good laboratory practice (glp) - chance and impediment...

New Chemicals and Food Residues

241

PS3-3 - 6135

Good Laboratory Practice (GLP) - chance and impediment for theregistration of new fumigants - Phosphine residues as an example

D. Klementz1, G.M. Kroos1, C. Reichmuth1

1 Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Stored Product Protection

(ISPP), Königin-Luise-street 19, 14195 Berlin, Germany, [email protected], +49 30 83042507

Abstract

Phosphine is a very effective fumigant tocontrol pest insects. Therefore the determinationof phosphine residues in treated cocoa beans ischosen as an example for a study according tothe principles of GLP and exemplify how thestandard requirements are concretely realized atthe Institute for Stored Product Protection. Forthe purpose of registration of a chemicalcompound for pest control data on the formationand decay rate of residues in treated productshave to be provided Europe-wide by applicantsaccording to GLP.

The installation of a GLP standard in a labrequires:

� defined organisation and responsibilities,� planned and supervised processes

according to written standard operationprocedures (SOPs),

� comprehensible studies.This quality ensuring system has been

established in Germany in 1990, currently about160 test facilities have been licensed. Since April2004 the Institute for Stored Product Protection,as the only public scientific institute dealing withaspects of storage and insect pest control inGermany owns a GLP permission for “thedetection of residues”.

The demand for GLP promotes high qualityof residue data on one side, but impedes on theother side strongly the introduction of newfumigants or the registration of new application

fields for an existing registered fumigant.Especially for small companies, it is difficult toinstall and keep the complex GLP infrastructureand the specific expertise. Similar constraintsapply to good experimental practice (GEP), thequality ensuring system for efficacy data.

The pros and cons of working with or withoutGLP standards in an analytical lab are discussed.

Key words: Good Laboratory Praxis (GLP),registration, new fumigants, phosphine, foodresidue.

Introduction

Since 1981 the Principles of GLP – based onregulations of the US Food and DrugAdministration - have been developed by anexpert group of the Organisation for EconomicCo-operation and Development (OECD) andthey should be applied to the non-clinical safetytesting of test items.

The principles of GLP have been developedto promote the

� protection of the consumer, animals andenvironment,

� quality and validity of non-clinical healthand environmental safety studies,

� quality of residue data,� provision of reliable data and� international mutual acceptance of data.

9th International Working Conference on Stored Product Protection

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Scope of GLP

The Principles of Good Laboratory Practice(GLP) are defined as rules and criteria for aquality system concerned with the organisationalprocess and the conditions under which non-clinical health and environmental safety studiesare planned, performed, monitored, recorded,archived and reported.

Chemicals control legislation in the OECDMember states requires a preventing riskassessment by testing chemicals to determinetheir potential hazards. The Principles of GLPhave been developed to promote the quality andvalidity of those test data used for determiningthe safety of active chemical substances andchemical products prior to their use.

The issue of data quality has an importantinternational dimension: If regulatory authoritiesin countries can have confidence in the qualityof test data developed by abroad test facilities,duplicat testing can be avoided and costs can besaved to government and sponsors of studies. Themutual recognition of these data by OECD andthe European Member States becomes importantfor data related to authorisation procedures wherenational or European authorities have to assessthe effects of chemicals relating to human healthand the environment. The principles for GLP alsosimplify this exchange of information betweenthe competent authorities and harmonise theprocedures.

The principles of GLP have been developedin the framework of the OECD and were firstpublished in 1981. Meanwhile a series ofdocuments on related issues have beenestablished. The OECD Principles of GLP (asrevised in 1997) laid down the scope of GLP:

� These Principles of GLP should be appliedto the non-clinical safety testing of test itemscontained in pharmaceutical products, pesticideproducts, cosmetic products, veterinary drugs aswell as food additives, feed additives, andindustrial chemicals. These test items arefrequently synthetic chemicals, but may be ofnatural or biological origin and, in somecircumstances, may be living organisms. The

purpose of testing these test items is to obtaindata on their properties and/or their safety withrespect to human health and/or the environment.

� Non-clinical health and environmentalsafety studies covered by the Principles of GLPinclude work conducted in the laboratory, ingreenhouses, and in the field.

� Unless specifically exempted by nationallegislation, these Principles of GLP apply to allnon-clinical health and environmental safetystudies required by regulations for the purposeof registering or licensing pharmaceuticals,pesticides, food and feed additives, cosmeticproducts, veterinary drug products and similarproducts, and for the regulation of industrialchemicals.

GLP regulations and requirements

In the Series on Principles of Good LaboratoryPractice and Compliance Monitoring thefollowing OECD papers are published (http://www.oecd.org):No 1: OECD Principles on Good Laboratory

PracticeNo 2: Revised Guides for Compliance

Monitoring Procedures for GoodLaboratory Practice

No 3: Revised Guidance for the Conduct ofLaboratory Inspections and Study Audit

No 4: Quality Assurance and GLP (revised 1999No 5: Compliance of Laboratory Suppliers with

GLP Principles(revised 1999)No 6: The Application of the GLP Principles to

Field Studies(revised 1999)No 7: The Application of the GLP Principles to

Short&Term Studies (revised 1999)No 8: The Role and Responsibilities of the Study

Director in GLP Studies (revised 1999)No 9: Guidance for the Preparation of GLP

Inspection ReportsNo 10: The Application of the Principles of GLP

to Computerised Systems(1995No 11: The Role and Responsibility of the

Sponsor in the Application of thePrinciples of GLP


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No 12: Requesting and Carrying Out Inspectionsand Study Audits in Another Country

No 13: The Application of the OECD Principlesof GLP to the Organisation andManagement of Multi-Site Studies

No 14: The Application of the Principles of GLPto in vitro Studies

All terms and definitions cities below arerequirements test management have to establishin an test facility:

I. Terms concerning the organisation of atest facility

� Test facility means the persons, premisesand operational unit(s) that are necessary forconducting the non-clinical health andenvironmental safety study

� Test facility management means theperson(s) who has have the authority and formalresponsibility for the organisation andfunctioning of the test facility according to thesePrinciples of GLP.

� Study Director means the individualresponsible for the overall conduct of the non-clinical health and environmental safety study.

� Quality Assurance Programme means adefined system, including personnel, which isindependent of study conduct and is designed toassure test facility management of compliancewith these Principles of GLP.

� Standard Operating Procedures (SOPs)means documented procedures which describehow to perform tests or activities normally notspecified in detail in study plans or testguidelines. SOPs should be available for, but notbe limited to, the following categories of testfacility activities.

1. Test and Reference Items Receipt,identification, labelling, handling,sampling and storage.

2. Apparatus, Materials and Reagents3. Record Keeping, Reporting, Storage, and

Retrieval4. Test System (where appropriate)5. Quality Assurance Procedures� Master schedule means a compilation of

information to assist in the assessment of workload

and for the tracking of studies at a test facility.

II. Terms concerning the Non-ClinicalHealth and Environmental Safety Study

� Non-clinical health and environmentalsafety study means an experiment or set ofexperiments in which a test item is examinedunder laboratory conditions or in the environmentto obtain data on its properties and/or its safety,intended for submission to appropriate regulatoryauthorities.

� Study plan means a document whichdefines the objectives and experimental designfor the conduct of the study, and includes anyamendments.

� Test system means any biological,chemical or physical system or a combinationthereof used in a study.

� Raw data means all original test facilityrecords and documentation, or verified copiesthereof, which are the result of the originalobservations and activities in a study. Raw dataalso may include, for example, photographs,microfilm or microfiche copies, computerreadable media, dictated observations, recordeddata from automated instruments, or any otherdata storage medium that has been recognisedas capable of providing secure storage ofinformation for a time period.

� Storage and Retention of Records andMaterials

All original documents citied above andactivities of the OECD concerning GLP areavailable from the OECD website: http://www.oecd.org/ehs/

Community legislation and legalregulations for the EuropeanAuthorisation of Plant Protectionproducts

The Member States of the EuropeanCommunity recognised that there was a need toprotect environment and to create standards toprotect consumers. For this reason, the firstCommunity environment legislation dealt with


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products and chemicals establishing registrationrequirements and procedures for most of theexisting chemicals.

Pesticides - plant protection products (PPP)and biocidal products (BP) - belonging to thesegroups need to be assessed and authorised beforethey can be placed on the market. In the case ofPPP the European Community has developed aregulatory framework, Directive 91/414/EEC of15 July 1991 concerning the placing of plantprotection products on the market – defining rulesand common standards for the authorisation ofPPP. The Directive requires risk assessments foreffects on health and environment and maximumresidues levels in food to be carried out, beforean active substance (existing or new) can be listedin the positive list of Annex I by the Commissionand before Member States authorise marketingand use of PPP. The legal provisions concerningGLP principles are summarized on the websiteof the European Union (EU) citied below: http://ec.europa.eu/enterprise/chemicals/legislation/glp/product/pesticides_en.htm.

� The introductions to annexes II and III tothe Directive require that tests regarding thesafety for human health and the environment foractive ingredients and plant protection productsmust be conducted in accordance with theprinciples laid down in Council Directive 87/18/EEC of 18 December 1986 on the harmonizationof laws, regulations and administrativeprovisions relating to the application of theprinciples of good laboratory practice and theverification of their applications for tests onchemical substances. Directive 2004/9/EC of theEuropean Parliament and of the Council of 11February 2004 on the inspection and verificationof GLP lays down the obligation of the MemberStates to designate the authorities responsible forGLP inspections in their territory for mutualacceptance of data. The Directive requires thatthe OECD Revised Guides for ComplianceMonitoring Procedures for GLP and the OECDGuidance for the Conduct of Test FacilityInspections and Study Audits must be followedduring laboratory inspections and study audits.

� Directive 2004/10/EC of the European

Parliament and of the Council of 11 February2004 on the harmonisation of laws, regulationsand administrative provisions relating to theapplication of the principles of GLP and theverification of their applications for tests onchemical substances requires from MemberStates to take all measures necessary to ensurethat laboratories carrying out safety studies onchemical products comply with the OECDPrinciples of GLP.

� Article 13 (1) of Directive 91/414/EECprovides that applicants for the authorisation ofa plant protection product have to submit adossier satisfying the requirements of AnnexesII and III which detail the data to be submitted.

� reference to test guidelines which intendsto ensure that the data submitted are scientificallyacceptable

� the circumstances in which studies haveor have not to be submitted

� the application of GLP� The Commission adopted temporary

derogations for the application of the principlesof GLP for residue trials started at the latest on31.12.1997 and for honeybee and beneficialarthropod testing started at the latest on31.12.1999 (Directives 93/71/EEC and 95/35/ECboth amending Directive 91/414/EEC; availablein EUR-Lex http://ec.europa.eu).

� Since the Directive provides in generalterms for the application of GLP to studies onthe properties and/or safety with respect to humanhealth or the environment, the StandingCommittee on Plant Health agreed that guidancedocument 7109/VI/94 rev. 6 contains the correctinterpretation of which individual studies ofAnnexes II and III, part A are related to humanhealth or the environment and therefore shouldbe subject to the GLP requirements.

Determination of phosphine in cocoabeans according to the GLP-guidelines

The phosphine residues were detectedaccording to a written study plan and based on a


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large number of SOPs dealing with descriptionsof organization, apparatures and methods.

Examples of used organisational SOPs are:� Documentation of any working step in the

lab,� Drawing up of study plans and final reports,� Management of data retrievals and

unchanged data,� Archiving and storage of documents and

papersExamples of used methodical SOPs are:� Handling with materials and reagents,� Handling with test and reference items,� Sample taking, sample taking systems and

sample transport,� Sample extraction and PH

3 detection,

� Checking, Excel program with examplefigures,

� Validation of the system “gas-chromatographwith mass selective detector”

Examples of used apparatus SOPs are:� Refrigerator, freezer, balance, gas-

chromatograph with mass selective detector

Aim

The use of GLP shall be demonstrated withthe determination of residue for registration of aphosphine releasing product. The Detia Gas Ex Bforte sachets should be tested for their suitabilityto be used for cocoa bean disinfestations.

In order to comply with the fixed maximumresidue limits (MRLs) for cocoa beans (0,01 mg/kg),applicants have to provide research results of theagent’s residues in treated raw cocoa accordingto GLP. It is then in the admission process for plantprotecting substances, e.g. when determiningwaiting times and ceiling values of the substanceto be applied, that these data are used for athorough risk assessment. Before the actual testin the fumigation station of the BBA in Berlin,Germany, a gas-proof steel chamber filled withraw cocoa was fumigated according to the dataof a potential applicant. Before fumigation andat fixed times after the process, samples weretaken and investigated for their residues ofphosphine.

Method development, method validationand storage stability studies

A large number of methodical SOPs weredeveloped to validate the PH

3 residue

determination method in cocoa beans. The aimswere:

� the detection of recovery rates,� the determination of the quantitative

detection limit,� the determination of the qualitative

detection limit,� the investigation of the linearity, accuracy

and precision of the phosphine detection and� the investigation of the storage stability of

phosphine residues in fumigated cocoa beans andstored at –18 °C.

Often the testing facilities do not develop amethod for the analytical work since the methodis always very specified. Prior to the analysis ofsamples a method had to be developed a methodto detect the PH

3 residues in cocoa beans. A test

procedure by Nowicki, recorded by Noack et.al.(1983) was applied, where PH

3 residues are

extracted from the samples by diluted sulphuricacid in a vacuum and their quantities determinedby a mass spectrometer.

Prior to analysis, the storage of treated cocoabeans was performed at –18 °C. Investigationof the storage stability of the phosphine residuesin fumigated cocoa beans revealed (Figure 1) thatno relevant decay occurred for 56 days of storageat this temperature. The average of the recoveryrate ranged within the required limits in Germanyof 70 % and 110 %.

Fumigation

Two packages of Detia Gas Ex B forte (weightper package: 9 g of substance, content: 66 %Mg3P2, equals 3 g PH3) were used for four bagsof raw cocoa in the fumigation process, as shownin Figure 2. Based on a volume of the fumigationchamber of 0.5 m³, the dose was 12.0 g/m³ ofthe applied PH

3. The dose was 18 g Mg3P2/

0.5 m3 and 6 g PH3/ 0.5 m3, respectively. With aweight of the treated cocoa of 100 kg, this


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corresponds to an application of 180.0 g of Mg3P2

per ton or 60.0 g of PH3 per ton of cocoa. Thefumigation time was 60 hours, whereassubsequent ventilation lasted 24 hours.

Surveillance of the fumigation parameter

The analytical detection of the fumigant

during the fumigation with Detia Gas Ex B fortewas carried out online with an IR-spectrometerMiran 1B (Wilks scientific corporation, Norwalk,USA), see Figures 3 and 4. Figure 5 shows thetemperature curve during the fumigation period.The average temperature was 19.2 °C.

Figure 1. Stability of phosphine residues in cocoa beans during storage after treatment.

Figure 2. Cocoa beans in the fumigation chamberbefore fumigation with Detia Gas Ex B forte.

Figure 3. Miran 1B (Wilks scientific corporation,Norwalk) in front of the fumigation chamber.

140

120

100

80

60

40

20

0

Recovery (%)

unfumigated beans with pure PH3 added to simulate 0.1 mg/kg (100 g cocoa beans in a 1.4-glass bulb)Dosage for the chamber fumigation 23.7 PH3/kg cocoa beans

0 7 14 28 35 56

Storage after fumigation (days)


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Detection of phosphine residues

A head space technique was used to determinethe phosphine contents in the cocoa beans. Theanalytical detection of PH

3 residues is performed

later on by gas-chromatography with massselective detector. As shown in Table 1 and inFigure 6, respectively the PH

3 contents in cocoa

beans were detected after 1, 3, 7, 10 and 14 daysafter a fumigation with Detia Gas Ex B forte. Thephosphine residues were in the range of0.051 mg/kg on day “1” after the fumigation end

and as soon as 10 days after storage, the residueshad dropped down to about 0.01 mg/kg. After14 days, the phosphine values were below thedetection limit of 0.01 mg/kg, the Europeanmaximum residue limit for these products.

The criteria for monitor the methods(requirements to recovery rates, relative standarddeviations and initial dose) described in theguideline “Methods of Residue Analysis forMonitoring” by Hänel and Siebers (1998) werecomplied with in the investigation of the samples.

Figure 4. Phosphine concentration during the fumigation period with Detia Gas Ex B forte.

Figure 5. Temperature curve in the fumigation chamber filled with cocoa beans during the fumigationwith Detia Gas Ex B forte.


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Table 1. PH3 contents in cocoa beans.Days after Mean value Absolutethe end of Sample PH

3 content PH

3 content standard variation

fumigation number (mg/kg) (mg/kg) deviation coefficient

Before fumigation UK1 nd nd not notUK2 nd calculated calculatedUK3 nd

1 P1_1 0.047 0.051 0.003 6.5P1_2 0.050P1_3 0.055

3 P2_1 0.028 0.030 0.002 6.8P2_2 0.033P2_3 0.030

7 P3_1 0.011 0.011 0.0 4.4P3_2 0.010P3_3 0.011

10 P4_1 0.011 0.011 0.0 4.4P4_2 0.011P4_3 0.010

14 P5_1 0.007 0.007 0.002 20.2P5_2 0.010P5_3 0.006P5_4 0.006P5_5 0.008

stability tests L1a 0.076 0.081 0.006 7.0L1b 0.089L1c 0.078

nd = not detectable.P1_1 = sample 1_1.L1a = stability sample added with 0.01 mg phosphine /kg cocoa beans.

MRL = maximal residue limit = 0.01 mg/kg

Figure 6. Phosphine content in cocoa beans after application of Detia Gas Ex B fort.


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The storage stability of the phosphine residuesin fumigated cocoa beans was investigatedsimultaneously to the detection of thesephosphine residues after the fumigation in thefumigation chamber. The storage stability resultspresented in Table 1, reveal a recovery rate of81 % ± 6 %. This recovery rate range within therequired limits in Germany of 70 % and 110 %.

Conclusion

This demand for GLP protects on one sidebut impedes on the other strongly the introductionof new fumigants or the registration of newapplication fields for an existing registeredfumigant. This is why a sufficient number ofthese labs in the individual member countries ismissing. This shortage is coursed by the limitednumber of GLP investigations, which require ahigh level of specific expertise and veryexpensive specific apparatus. The limited numberdepends strongly on the amount of money, whichcan be earned by this business.

The biggest advantages of working under theGLP guidance are:

� better comprehensibility,� increasing protection of the consumer,

animals and environment,� increasing quality and validity of studies,� increasing quality of residue data,� standardisation of methods and procedures,� introduction of a high level of specific

expertise and� international mutual acceptance of data.

References

Hänel, R., Siebers, J., 1998. Leitlinie:Rückstandsanalysenmethoden für dieÜberwachung. “Methods of Residue

Analysis for Monitoring”, Berichte aus derBiol. Bundesanstalt f. Land- undForstwirtschaft.

Kroos, G.M., Pérez, G.H. Food Safety Hazardsfrom Contaminating Organisms in DurableStored Products; Appendix ‘EuropeanLegislation concerning food safety’ In:Health risks and safety hazards related tothe occurrence of pest organisms in storedproducts: Guidelines for Risk Assessment,Prevention and Management; Council ofEurope, Public Health Committee, Ad-hocGroup on Stored Product Protection; inprint.

Noack, S., Reichmuth, C., Wohlgemuth, R.,1983. PH

3-Rückstände bei

Vorratsschutzbegasungen in Abhängigkeitvon Konzentration, Einwirkungszeit undLagerdauer nach der Begasung. Zeitschriftfür Lebensmittel- Untersuchung und –Forschung 177, 87-93. J.F. BergmannVerlag.

OECD series on principles of Good LaboratoryPractice and compliance monitoring,Number 1: OECD Principles on GoodLaboratory Practice (as revised in 1997;ENV/MC/CHEM, 98, 17

Official Journal of the European CommunitiesL 230, 18/8/91, p. 1

Official Journal of the European CommunitiesL 15, 17.1.1987, p. 29

Official Journal of the European CommunitiesL 50, 20.2.2004, p. 28

Official Journal of the European CommunitiesL 50, 20.2.2004, p. 44–59

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