AU653708B2 - Biological cryopreservation - Google Patents

Description

WO 91/01637 PCT/GB90/01268 1 BIOLOGICAL CRYOPRESERVATION This invention relates to the cryopreservation of eggs belonging to the phylum Mollusca, and in particular to the cryopreservation of unfertilised eggs, so that they may be used outside the breeding season, for example in hatcheries or for use in bioassays.

Various assays and techniques are known for the detection and quantification of pollutants in water, especially sea water. Bioassays employ living matter and usually expose a living organism to the pollutant at a particular stage in its development, which is then monitored. Such bioassays are already known from the following art: C.E. Woelke, "Development of a receiving water quality bioassay criterion based on the 48hr Pacific oyster (Crassostrea gicas) embryo", Washington Dept. of Fisheries Technical Report 9, 1-93(1982); "Conducting static acute toxicity tests with larvae of four species of bivalve molluscs", Designation E724-80, Annual Book of ASTM Standards, American Society for Testing and Materials, 1-17 (1980); A.R.D. Stebbing et al, "The Effects of Stress and Pollution on Marine Animals", Bioassay, New York, 133- 140 (1985); and J.E. Thain and J. Watts, "The use of a bioassay to measure changes in water quality associated with a bloom of Cryodinium aureolum, Hulburt Rapp. P-v Reun, Cons.Int.Explor.mer 187, 103-107 (1987).

WO 91/01637 PCI/GB90/01268 2 The cryopreservation of sperm is disclosed in the following documents: J.E. Lannan, Genetics 68:599-601 (1971), Experimental self-fertilisation of the Pacific Oyster Crassostrea VirQinica; and J.B. Hughes, Cryobiology 10:342-344 (1973), An examination of eggs challenged with cryopreserved spermatazoa of the American Oyster, Crassostrea Virginica; and S.R. Zell, M.H. Barnford and H. Hidu, Cryobiology 16:448-460 (1979), Cryopreservation of the spermatazoa of the American Oyster, Crassostrea Virginica.

In a modified assay sperm alone is exposed to the pollutant and thereafter used to fertilise eggs, see P.A. Dinnel et al, "Improved methodology for a sea urchin sperm cell bioassay for marine waters", Arch.Environ. Contam.Toxicol. 16, 23-32 (1987) Sperm is used to fertilise eggs and then two parameters are recorded, namely the percentage fertilisation and the pattern of development of the surviving embryos.

One known bioassay for water pollutants monitors the development of molluscan embryos from fertilisation to the prodissoconch stage (straight-hinge larvae), see Annual Book of ASTM standards, supra. However, such assays are limited in that they can only be performed at those times of the year when the molluscs concerned are in a reproductive condition. The use of WO 91/01637 PCT/GB90/01268 3 some laboratory conditioning techniques to induce unseasonal reproduction can extend availability but this is at the expense of cost and inconvenience. Thus the basic problem of inadequate availability of molluscan embryos outside the breeding season remains.

Furthermore, the use of bioassays is restricted to locations where reproductive molluscs are available, which precludes its use in organisations and places that do not have the appropriate wet facilities.

The present invention seeks to overcome or at least mitigate these problems, and seeks to allow bioassays to be conducted conveniently all year round and without a significant increase in costs.

Therefore, according to a first aspect of the present invention there is provided a process comprising cryopreserving unfertilised eggs of the phylum Mollusca. By cryopreserving (freezing) the eggs they can be stored for long periods of time, sufficient for survival of the eggs through the unseasonal periods of the year. The eggs can then be thawed at a convenient time prior to use in a hatchery or a bioassay.

By this process the availability of eggs can be extended to any time of the year, and in particular to these periods when the eggs are not naturally abundant.

In addition, the present invention may provide egg stocks for hatcheries out of season, the capability to extend the stocking season and allow increased control of production cycles.

The process of the present invention may have the WO 91/01637 PCT/GB90/01268 4 additional advantage of providing a level of comparability and cross-referencing (such as between bioassays) that is not possible with prior art techniques which use fresh eggs each time. Thus the present invention allows one to work with the same batch of eggs in assays over a long period of time, even years, since the cryopreservation method allows a batch of eggs to be stored for such a time, whereas prior art techniques allow only a few hours in which to obtain consistent experimental results. In addition, the process of cryopreservation of the present application and the advantages that may result regarding storage allow eggs (for example from the same batch) be transported, such as for export, worldwide.

Further benefits of cryopreservation and storage of eggs may be realised in the form of conservation of genetic material and the maintenance of disease-free stocks. In addition the invention may assist in the protection of valuable stocks of eggs against disease or pollution and may allow for the maintenance of genetically valuable eggs such as the collection and storage of exotic species, foreign species and certified disease-free populations.

Particularly suitable eggs are those belonging to the class Bivalvia (Pelecypoda), for example the subclass Lamellibranchia, such as the super order Pteriomorphia, Heterodonta or Palaeoheterodonta such as the orders Ostridae, Mytilidae, Pectimidae or Veneridae. Preferred eggs are from the genera Mytilus, Ostrea, Patinopectin, Arctopecten, Venus, Mercenaria, Tapes or Crassostrea, such as mussels, molluscs and oysters can WO 91/01637 PCT/GB90/01268 be used, especially eggs of the genus Crassostrea.

Particularly preferred species include: Crassostrea gigas Pacific oyster Tapes hillipinarum Manila clam Crassostrea virginica American oyster Mercenaria mercenaria Hard clam Tapes decussata Native palourde Argopecten irradians Bay scallop Generally the eggs are cryopreserved in any suitable aqueous medium which advantageously contains a cryoprotectant. However, the aqueous medium is preferably not saline. The eggs may be suitably removed from the animal, for example by surgical excision,before they are exposed to sea water. Thus, for example, the eggs are removed before natural spawning and placed directly into a cryopreservation medium. If the eggs are liberated into sea water before addition to the medium then this may lead to low viability following freezing and thawing. It is speculated that this is due to changes in permeability of the egg membrane which occurs on exposure to sea water which may result in a lowered permeability to the cryoprotectant. The excised female gonadal material which may contain other debris as well as the eggs, may need to be purified before cryopreservation, for example by filtration.

There is no precedent for selecting eggs at this stage, prior to release, since the eggs are arguably 'unready' WO 91/01637 PCT/GB90/01268 6 for fertilisation at this stage. .Furthermore, there is no precedent for keeping eggs away from the medium in which they would normally continue their development (that is to say sea water) and still be receptive to fertilisation.

Prefered methods of cryopreservation are disclosed in EP-A-0246824 and International Patent Application entitled "Nucleation of Ice" filed on 13th August 1990 in the name of Cell Systems Ltd., although any suitable cryoprotection protocol may be employed that ensures survival of a sufficient proportion of the eggs for the protocol to be practical. Thus the eggs are preferably cryopreserved in an aqueous medium in contact with an organic solid. The solid may act as a nucleating agent which causes water in the aqueous medium to be nucleated at or near the freezing point of the medium, to minimise damage to the eggs. Suitable organic solids include steroids, amino acids, oligomeric or polymeric amino acids, and polyhydroxylated compounds.

Cholesterol is especially preferred, especially if it has been crystallised from either methanol or acetic acid. Cholesterol crystallised from methanol is the ice nucleator of choice.

The organic solid may be added to the aqueous medium for example at a concentration of from 0.0001 to 0.001 g/ml, such as 0.2mg/ml or above. Preferably the medium contains from 0.25 to 1.5 mg/ml of cholesterol, optimally from 0.75 to 1.25 mg/ml. However, conveniently the organic solid can be coated on to a substrate that is in contact with the aqueous medium.

The substrate will usually be a vessel in which -1-V WO 91/01637 PCT/GB90/01268 7 cryopreservation takes place, such as an ampoule, straw, bag or tube. The organic solid may also be provided on polymer beads for example acrylic beads, such as are available from Bio-Rad, eg. Bio-Beads Sm7.

Coating densities of the organic solid on the substrate are suitably at least 0.0007 mg/mm 2 for example within a range 0.001 to 0.1 mg/mm 2 with about 0.0035 mg/mm 2 being optimal.

The aqueous medium may contain additional soluble components (such as a cryoprotectant, sugar and/or salt at a concentration range of from almost zero concentration (infinite dilution) up to high concentrations provided that there is still free water available to be frozen. Preferably the aqueous medium is provided with a cryoprotectant, for example glycerol and/or dimethyl sulphoxide. Suitably the cryoprotectant is present in an amount of from 1 to for example from 5 to 15% v/v, such as about The aqueous medium may also contain a sugar, such as mannitol and/or trehalose, for example at a concentration of from 0.1M to 10.0M, more preferably from 0.8M to 1.2M, with about 1.OM being optimal.

To ensure cryopreservation the eggs should be cooled to at least -30 0 C. However, once cooled the eggs can then be transferred to liquid nitrogen for long term storage if desired. Short term storage can be achieved in deep freezes at about -80 0 C. The preferred storage temperature is below -135 0 C, below the glass transition temperature of water, which can be attained in mechanical refrigerators. This is preferably achieved WO 91/01637 PCT/GB90/01268 8 using liquid nitrogen, which has a boiling point of -196 0 C. It should be noted that the eggs should be cooled with care; rapid cooling can be fatal. The cooling protocol optimally includes at least one isothermal hold, and preferably two or three, each of which usually lasts from 1 to 10 minutes, such as from 4 to 8 minutes. The eggs will usually be cooled starting from room temperature, for example about 20 0

C.

The preferred cooling protocol is as follows: optionally cooling the eggs at a rate of from 2 to 6 0 C/minute to a temperature of from +3 to -5 0

C;

S(b) optionally maintaining the eggs at about that temperature for from 4 to 6 minutes; cooling the eggs at a rate of from 2 to 6 0 C/minute to a temperature of from -15 to 0

C;

maintaining the eggs at about that temperature for from 3 to 7 minutes; further cooling the eggs at a rate of from 2 to 60C/minute to a temperature of from -30 to 0

C;

optionally maintaining the eggs at that temperature for up to 2 minutes; and optionally plunging the eggs into liquid nitrogen.

WO 1/01 637 PCT/GB90/01268 9 The most preferred cooling protocol comprises: cooling eggs of the phylum Mollusca, at a rate of about 3 0 C/minute to a temperature of about g0C; maintaining the eggs at about 0° C for about minutes; further cooling the eggs at a rate of about 3°C/minute to a temperature of about maintaining the eggs at a about that temperature for about 5 minutes; cooling the eggs at a rate of about to a temperature of about -35 0

C;

maintaining the eggs at about -35 0 C for no longer than one minute; and plunging the eggs into liquid nitrogen.

According to a second aspect of the present invention there is provided cryopreserved unfertilised eggs of the phylum Mollusca. Prefered features and characteristics of the second aspect of the invention are as for the first mutatis mutandis. The invention also extends to providing cryopreserved unfertilised eggs of the phylum Mollusca, the process comprising a process of the first aspect and subsequent thawing the eggs.

WO 91/01637 PCT/GB90/01268 The invention in its broadest terms contemplates various bioassays for the detection of, eg. pollutants.

A third aspect of the present invention relates to a method of conucting a bioassay, the method comprising contacting cryopreserved unfertilised eggs of the phylum Mollusca or larvae resulting from the fertilisation of a cryopreserved unfertilised egg of thie phylum Mollusca, with a sample to be assayed.

Thus it will be appreciated that the bioassay may be conducted on the cryopreserved eggs themselves, or the eggs fertilised and the bioassay conducted on the resultant larvae.

Thus a preferred method for conducting a bioassay comprises: cryop.eserving unfertilised eggs of the phylum Mollusca; thawing the eggs; optionally fertilising the eggs to produce larvae of the phylum Mollusca; contacting a sample to be assayed with the eggs, or if is conducted with the larvae.

Although once the eggs are fertilised they develop through several stages, inducing embryo, veliger and WO 91/01637 PCT/GB90/01268 11 prodissoconch stages, the term "larvae" here is intended to cover all these stages; that is, the organism resulting from the fertilisation of egg with sperm, from the moment of fertilisation onwards.

The bioassly (contact with the sample) preferable lasts for no longer than 48 hours, since there is a danger that the larvae will starve without feeding. Preferred larvae for bioassays are 48 hours old, or in the prodissoconch 1 stage (ie. post-trocophone stage).

Usually both the median lethal concentration (LC50) and median effective concentration (EC50), based on abnormal shell development, are measured.

During the bioassay the larvae are preferably kept in an aqueous medium, such as saline, suitably at from to 25 0 C. The aqueous medium is preferably aerated.

Advantageously the medium is agitated frequently, preferably with a perforated plunger to avoid damaging the larvae.

The density of the larvae should preferably be below 100 per ml, and suitably above 1 per ml. Preferred ranges are from 10 to 50 per ml, optionally from 15 to per ml.

Bioassays assay by biological function, productivity, development or performance to determine the effect of a substance or subtances in the sample or conditions presented to the biological material involved in the bioasay. The sample may comprise a toxicant or WO 91/01637 I' /GB90/01268 12 pollutant, or toxicant although bioassays are not limited to substances which may ha" a negative effect on biological activity. Bioassays can be used to determine the effects (if any) of various additives, for example paint additives, on various life forms which might be relevant for paint that is to be painted on oil rigs.

The sample may be taken from the environment suspected of being polluted, for example sea water, optionally diluted, or it may be a suspension of a sample, usually in water. The terms "pollutant" and "toxicant" include any substance that may be considered harmful, noxious, dangerous or even toxic to any living matter especially wildlife and humans, and not necessarily harmful, noxions, dangerous or toxic only to unfertilised eggs of the phylum Mollusca. However, it will be appreciated that the term "sample" not only encompasses pollutants, but any substance whose effect on biological activity is to be investigated, including positive effects. Indeed, some bioassays are not conducted with environmental samples and so "sample" here includes analytical or reagent grade chemicals that mey be obtained commercially. The exact practical procedure of such bioassays will be well known to those skilled in the art.

The unfertilised eggs will have suitably been first cryopreserved in accordance with the first aspect of the present invention. In a preferred embodiment a method of conducting a bioassay comprises: cryopreserving unfertilised eggs of the WO 91/01637 PCT/GB90/01268 phylum Mollusca; thawing the unfertilised eggs; fertilising the eggs to produce larvae; and contacting the larvae with a sample to be assayed, for example one suspected of comprising a pollutant or toxicant.

Any suitable cryopreservation and thawing protocol may be employed provided that sufficient unfertilised eggs survive for the protocol to be practical. Thawing may take place at a temperature of from 15 to 25 0 C, for example about room temperature, either in air or suitably in a liquid medium such as water. The eggs may be filtered gently, for example on a 15 micron filter, before use in the bioassay.

Thus for preference the bioassay is conducted on larvae of the phylum Mollusca. Thus in order to allow such bioassays to be conducted outside of the breeding season the fertilising sperm is for preference also cryopreserved. The cryopreservation techniques described for the first aspect in relation to eggs are applicable to sperm mutatis mutandis However, a particularly preferred cooling protocol for the sperm is to add the sperm to the aqueous medium (preferably 1.OM mannitol, 1.OM trehalose, 15% v/v DMSO) in a volume ratio of about 1:1. The mixture may then be placed in a 0.5ml straw and cholesterol crystallised from methanol (eg. 0.2mg/ml) added. This may then be cooled from room temperature to -100 0 C at a rate of from 40 to 60 0 C/minute, such as about 50 0 C/minute. It WO 91/01637 PCT/GB90/01268 14 may then be plunged into liquid nitrogen. The sperm is preferably thawed by immersion in, eg. water, at about 0 C. This protocol may give 88% and abov fertilisation rates when compared with unfrozen controls.

Thus in a particularly preferred embodiment a method of conducting a bioassay comprises: separately cryopreserving unfertilised eggs and sperm of the phylum Mollusca: thawing the eggs and sperm; fertilising the eggs with the sperms to give larvae of the phylum mollusca; contacting the larvae with a sample to be assayed.

A fourth aspect of the present invention relates to a bioassay kit comprising cropreserved unfertilised eggs of the phylum Mollusca and means for contacting a sample with the unfertilised eggs or larvae resuting from fertilisation of the eggs. Thus the kit may comprise one or more containers or wells in which the eggs or lavae and sample can be brought into contact with each other. The kit may also possess a surface at least partially coated with an organic solid ice nucleator as described in the first aspect (such as on the inside of a container or well). The kit preferably also comprises cryopreserved sperm. This allows WO 91/01637 PCT/GB90/01268 bioassays to be conducted whenever and wherever it is convenient to do so, without having to rely on the availability of either eggs or sperm.

Other preferred features and characteristics of the fifth aspect are as for previous aspects mutatis mutandis.

The invention will now be described by way of example, which is profided by way of illustration and not to be contsrued as being limiting to the scope of the present invention.

Example Mature animals of the species Crassostrea gcrias were selected from a laboratory maintained and conditioned population that had been established as being in reproductive condition. The animal had its external surfaces blotted dry and was then opened. All internal fluids were blotted dry and the gonadal tissue exposed.

ThL outer membrane of the gonad was pierced with a clean, fine-bore glass pasteur pipette and the enclosed material removed gently by the application of negative pressure. Material was extracted until it was no longer easy to fill the pipette without marked mechanical agitation.

The gonadal material was transferred to a dry petri dish during collection and the final gonadal suspension WO 91/01637 PCT/GB90/01268 16 layered onto a 15 micron seive and, as far as practicable, the ovarian fluid removed. The eggs were then placed directly into the appropriate cryoprotective agent (CPA).

CPA: 0.85M Trehalose and 15%v/v DMSO in distilled water.

Eggs were loaded at between 0.5 to 1.0 million per ml into 0.5ml plastic straws coated with cholesterol. At exactly 10 minutes after the introduction of the eggs into the CPA the cooling protocol was initiated. The protocol was: Cool at 3 0 C per minute from 20 0 C to OOC; Hold at 0 C for 5 minutes; Cool at 3°C per min from 0 C to -200C; Hold -20 0 C for 5 minutes; Cool at 3 0 C per minute to -35 0

C;

Hold at -35 0 C for no more than 1 minute; and Plunge into liquid nitrogen.

Thawing was by totally immersing the straw in water at

C.

When the last region of ice had just disappeared the end of the straw was snipped off and the contents blown into 0.5ml of sea water at room temperature. After sec lml of sea water was added and left for 5 min.

Then a further Iml of sea water was added every 5 min up to a volume of 10ml. The eggs were pipetted onto a micron filter and then resuspended, above the filter, in clean sea water for 1 min with very gentle agitation. The eggs were then taken off the filter and WO 91/01637 PCT/GB90/01268 17 resuspended in 200ml of clean sea water.

The eggs were left to rehydrate for a minimum of minutes prior to insemination of 25 0 C in filtered seawater.

Using eggs of Crassostrea gigas, 55% ofthe intact, mature oocytes that had recovered from cryopreservation were fertilised using fresh spermatazoa stripped from an approprately conditioned male. The derived embryos were cultured in filtered, UV treated seawater at 25 0

C

for 24 hours. Development to D-larvae was comparable for control material, where eggs were unfrozen, and experimental material where eggs were cryopreserved.

Claims (16)

1. A process comprising cryopreserving unfertilised eggs of the phylum Mollusca in a non-saline aqueous medium.

2. A process as claimed in claim 1 wherein the eggs belong to the genus Crassostrea.

3. A process as claimed in claim 1 or 2 wherein the aqueous medium is in contact with solid cholesterol.

4. A process as claimed in claim 3 wherein the cholesterol is coated on a substrate.

5. A process as claimed in any preceding claim wherein the eggs are cooled to a temperature of at least -30 0 C.

6. A process as claimed in any preceding claim comprising: optionally cooling the eggs at a rate of from 2 to 6 0 C/minute to a temperature of from +3 to optionally maintaining the eggs at about that temperature for from 4 to 6 minutes; cooling the eggs at a rate of from 2 to to a temperature of from -15 to -250C; maintaining the eggs at about that temperature for from 3 to 7 minutes; further cooling the eggs at a rate of from 2 to 6C/minute to a temperature of from -30 to optionally maintaining the eggs at that temperature for up to 2 minutes; and optionally plunging the eggs into liquid nitrogen. S.7. A process as claimed in claim 6 comprising: cooling the eggs at a rate of about 30C minute to a temperature of about 0 C; 30 maintaining the eggs at about 0 C for about 5 minutes; further cooling the eggs at a rate of about 7°C/minute to a temperature of about -200C; maintaining the eggs at about that temperature for about 5 minutes; 19 cooling the eggs at a rate of about 3 0 C/minute to a temperature of about -35 0 C; maintaining the eggs at about -35 0 C for no longer than one minute; and plunging the eggs into liquid nitrogen.

8. A process as claimed in claim 6 further comprising thawing the eggs.

9. Cryopreserved unfertilised eggs of the phylum Mollusca which are, or have been, cryopreserved according to a process as claimed in any preceding claim. Cryopreserved eggs according to claim 9 which belong to the class Bivalvia.

11. A method of conducting a bioassay comprising: cryopreserving unfertilised eggs of the phylum Mollusca; thawing the unfertilised eggs; fertilising the eggs to produce larvae as herein before defined of the phylum Mollusca; and I contacting the larvae with a sample to be assayed.

12. A method as claimed in claim 11 wherein the eggs or larvae and sample are contacted for no longer than 48 hours and/or the larvae are at, or beyond, the prodissoconch 1 stage.

13. A method as claimed in claim 11 wherein the larvae are kept in saline at a temperature of from 15 to 25 0 C. :14. A method as claimed in claim 13 wherein the saline is frequently agitated using a perforated plunger.

15. A method as claimed in claim 14 wherein the density of the larvae is from 15 to 30 per mL saline. 30 16. A method as claimed in claim 11 wherein the cryopreservation is by a process as claimed in any of claims 1 to 8 and/or where the cryopreserved unfertilised eggs used are according to claim 9 or 20

17. A bioassay kit comprising cryopreserved unfertilised eggs of the phylum Mollusca according to claim 9 or 10 and means for contacting a sample with the unfertilised eggs or larvae as hereinbefore defined resulting from fertilisation of the eggs.

18. A bioassay kit as claimed in claim 17 additionally comprising cryopreserved sperm. DATED this 9 day of August 1994 CELL SYSTEMS LIMITED Patent Attorneys for the Applicant: F.B. RICE CO. ,i e i C o*o* INTERNATIONAL SEARCH REPORT 1. CLI.8IpI~jATFON Of 29UU.1CY M*TfTW fit several ~eooflagtilo gvMouia spoly, Indlftalo At" 4 AuuWaln# to Matttlanli Patint CiiaatftCAtbn UP~C) Ur1 to inhoMital CjAa&flJr~tmn bug i110 IPCS A 01 X 1/02, G 01 N 33/50 if. P19LOS5A1C~ clanitimlla SWAMnttiu I~r~ Documentaion~ 54:Mo other thn~l Min0mul" Ocuumntlian the 104111i thitt 09611 DIGUFMmntoWo an, t.lIdUe In the Pfoldl Ovarche.' Ilt. DoauUMI CON410111119TO 1 11119LSVAN 1 14 Categoryp ho of Datauif^ 01OoumM wilth Indleaion what. hoorollie, of the wilavsyt pasie,2 Itelaill toC liti No0., 'C BiOlOgical Abstrcts? voluine 52, nlo. M~ay 1971, (PhiladelPhia, PA, US) L.1. Rebhun et al.. "Electron mi1croscope studies of frozen-substituted nain6 eggs: 1, Conditions for Iavoidance of i.ntracell1ular ice see page 5574, abstracot no. 55373 &Amter J Ariat, 130(l), 1-15,1971 A FEP, A, 0246824 (CE.LL, SYSTBME LTD) November 197? (cited i.n the Application) dpecia w8a~o8 *1 eft" detomets 0b Tr laer, gelumel&M puAlCIsoll Mt I" Inomtiobnal l ne INt A" :*Gvvman defining ttrio -bnbFIu St1A of the 5ft whl~h If AM!pfolt M S tt'i InMfIplie oit t.icy atli hue aonuilrm to be arl Pa lauI8! ro"~ cr~te..t"eiV" h hiWIt rifhr udaltil orIN? detsr% kin ouill e anI01UOh alAterth In tdfflu"~ -X docupent of var"uuar ra1.vencal ill. claimerd Invoiafl ill, MAN. =6s toi~e 14 at hO e sawaie to LP desumeim wdlIch MAma~ 1w Cattai on piotyornli? at v to n ilnentiveao witich Is eltol to @a &ahsh "h pUhIuIOOj0 qetip bE anm wy deunw IOi lO OVMCII etVme the ebatneC Invenla~n OltatinCl514 4 1 C101 Mit. h fala W lI l aeo1110 be comniderod to In-uIva in hIree 0 WI when the WO ue~t~fint Iftortia to ant' 1ieaweeruse o, 61hibia4l1 19 AOlMn to vamblnmti wth Oat oe O the~l01%r sur-h 600"i Ottil he Monte, 4Cu fOMLMMIZ beinlg &Oftvit to a farCailt IC1d -P1 faument publhat %tior %a thS WtnilAitatflin 1e blt In 11" GV lae n th 11,11 844. clam%" A -V ecgmqi ms. .w4 oft, tt. ft~i" faift IV, 81N PICRTICIN Ce 04 W1 AtC40OWUD ofptua 04o Intaptiehewal Awwagmn Date 01 Mailig of this IntapiaaJlegi w s eot 14 November 1990 4DCT triaffilflt IMAt~ngilhn t l~~t L SURPAW PATENT OFFCE Farm IfCTIIS5igiO (seand Iisaif wJ~am" loul ~1 ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. GB 9001268 SA 39520 ihis annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 27/11/90 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) date EP-A- 0246824 25-11-87 AU-B- AU-A- JP-A- 598260 7299087 63287479

21-06-90 19-11-87

24-11-88 For more details about this annex see Official Journal of the European Patent Office, No. 12/82 2 For more details about this annex :see Official Journal of the European Patent Office, No. 12/82