SIST EN 1186-4:2002

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Materiali in predmeti v stiku z živili - Polimerni materiali - 4. del: Preskusne metode za celotno migracijo v olivno olje z migracijsko celicoWerkstoffe und Gegenstände in Kontakt mit Lebensmitteln - Kunststoffe - Teil 4: Prüfverfahren für die Gesamtmigration in Olivenöl mittels ZelleMatériaux et objets en contact avec les denrées alimentaires - Matiere plastique - Partie 4: Méthodes d'essai pour la migration globale dans l'huile d'olive en celluleMaterials and articles in contact with foodstuffs - Plastics - Part 4: Test methods for overall migration into olive oil by cell67.250Materiali in predmeti v stiku z živiliMaterials and articles in contact with foodstuffsICS:Ta slovenski standard je istoveten z:EN 1186-4:2002SIST EN 1186-4:2002en01-september-2002SIST EN 1186-4:2002SLOVENSKI
STANDARDSIST ENV 1186-4:19971DGRPHãþD



SIST EN 1186-4:2002



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 1186-4April 2002ICS 67.250Supercedes ENV 1186-4:1994English versionMaterials and articles in contact with foodstuffs - Plastics - Part4: Test methods for overall migration into olive oil by cellMatériaux et objets en contact avec les denréesalimentaires - Matière plastique - Partie 4: Méthodesd'essai pour la migration globale dans l'huile d'olive encelluleWerkstoffe und Gegenstände in Kontakt mit Lebensmitteln- Kunststoffe - Teil 4: Prüfverfahren für dieGesamtmigration in Olivenöl mittels ZelleThis European Standard was approved by CEN on 4 January 2002.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2002 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 1186-4:2002 ESIST EN 1186-4:2002



EN 1186-4:2002 (E)2ContentspageForeword.31Scope.52Normative references.53Principle.54Reagents.65Apparatus.76Preparation of test specimens.97Procedure.98Expression of results.169Test report.17Annex A (normative)
Determination of the suitability of olive oil as the fatty food simulant and oftriheptadecanoin as the internal standard.18Annex B (normative)
Determination of the need for sample conditioning.20Annex C (normative)
Determination of the need for sample conditioning and determination of themass of moisture sensitive test specimens, by vacuum drying.21Annex D (normative)
Determination of change in moisture content of test specimens by measurementof the transfer of water to, or from olive oil, by Karl Fischer titration.23Annex E (informative)
Typical chromatograms and calibration graph.25Annex F (informative)
Precision data.28Annex ZA (informative)
Relationship of this European Standard with Council Directive 89/109/EECand Commission Directive 90/128/EEC and associated Directives.29Bibliography.31SIST EN 1186-4:2002



EN 1186-4:2002 (E)3ForewordThis document EN 1186-4:2002 has been prepared by Technical Committee CEN/TC 194 "Utensils in contact withfood”, the secretariat of which is held by BSI.This European Standard shall be given the status of a national standard, either by publication of an identical text orby endorsement, at the latest by October 2002, and conflicting national standards shall be withdrawn at the latestby October 2002.This document supersedes ENV 1186-4:1994.This European Standard is one of a series of methods of test for plastics materials and articles in contact withfoodstuffs.This document has been prepared under a mandate given to CEN by the European Commission and the EuropeanFree Trade Association, and supports essential requirements of EC Directive(s).For relationship with EC Directive(s), see informative annex ZA, which is an integral part of this document.At the time of preparation and publication of this standard the European Union legislation relating to plasticsmaterials and articles intended to come into contact with foodstuffs is incomplete.
Further Directives andamendments to existing Directives are expected which could change the legislative requirements which thisstandard supports.
It is therefore strongly recommended that users of this standard refer to the latest relevantpublished Directive(s) before commencement of any of the test or tests described in this standard.EN 1186-4 should be read in conjunction with EN 1186-1.Further Parts of this standard have been prepared concerned with the determination of overall migration fromplastics materials into food simulants.
Their titles are as follows:EN 1186 Materials and articles in contact with foodstuffs- Plastics-Part 1Guide to the selection of conditions and test methods for overall migrationPart 2Test methods for overall migration into olive oil by total immersionPart 3Test methods for overall migration into aqueous food simulants by total immersionPart 5Test methods for overall migration into aqueous food simulants by cellPart 6Test methods for overall migration into olive oil using a pouchPart 7Test methods for overall migration into aqueous food simulants using a pouchPart 8Test methods for overall migration into olive oil by article fillingPart 9Test methods for overall migration into aqueous food simulants by article fillingPart 10Test methods for overall migration into olive oil (modified method for use in cases whereincomplete extraction of olive oil occurs)Part 11Test methods for overall migration into mixtures of
14C-labelled synthetic triglyceridePart 12Test methods for overall migration at low temperaturesSIST EN 1186-4:2002



EN 1186-4:2002 (E)4Part 13Test methods for overall migration at high temperaturesPart 14Test methods for 'substitute tests' for overall migration from plastics intended to come intocontact with fatty foodstuffs using test media iso-octane and 95 % ethanolPart 15Alternative test methods to migration into fatty food simulants by rapid extraction into iso-octane and/or 95 % ethanolAnnexes A, B, C and D to this standard are normative where applicable. The annexes E and F are informative.According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain,Sweden, Switzerland and the United Kingdom.SIST EN 1186-4:2002



EN 1186-4:2002 (E)51 ScopeThis Part of this European Standard specifies test methods for the determination of the overall migration into fattyfood simulants, from one surface only of plastics in the form of sheet and film at temperatures above 20 °C and upto, but not including, 100 °C for selected times.This method is most suitable for plastics in the form of films and sheets, but is particularly applicable to thosematerials consisting of more than one layer or surfaces that differ in their migration characteristics, which have tobe tested with food simulant in contact with the surface which is intended to come into contact with foodstuffs.NOTEThis test method has been written for use with the fatty food simulant, olive oil.
The test method can also be usedwith appropriate modifications with 'other fatty food simulants ' called simulant D - a synthetic mixture of triglycerides, sunfloweroil and corn oil.
These other fatty food simulants will produce different chromatograms for the simulant methyl esters to those ofthe methyl esters of olive oil.
Select suitable chromatogram peaks of the methyl esters of the other fatty food simulants for thequantitative determination of the simulant extracted from the test specimens.The test method described is applicable to most types of plastics, although there are some plastics for which it isknown not to be applicable.2 Normative referencesThis European Standard incorporates by dated and undated reference, provisions from other publications.
Thesenormative references are cited at the appropriate places in the text, and the publications are listed hereafter. Fordated references, subsequent amendments to and revisions of any of these publications apply to this EuropeanStandard only when incorporated in it by amendment or revision.
For undated references the latest edition of thepublication referred to applies (including amendments).EN 1186-1:2002, Materials and articles in contact with foodstuffs – Plastics – Part 1: Guide to the selection ofconditions and test methods for overall migration.ISO 648, Laboratory glassware - One mark pipettes.ISO 4788, Laboratory glassware – Graduated measuring cylinders.3 PrincipleThe overall migration from a sample of the plastics is determined as the loss in mass per unit of surface areaintended to come into contact with foodstuffs.The selection of the conditions of test will be determined by the conditions of use, see clauses 4, 5 and 6 ofEN 1186-1:2002.Test specimens of known mass are exposed in a cell to olive oil for the exposure time, at temperatures above20 °C and below 100 °C, then taken from the cell, blotted to remove oil adhering to the surface, and reweighed.The specimens will usually retain absorbed olive oil that extracted and determined quantitatively by means of gaschromatography after conversion to methyl esters.
Methylation is carried out by reacting a borontrifluoride/methanol complex with fatty acids formed by hydrolysing the oil with potassium hydroxide.
An internalstandard, triheptadecanoin, is added prior to the extraction of the absorbed olive oil from the test specimens.
Thisensures that any active or extractable components of the plastics react with the internal standard, as well as withthe extracted olive oil. The internal standard is also subjected to the hydrolysis and methylation reactions, providingcompensation for any inefficiencies in the hydrolysis and methylation processes.Migration into the olive oil is calculated by subtracting the mass of olive oil retained by the test specimen from theSIST EN 1186-4:2002



EN 1186-4:2002 (E)6mass of the test specimen after removal from the olive oil, then subtracting this mass from the initial mass of thespecimen.The total loss in mass is expressed in milligrams per square decimetre of surface area of the specimen and theoverall migration is reported as the mean of a minimum of three determinations on separate test specimens.To allow for inaccuracies which may arise during the procedure and which may be difficult to detect, due forexample to contamination or loss of oil during the sample handling stages, four determinations are carried out onthe sample allowing for the result from one specimen to be discarded.This method includes variations that applicable to certain plastics.NOTEBefore starting a migration exercise, the test sample should be examined for the presence of components interferingin the determination of the amount of olive oil extracted, see 7.1.
If an unacceptable amount of interference is present thensuitability of one of the 'other fatty food simulants' should be examined, see annex A and 9.3 and 9.5 of EN 1186-1:2002.
If aninterference is present which would interfere with the triheptadecanoin internal standard an alternative internal standard shouldbe used, see annex A, and 9.3 of EN 1186-1:2002.4 ReagentsNOTEAll reagents should be of recognized analytical quality, unless otherwise specified.4.1Olive oil, reference simulant D, as specified in 5.2 of EN 1186-1:2002.4.2Extraction solvent (see 10.1 of EN 1186-1:2002).4.2.1For non-polar plastics, such as polyethylene and polypropylene:- Pentane 98 % boiling point 36 °C.For polar plastics, such as polyamide and polyacetal:- 95/5 by volume azeotropic mixture of pentane 98 % and ethanol 99 %.NOTE 1Pentane is a very volatile and highly flammable solvent.
Care should therefore be taken when handling this solventto prevent contact with sources of ignition.
Ethanol is also a flammable solvent.
It is not recommended that extractions witheither pentane or the pentane/ethanol mixture be left unattended, particularly overnight.NOTE 2Due to the low boiling points of these solvents, cooled condenser water can be required to prevent undue loss of thesolvent from the condenser.or4.2.2Other suitable solvent.NOTE 1In previous methods for determining overall migration into olive oil the extraction solvent used has been 1,1,2-trichloro-trifluoroethane.
For environmental reasons the use of this solvent should be avoided where possible, see 9.1 of EN1186-1:2002.
Experience has shown that this solvent, although effective for most plastics requires longer periods of extraction.NOTE 2Some solvents can contain non-volatile substances which, after hydrolysis and methylation processes, produce gaschromatography peaks with retention times similar to the retention times of olive oil methyl esters and methyl heptadecanoatefrom the internal standard.
Solvents found to contain such substances should be redistilled before use.4.3Internal standard, triheptadecanoin (glyceryl trimargarate) CAS No. 2438-40-61) of a quality such that theproducts from hydrolysis and methylation processes do not contain substances giving detectable gas
1) The source of this is the Chemical Abstracts published by the American Chemical Society.SIST EN 1186-4:2002



EN 1186-4:2002 (E)7chromatography peaks (see 9.3 of EN 1186-1:2002) with similar retention times to the olive oil methyl ester peaks.Prepared as a solution containing 2,0 mg/ml in cyclohexane.4.4Potassium hydroxide solution, 11,0 g/l in methanol.4.5Boron trifluoride, methanol complex, approximately 150 g/l BF3.4.6n -Heptane.4.7Sodium sulfate.4.7.1Sodium sulfate, anhydrous, Na2SO4.4.7.2Sodium sulfate, saturated solution.4.8Diethyl ether.4.9Karl Fischer solvent, commercially prepared, methanol and chloroform based, water capacity of 5 mg/ml.4.10Karl Fischer titrant (for volumetric apparatus only), commercially prepared, water capacity of 2 mg/ml.5 Apparatus5.1Cutting slab, clean smooth glass, metal or plastics slab of sufficient area to prepare test specimens,250 mm ´ 250 mm is suitable.5.2Tweezers, stainless steel, blunt nosed.5.3Cutting implement, scalpel, scissors, sharp knife or other suitable device.5.4Rule, graduated in millimetres, and with an accuracy of 0,1 mm.5.5Analytical balance capable of determining a change in mass of 0,1 mg.5.6Cells, type A as shown in Figure C.3 of EN 1186-1:2002.NOTEFor details of equivalent cells see 7.3 of EN 1186-1:2002.5.7Conditioning containers, for conditioning test specimens at 50 % ± 5 % relative humidity and 80 % ± 5 %relative humidity at 20 °C ± 5 °C.NOTEFor 50 % relative humidity, 43 % w/v sulphuric acid solution in water is suitable and for 80 % relative humidity, 27 %w/v sulphuric acid solution is suitable. The solutions should be freshly prepared by adding a weighed amount of acid to asuitable volume of water, cooling to room temperature and making up to the required volume. It is recommended that relativehumidity and temperature be maintained during the conditioning period.
Therefore the containers should be placed in athermostatically controlled room or oven, at a temperature of approximately 20 °C, the set temperature should not vary by morethan ± 1 °C.5.8Glass tubes, ground neck and stoppers, for retaining the olive oil.
Tubes with an internal diameter ofapproximately 35 mm and length in the range of 100 mm to 200 mm, excluding the ground neck, see 7.2 ofEN 1186-1:2002, have been found to be satisfactory.5.9Thermostatically controlled oven or incubator capable of maintaining the set temperature, within thetolerances specified in Table B.2 of EN 1186-1:2002.5.10Filter paper, lint-free.5.11Anti-bumping beads.SIST EN 1186-4:2002



EN 1186-4:2002 (E)85.12Soxhlet type extractors, capable of holding test specimens on the supports, with 250 ml or 500 ml roundbottom flasks to fit.NOTEAlternative extractors capable of satisfactorily extracting absorbed olive oil from the test specimens can be used.5.13Water bath, capable of holding the flasks of soxhlet type extractors (5.12)5.14Rotary evaporator or distillation apparatus, for evaporation and collection of the extraction solvent.NOTEArtificially cooled water can be necessary for efficient condensation of a low boiling point solvent.5.15Steam bath or water bath.5.16Flasks, 50 ml, long neck with condensers to fit, for methyl ester preparations.5.17Measuring cylinders, complying with the minimum requirements of ISO 4788, 500 ml, 250 ml, 100 ml, 25 ml,and 10 ml.
A 10 ml graduated syringe may be used in place of the 10 ml measuring cylinder.5.18Pipettes, complying with the minimum requirements of ISO 648, 5 ml and 10 ml.5.19Glass beads, 2 mm to 3 mm in diameter or glass rods, 2 mm to 3 mm in diameter and approximately100 mm long (see 7.2 of EN 1186-1:2002).5.20Gas chromatograph, with flame ionisation detector equipped with an appropriate column.
When using apolar column, the major peaks of olive oil, such as C16:0, methyl hexadecanoate (methyl palmitate), C16:1, methyl9-hexadecenoate (methyl palmitoate), C18:0, methyl octadecanoate (methyl stearate), C18:1, methyl 9-octadecenoate (methyl oleate), C18:2, methyl 9,12-octadecadienoate (methyl linoleate) and the internal standardC17:0, methyl heptadecanoate (methyl margarate) shall demonstrate baseline separation. Optionally, a non-polarcolumn can be used which shall give baseline separation of the methyl esters with 16 and 18 carbon numbers andthe internal standard with 17 carbon number.NOTEThe following columns have been found to be suitable:- Column 1, polar column, WCOT fused silica column, length 50 m, internal diameter 0,25 mm, coated with a 0,21micrometre film of cyanopropyl silicone;- Column 2, non polar column, BP1, length 25 m, internal diameter 0,32 mm, with a 1 micron film thickness;- Column 3, polar column, stainless steel column 2 mm to 3 mm internal diameter and 2 m to 3 m length with a packingof 10 % to 20 % by mass of polyestersuccinate on a stationary phase of diatomaceous earth 80 mesh to 100 mesh.5.21Glass tubes with ground glass necks and stoppers, of a volume of approximately 10 ml, for storing theheptane layer if necessary.5.22Vacuum oven or vacuum desiccator, capable of maintaining a temperature of 60 °C ± 2 °C.
The vacuumoven or vacuum desiccator shall be equipped with or connected to a vacuum pump capable of achieving a vacuumof 1,3 kPa or less.
The vacuum pump shall be provided with a time controller to switch on the vacuum pump everyhour for 15 min.NOTEIf a vacuum oven is not available, a vacuum desiccator placed in an oven at 60 °C can be used.5.23Desiccator containing self indicating silica gel or anhydrous calcium chloride.5.24Balance, capable of determining a change of mass of 10 mg.5.25Disposable plastic syringes with luer fitting. 1 ml or 10 ml size.5.26Wide gauge luer needles (80 mm ´ 1,2 mm).SIST EN 1186-4:2002



EN 1186-4:2002 (E)95.27Karl Fischer apparatus, either an automated volumetric titrator, or an automated coulometric titrator.
TheKarl Fischer titrator shall be capable of measuring the water content of the simulant with a precision
(standarddeviation) of 10 mg/kg or less (equivalent to 1 mg/dm2 plastic).
An automated volumetric or coulometric instrumentshall be used.
Manual titration procedures do not give the required accuracy or precision.6 Preparation of test specimens6.1 GeneralIt is essential that test specimens are clean and free from surface contamination (many plastics can readily attractdust due to static charges). Before preparing test specimens, remove any surface contamination from the sampleby gently wiping it with a lint-free cloth, or by brushing with a soft brush. Under no circumstances wash the samplewith water or solvent. If it is specified in the instructions for use of the article that it should be washed or cleanedbefore use see 8.1 of EN 1186-1:2002.
Minimise handling of the samples and, where necessary, wear cottongloves.When preparing test specimens measure the surface area according to 8.3 of EN 1186-1:2002.6.2 Number of test specimensSeven test specimens are required for samples, in the form of thin films, sheets and flat sections cut fromcontainers or similar articles.These test specimens are utilized as follows:a) four test specimens for the migration test;b) two test specimens to check for possible loss of volatiles;c) one test specimen to determine the suitability of olive oil as the fatty food simulant and triheptadecanoinas the internal standard (see annex A);If the conditioning test in annex C is used, one additional test specimen is required.NOTEThe two test specimens, b), are used to check whether the sample losses mass from the evaporation of volatiles,such as solvents, during the test period.
If the vacuum drying procedure in annex C is used these test specimens are notrequired as during the vacuum drying any volatiles will have been removed from the test specimens.If previous testing has established that interference in the gas chromatography procedure is unlikely and annex A isomitted, one fewer test specimen will be required.A minimum of three valid test results is required to calculate the mean.
Testing in triplicate is allowed but in thiscase if one test result is invalid repeat the entire procedure.6.3 Cutting test specimensLay the sample on the cutting slab (5.1) with the surface to be in contact with the olive oil uppermost.
Take the ringfrom the cell type A (5.6) and place on the test sample.
Cut out the test specimen by cutting round the outer edgeof the ring, using the cutting implement (5.3)7 Procedure7.1 GeneralDetermine the applicability of the method by carrying out the procedure described in annex A.
If prior tests haveestablished that the method is applicable then annex A may be omitted.SIST EN 1186-4:2002



EN 1186-4:2002 (E)10Before weighing, discharge any build up of static electricity with an antistatic gun or other suitable means.7.2 Initial weighing of test specimens7.2.1Determine the need for conditioning of the test specimens by carrying out the procedure described inannex B or in annex C.
If prior tests have established that sample conditioning is not required then annex B andannex C may be omitted.
If prior tests have established that the procedure described in annex D is applicable tothe sample, then annex B or annex C may be omitted.7.2.2If the tests described in annex B or annex C show that conditioning is not necessary, determine and recordthe mass of each test specimen.7.2.3If the tests described in annex B or annex C show that conditioning is necessary, follow the directions inthe relevant annex to determine the initial mass of the sample.7.2.4If the tests described in annex B show that conditioning is necessary, but constant mass cannot beachieved within 5 days then carry out the conditioning procedure described in C.3.1 or annex D.NOTE 1Long conditioning periods are not satisfactory due to oxidation of the olive oil which can occur upon prolongedconditioning.NOTE 2The conditioning procedures described in annex C and annex D can be used if it has been established that theseprocedures are more suited to the polymer type under test.7.3 Exposure to food simulantTake four type A cells (5.6), mark them for identification purposes.
Place in the thermostatically controlled oven orincubator (5.9), which is set at the test temperature and leave until the test temperature has been attained.Take five glass tubes (5.8), measure 125 ml ± 5 ml of olive oil (4.1) into each tube by measuring cylinder andstopper the tubes.NOTE 1If the procedure described in annex D is used, it can be necessary to dry all of the olive oil used for the migrationtest, see D.3.2.Alternatively mark the tubes for a volume of 125 ml and fill with olive oil to the mark.
Place into one of the tubes athermometer or thermocouple and stopper the tubes.
Two extra tubes with a minimum of 50 ml of olive oil arerequired as blank simulant, if the procedure in annex D is used.
Place the five or seven tubes, and two emptytubes, in the thermostatically controlled oven or incubator (5.9) set at the test temperature.
Leave until the olive oilhas attained the test temperature, using the thermometer or thermocouple to monitor the temperature.Remove the cells from the thermostatically controlled oven or incubator, dismantle the cells and place on the baseof each cell one of the test specimens.
Reassemble the cells, ensuring that the clamping screw wheel is welltightened down.Remove four tubes containing 125 ml of olive oil from the thermostatically controlled oven or incubator orrefrigerator and transfer the olive oil from each tube to each of the cells through the filler hole.
Remove thethermometer or thermocouple from the tube and insert, if applicable see NOTE 5, in one of the cells and replacethe filler plugs.NOTE 2The olive oil in the fifth tube is used as a reference standard in constructing the calibration graph (see 7.6.2.2) and ifthe procedure in annex D is used, as the third blank sample for Karl Fischer titrations.
The olive oil in the sixth tube is used tocheck the temperature of the oil.
If glass beads or glass rods have been used to raise the level of the olive oil to achieve totalimmersion, then similar glass beads or glass rods should be added to the sixth tube.Remove the two empty tubes from the thermostatically controlled oven or incubator or refrigerator and place ineach tube one of the remaining two test specimens and stopper.SIST EN 1186-4:2002



EN 1186-4:2002 (E)11NOTE 3These two test specimens are used to check whether the sample loses mass from the evaporation of volatiles, suchas water, solvents and oligomers, during the test period.
If the vacuum drying procedure in annex C is applicable these testspecimens are not required as during the vacuum drying volatiles will have been removed from the test specimens.Replace the four cells and the two tubes in the thermostatically controlled oven or incubator set at the testtemperature. This part of the operation should be carried out in the minimum time to prevent undue heat loss fromthe cells and olive oil.
Observe the temperature of the thermostatically controlled oven or incubator or the olive oil(see NOTE 5) in the one of the cells and leave the cells and tubes for the selected test period, taking into accountthe tolerances specified in Table B.1 of EN 1186-1:2002, after the olive oil in the cell has reached a temperaturewithin the tolerance specified in Table B.2 of EN 1186-1:2002.NOTE 4Annex B of EN 1186-1:2002 includes tolerances on a wide range of contact times and contact temperatures.
All ofthese contact times and contact temperatures are not necessarily relevant to this Part of the standard.NOTE 5For exposure times of 24 h or more it is acceptable to monitor the temperature of the airbath of the thermostaticallycontrolled oven or incubator or refrigerator, instead of the temperature of the simulant.Take the cells and tubes from the oven or incubator and immediately remove the
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