitable amounts for use in food and pharmaceutical packaging range from 0. 1 to 25 weight %.

Suitable amounts for use in paints and other coating applications range from 0. 1 to 25 weight %.

At present, it is not understood why the zinc oxide of the present invention is such an efficient biostatic agent.

The smallest structural dimension of the microorganisms used in the studies upon which this invention is based is typically around 5 Am. This approximates to the diameter

of the zinc oxide used in the studies. However, the majority of pores of the zinc oxide have diameters two orders of magnitude smaller than the microorganisms under study, rendering them inaccessible to those microorganisms.

This means that up to around 90%, and typically up to around 99%, of the total surface area of the zinc oxide particles is inaccessible to the microorganisms to be treated, leaving only the small external surface area, which can be as little as 1% of the total surface area, accessible to those microorganisms. This renders the enhanced biostatic activity of the zinc oxide of the present invention particularly surprising.

The present invention is now further illustrated by the following Examples, and by reference to the accompanying drawings.

Examples Example 1-Qualitative Assessment of the Effect of Zinc Oxides on Microorganism Growth The two zinc oxides used were Zinc Oxide BP and a zinc oxide according to the present invention, Decelox Bio.

Zinc Oxide BP is a high purity zinc oxide tested to the British Pharmacopoeia specification, and having a typical specific surface area of 5 m2/g, as measured by gas adsorption and the BET method. Assuming that the primary particles are spherical in shape and have a particle density of 5. 606, this corresponds to a primary particle size of around 0. 2 hum. Zinc oxide BP is available from Elementis Pigments Limited.

As mentioned above, Decelox Bio is also available from Elementis Pigments Limited. Scanning electron microscopy and direct particle size measurement, by laser diffraction techniques, support the presence of particles having an average size of around 4 Am. If the particles are assumed to be spherical, this corresponds to an external surface area of 0. 27 m2/g. However, measurement of specific surface area by gas adsorption and the BET method indicates a typical value of 80 m2/g, such that greater than 99% of

the surface area of this zinc oxide can be attributed to internal porosity. A further gas adsorption technique, the BJH method for determination of pore area distribution, reveals the material to be porous and that approximately 60% of, the surface area is attributed to pores having a diameter of 20 nm or below.

Both types of zinc oxide were ultrasonically dispersed at levels up to 25% (w/v) in an equal volume mix of filter sterilised distilled deionised water containing 2% (v/v) Dispexs HDN (supplied by Ciba Specialty Chemicals) and polyethylene glycol (PEG) 200 (supplied by Ellis and Everard).

Sterile growth media, containing zinc oxide at 1% (w/v) andlDispexs HDN at 0. 1% (v/v), were prepared by adding, and mixing thoroughly, the appropriate amount of zinc dxide dispersion to sterile YPD media containing yeast extract (supplied by Oxoid), trypticase peptone (supplied by BBL), agar (supplied by Mikrobiologie) and glucose AnalaR, (supplied by BDH). 25 ml aliquots of the resulting mixtures were poured into separate sterile Petri dishes and allowed to solidify. The media prepared in this way were used immediately, or stored at 4°C until ready to use.

A control plate was prepared in a similar manner without the presence of zinc oxide, by mixing together filter sterilised water containing 0. 1% Dispex HDN with sterile YPD media.

Inoccula of microorganisms N. crassa (red bread mould, Phillip'Harris H51900/9) and A. oryzae (Phillip Harris 52020/7) were prepared by inoculating on to a YPD plate, incubating at 30°C for 48 hours, and then placing 5 ml of 0. 85% l (w/v) NaCl on to the surface of the plate. In both cases, the surface was rubbed with a sterile bent glass rod to suspend the microorganisms in the saline, and the resulting suspension was transferred to a sterile tube and adjusted to be equivalent to a 0. 5 McFarland Turbidity Standard.

Confluent growth on YPD control plates was achieved using N. crassa at 0. 5 McFarland Turbidity Standard and A. oryzae at a 1 in 10 dilution of the 0. 5 McFarland Turbidity Standard. The growth media containing the different zinc oxides were surface inoculated with N. crassa and A. oryzae in an amount which, on the control plates, gave near-confluent growth. The inoculated media were then incubated at 30°C for 48 hours, and microorganism growth was estimated visually taking into account the surface area covered and the density of growth.

The results, in terms of percentage growth inhibition, are reported in Table 1 below.

Table 1 Zinc Oxide N. crassa A. oryzae % inhibition % inhibition 1 % BP 60 40 1 % Decelox Bio >80 80 In the presence of 1% (w/v) of each of the two zinc oxides, N. crassa showed greater than 50 % inhibition of growth. There was a differential inhibition with the most effective inhibitor being Decelox Bio. A. oryzae was more resistant to the presence of zinc oxide, showing less inhibition than N. crassa in both cases, but growth of A. oryzae was also significantly inhibited. Decelox Bio was significantly more effective than Zinc Oxide BP in inhibiting the growth of the two test microorganisms.

Example 2-Quantitative Determination of the Inhibitory Effects of Zinc Oxides Example 1 was repeated using a smaller inoccula size to determine the effect of zinc oxide concentration on the number of colonies formed. Specifically, 100 Al N. crassa was used as the inocculm at 10-1 and 10-2 dilutions of the 0. 5 McFarland Turbidity Standard, and compared with the usual level of 10°. The results are presented in Table 2, below, in terms of the number of colonies formed.

Table 2 Zinc Oxide Percent Zinc Oxide in Medium Control (0%) 0. 0625 % 0. 25 % 1 % 5 % BP 49 30 23 26 6 Decelpx Bio 49 30 12 7 0 L The results demonstrate that there is a dramatic reduction in the number and the size of N. crassa colonies formed at concentrations of Decelox Bio above 0. 06 up to 5% (w/v). At a loading of 5% (w/v) no colonies were apparent after 4 days incubation at 30°C.

Example 3-Further Investigation of Quantitative Inhibitory Effects of Zinc Oxides Agar'plates containing various concentrations of Decelox Bio were prepared as described in Example 1. A well was made in the centre of each agar plate using a sterile corer to remove an 18 mm diameter section of agar.

Inoccula were prepared by mixing together a 1 ml aliquot of a suspension of microorganisms equivalent to a 0. 5 McFarland Turbidity Standard with 9 ml of molten, tempered, agar. A sample of inocculum was placed in the well in the centre of each plate, and the plates incubated at 30°C for 15 days. Measurement of the growth of the microorganisms was determined as total diameter of colony minus 18 mm.

The colony diameter was measured at intervals.

The effect of growing the two test microorganisms, N. crassa and A. oryzae, in the presence of various concentrations (0. 0625% ; 0. 25% ; 1% and 5%) of each of Decelox Bio and Zinc Oxide BP for 5 days was observed.

In all of the tests carried out there was a difference in the, susceptibilities of the two microorganisms to the effects of zinc oxide. N. crassa was found to be highly susceptible to zinc oxide treatments. However, A. oryzae was found to be more resistant in all cases.

Growth of N. crassa was strongly inhibited at each loading of zinc oxide compared to the control plate. Some growth was present, particularly at the lowest loading of 0. 0625%, but even at this level of Decelox Bio, a 77% inhibition was seen. A loading of 0. 25% Decelox Bio gave 98% inhibition of growth and greater concentrations of Decelox Bio resulted in complete inhibition of growth.

Similar results were obtained for plates inoculated with A. oryzae. In this case more growth was evident at a lower loading of zinc oxide. In the case of Decelox Bio, 42% inhibition of growth compared to the control was exhibited after 5 days incubation, by a 0. 0625% loading ; 87% by 0. 25% Decelox Bio ; and complete inhibition by 1% and 5% Decelox Bio.

The differential ability of Decelox Bio to inhibit mould growth was more evident when cultures were incubated for longer periods. Decelox Bio completely inhibited the growth of N. crassa at concentrations of 1% and 5% for the full incubation period of 15 days. Fungal growth was present at concentrations of 1% and 5% Zinc Oxide BP.

This observation means that quantification of the differential inhibitory activity of the two zinc oxide preparations is difficult, as there is not a linear relationship between concentration and inhibitory power.

Similar diameter colonies grew in the presence of 0. 25% of each of the zinc oxides ; at this concentration Decelox Bio about 50% more effective as a growth inhibitor than Zinc Oxide BP. However at a concentration of 1% or greater Decelox Bio completely inhibited growth, while the Zinc Oxide BP preparation allowed growth. At concentrations of 1% and 5%, under these growth conditions, Decelox Bio is an infinitely better inhibitor of the growth of N. crassa than Zinc Oxide BP.

Similar results were obtained in the case of A. oryzae. Growth was present at a loading of 1% of each of the zinc oxides but completely inhibited in the presence

formulation at a 5% loading. The diameter of the A. oryzae colony which grew on 1% Decelox Bio was approximately one third the size of that which grew on Zinc Oxide BP. At concentrations of 5%, Decelox Bio was an infinitely better inhibitor of the growth of A. oryzae than Zinc Oxide BP.

Example 4-Inhibitory Effect of Triclosan and Zinc Oxide in Combination Agar plates containing a number of concentrations of Triclosan (Irgasan DP 300, supplied by Ciba), were prepared in the presence and absence of 1% Decelox Bio. The plates were surface inoculated as in Example 1, and incubated for 5 days at 30°C. The inoculum used gave confluent growth on control plates containing no Triclosan or ZnO.

The results observed, in terms of surface area coverage and number of colonies, are reported in Table 4, below.

The test microorganisms exhibited similar Minimum Inhibitory Concentrations (MIC's ; the minimum concentration of a compound at which all growth of a particular microorganism is inhibited) when grown in the presence of Triclosan alone. A. oryzae had an MIC of 0. 0025% (25 ppm) Triclosan and N. crassa had an MIC of 0. 00125% (12. 5 ppm).

In the presence of Triclosan and 1% Decelox Bio the complete inhibition of growth of A. oryzae occurred in the plate containing 0. 0025% Triclosan/1% Decelox Bio, so the combination of chemicals was no more efficient at completely inhibiting growth of this microorganism than Triclosan alone. The amount of growth seen in plates containing Decelox Bio in addition to Triclosan was less than the growth seen at equivalent concentrations of Triclosan alone. On the plate containing 0. 00125% Triclosan/1% Decelox Bio this was evident in a reduction of the number of colonies to approximately one quarter of the number on the plate containing half that concentration of Triclosan.

Table 4 YPD media containing: N.crassa A.oryzae 0.000075% Triclosan (T) ++++ ++++ 0.00015% T +++ ++++ 0.0003% T +++ (4 colonies) +++ 0.0006% T +++ (1 colony) +++ 0.0012% T 0 ++ 0.0025% T 0 0 0.000075% T/1% Decelox Bio + (21 colonies) +++ 0.00015% T/1% Decelox Bio + (4 colonies) +++ 0.0003% T/1% Decelox Bio + (1 colony) ++ 0.0006% T/1% Decelox Bio 0 ++ 0.0012% T/1% Decelox Bio 0 + (25% of colony numbers) 0.0025% T/1% Decelox Bio 0 0 YPD control ++++ ++++ 1% Decelox Bio only + (20 colonies) 3+ Symbol Key :<BR> 0 No apparent growth on sample.<BR> <P>+ No growth originating from the centre of the sample ; <20% sample coverage.<BR> <P>++ No growth originating from the centre of the sample ; 20-50% sample<BR> coverage.<BR> <P>+++ Greater than 50 % of sample surface covered.<BR> <P>++++ Sample completely covered.

The. effect of Decelox Bio in combination with Triclosan was more pronounced on the growth of N. crassa ; the MIC in the absence of Decelox Bio was 0. 00125% and this was reduced to 0. 00062 when 1% Decelox Bio was present. In addition, the number of colonies that grew was much reduced. On the plate containing 0. 000075% Triclosan and 1% Decelox Bio only 21 colonies grew ; this was mirrored by the control plate which contained 1% Decelox Bio only (20 colonies} and confirmed the results reported in Example 2.

Example 5-Inhibition of Growth on Paint and Polymer Samples Paint and polymer film samples covering an area of 2 cm2 were prepared. The paint film samples were of 100 pm dry film thickness and supported on Leneta polymer sheet.

The polymer film samples were 200 um thick. The samples were sterilised by immersion in 70% ethanol for 5 minutes and air dried. Each sample was applied to the surface of a cushion of agar in a Petri dish ; the agar contained no nutrients. A microorganism inocculum, prepared as in Example 1, was mixed with 4 ml of molten YPD agar and added to the surface of the paint or polymer sample. The only nutrients available to the microorganism were, therefore, in the top YPD layer. a) Paint samples Paint samples were prepared according to the formulations described in Tables 5 and 6, below. In each Table amounts of components are given in g, and the weight % of zinc oxide based on the resin solids in the dried paint film is also given. The A. 1-K. l series was an emulsion paint with a pigment volume concentration of 50% ; the A. 2-K. 2 series was an exterior silicone emulsion paint.

In the emulsion paint (A. l-K. 1), the"mill base" consisted of 14. 9 parts by weight (pbw) water, 0. 10 pbw Calgons N, 0. 10 pbw Parmetols A26, 0. 10 Pigmentverteilers A,. 0. 30 : pbw Agitan° 280, 5. 80 pbw Kronoss 2190, 9. 60 pbw Omyacarbs 2 GU, 11. 70 OmyacarbX 5 GU, 5. 80 Omya Hydrocarb°,

3. 40 pbw Talkum IT Extra@, 3. 80 pbw Socals P2, 1. 50 pbw aluminium silicate P820 and 0. 80 pbw Texanol@. The"mill base"for the exterior silicone emulsion paint (A. 2-K. 2) consisted of 20. 00 pbw water, 0. 40 Bentones LT, 0. 20 pbw Agitant E255, 0. 20 pbw Acticides SPX, 0. 05 pbw Calgons N, 0. 30 pbw Lopons 890, 12. 00 pbw Kronoss 2190, 5. 00 pbw Omyacarb 2 GU, 10. 00 pbw Omyacarbs 5 GU, 5. 00 pbw Talkum ASE 10, 5. 00 pbw Plastorits 000 and 0. 10 pbw 10% sodium hydroxide.

Table 7 shows the results of 5 days incubation of N. crassa and A. oryzae in the presence of paint samples ; further incubation did not result in further growth of the microorganisms, probably due to the limited nutrients available to the microorganisms.

The formulation of each set of paint samples appeared to modulate the efficacy of the inhibitory properties of the different zinc oxides ; more growth of each of the microorganisms was generally present in the A. 2-K. 2 series, particularly at lower loadings. In both formulations, however, there was a clear dose-dependant effect on growth ; higher concentrations of Zinc Oxide resulted in more inhibition and therefore reduced growth.

As in previous examples, N. crassa was seen to be more susceptible to inhibition by Zinc Oxide than A. oryzae.

Table 5 Sample Sample Sample Sample Sample Sample Sample A.1 B.1 C.1 D.1 E.1 F.1 K.1 Mill base 232.6 232.6 232.6 232.6 232.6 232.6 232.6 zinc Oxide BP 0.16 0.64 3.2 --- --- --- --- Decelox Bio --- --- --- 0.16 0.64 3.2 --- Water 38.04 37.56 35.00 38.04 37.56 35.00 38.2 Acronal 290 D 128.4 128.4 128.4 128.4 128.4 128.4 128.4 AMP 90 0.8 0.8 0.8 0.8 0.8 0.8 0.8 Benaqua 1000 0.25 0.25 0.25 0.25 0.25 0.25 0.25 ZnO based on 0.25 % 1.0 % 5.0 % 0.25 % 1.0 % 5.0 % --- resin solids [%]

Table 6 Sample Sample Sample Sample Sample Sample Sample A.2 B.2 C.2 D.2 E.2 F.2 K.2 Mill base 273.0 273.0 273.0 273.0 273.0 273.0 273.0 Zinc Oxide BP 0.16 0.64 3.2 --- --- --- --- Decelox Bio --- --- --- 0.16 0.64 3.2 --- Water 30.84 30.36 27.80 30.84 30.36 27.80 31.00 Acronal S 716 44.0 44.0 44.0 44.0 44.0 44.0 44.0 Wacker BS 2143 42.0 42.0 42.0 42.0 42.0 42.0 42.0 Wacker BS 1306 4.0 4.0 4.0 4.0 4.0 4.0 4.0 Rheolate 278 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Lusolvan FBH 4.0 4.0 4.0 4.0 4.0 4.0 4.0 ZnO based on 0.25 % 1.0 % 5.0 % 0.25 % 1.0 % 5.0 % --- resin solids [%]

Table 7 Sample Code Zinc Oxide based on A. oryzae N. crassa resin solids (%) A. 1 0. 25 BP ++ +++ B. 1 1. 0 BP + + C. 1 5. 0 BP + 0 D. 1 0. 25 Decelox Bio ++ ++ E. 1 1. 0 Decelox Bio ++ + F. 1 5. 0 Decelox Bio + 0 K. 1 No Zinc Oxide +++ ++++ A. 2 0.36 BP +++ ++++ B. 2 1. 43 BP ++ + C. 2 7. 15 BP ++ + D. 2 0. 36 Decelox Bio +++ +++ E. 2 1. 43 Decelox Bio ++ ++ F. 2 7. 15 Decelox Bio ++ K. 2 No Zinc Oxide +++ ++++ The key to the symbols used is the same as that for Table 4. b) Polymer samples Low density polyethylene (LDPE) films, 200 Hm thick, were prepared, containing 0. 3, 1 and 5 weight % Decelox Bio. Film without the addition of zinc oxide was also prepared. The film containing no zinc oxide supported less vigorous growth than the paint sample controls. Both microorganisms failed to completely cover the polymer sample with growth following 5 days incubation. The effects of including different loadings of Decelox Bio in the polymer. sample are reported in Table 8, below.

Table 8 Decelox Bio A. oryzae N. crassa No ZnO, control +++ +++ 0. 3% ++ ++ 1% ++ + 5% + 0

The key to the symbols used is the same as that for Table 4.

Example 6-UV absorbing properties A further advantage of the zinc oxide of the present invention when incorporated into plastics or paints is that it shows a surprisingly enhanced W absorption compared with an aqueous dispersion of the zinc oxide. This is illustrated in Figure 1.

Figure 1 shows W/visible spectra of Decelox Bio, a zinc oxide according to the present invention, in different media.

W/visible spectrum (A) was obtained from a 2% loading of Decelox Bio in water, dispersed with the aid of an ultrasonic probe and placed in a 100 Um path length measuring cell. W/visible spectrum (B) was obtained from a 1% loading of Decelox Bio in a 200 Am polythene film.

In both cases, the product of the loading and path length is equal, hence for similar performing materials, the absorption curves should be similar. However, better UV absorption was observed in the polyethylene film sample.