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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code CTD 0203

2. Project title

Conjugated Residue Behaviour: Impact on Human Health Assessment

3. Contractororganisation(s)

SyngentaJealotts Hill Research StationBracknellBerkshireRG42 6EY     

54. Total Defra project costs £ 402,066.00 ex VAT

(agreed fixed price)

5. Project: start date................ June 2004

end date................. May 2007

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.The aim of the project was to conduct a literature review and subsequent programme of research to enable more informed decisions to be made on the regulation of pesticide conjugates and bound residues. The principal requirements were to achieve an improved understanding of the behaviour of common pesticide conjugates and bound residues under conditions found in the gastrointestinal tract (GIT) of humans and livestock species.

Following a critical appraisal of literature data published from 1970 onwards, a practical study was designed to test existing assumptions and to address knowledge gaps. The test materials for the study comprised twelve glucose conjugates (β-D-glucosides) representing Phase II plant metabolites and two typical bound residues produced by spray application of pentachlorophenol and 3,4-dichloroaniline to wheat grown under agronomic conditions. The principal investigations were as follows:-

Conjugates Stability under the pH and enzymatic conditions of the human and ruminant GIT. Propensity for permeability in the intestine (using CACO-2 cell model). Stability to bovine rumen microflora and human GIT microflora. In silico predictions of physicochemical properties and in vivo behaviour. In vivo oral bioavailability in the rat.

Bound Residues Characterisation of the nature of the binding, including an assessment of characterisation methods. Solubilisation/release under pH and enzymatic conditions found in the GIT. Solubilisation/release by bovine rumen microflora and human GIT microflora. In vivo oral bioavailability in the rat.

The most significant results arising from experiments with conjugates are summarised:-

On the basis of the conjugates examined in this study, it was evident that the β-D-glucosides of simple phenols and benzyl alcohol were stable to the pH of the human and ruminant GIT.

These conjugates were also stable to the digestive enzymes present in the human stomach and small intestine.

β-D-Glucosides of simple anilines were unstable at the pH of the human stomach and degraded. The rate of degradation could be correlated with the basicity of the nitrogen atom in the free exocon.

The β-D-glucoside of benzoic acid underwent acyl migration, a process which is more widely

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recognised for glucuronides. The benzoic acid exocon was not released from the conjugate under the pH and enzymatic conditions of the human and ruminant GIT.

The CACO-2 model did not prove useful for predicting the permeability of the conjugate. At physiological concentrations the model may be more accurate but still not suitable for regulatory use without extensive validation work and the use of radiolabelled conjugates.

The β-D-glucosides of phenol, benzyl alcohol and benzoic acid were degraded almost instantaneously by the microflora present in bovine rumen fluid, indicating that the free exocons should be available in the GIT of ruminant species.

The β-D-glucosides of phenol, benzyl alcohol and benzoic acid were degraded almost instantaneously by human colonic microbiota, indicating that exocons should be released in the colon.

In vivo studies in the rat demonstrated that the β-D-glucosides of phenol and benzyl alcohol were absorbed and excreted unchanged in the urine. They were not subject to first pass deconjugation or other metabolism in the intestine, intestinal wall or liver.

In silico models may have potential for predicting permeability and bioavailability but need to be "trained" with appropriate data sets for conjugates to improve their validity.

Further work is recommended in the following areas:-

Carry out an in vivo rat study of the β-D-glucoside of benzoic acid to study the effect of acyl migration on absorption and metabolism.

Investigate the absorption and excretion of higher molecular weight conjugates. The current data set with two relatively low molecular weight compounds may be anticipated to represent the worst case scenario, hence it would be interesting to assess whether other compounds would be absorbed as readily and whether they would be metabolised prior to excretion. If higher molecular weight conjugates were not absorbed directly, their degradation in the colon and potential for absorption as exocon moieties, should also be examined.

Investigate options for further development of the in silico models including an assessment of any new models which become available through both commercial and academic organisations.

Experiments with bound residues derived from pentachlorophenol and 3,4-dichloroaniline demonstrated the following:-

Very thorough multi-stage solvent extraction of the wheat was needed to ensure that the residual debris represented a true unextractable residue.

The literature methods used for the characterisation of bound residues were generally found to be time consuming and difficult to reproduce. They do not necessarily indicate the true nature of molecular binding to macromolecules and results could be misleading. It was, nevertheless, clear that the pentachlorophenol and 3,4-dichloroaniline residues differed significantly in their nature of association with plant macromolecules.

Both the pentachlorophenol and 3,4-dichloroaniline bound residues had low bioavailability in the rat. In vitro tests tended to solubilise the bound residues, especially the pentachlorophenol sample, to an

extent which, in the absence of in vivo data, may have led to an overestimate of the likely bioavailability. Solubilisation of bound residues does not necessarily equate with bioavailability.

It would be of interest to conduct an assessment of in vitro gastrointestinal models to determine whether they could provide an accurate prediction of the bioavailability of bound residues and offer a reliable substitute for animal experiments.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings;

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possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

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Michael W. Skidmore, Jill P. Benner, Cathy Chung Chun Lam, James D. Booth, Terry Clark, Alex J. Gledhill, Karen J. Robert, August 2007; Bioavailability of Common Conjugates and Bound Residues.in Pesticide Chemistry: Crop Protection, Public Health, Environmental Safety, Ed. Hideo Ohkawa, Hisashi Miyagawa, Philip W. Lee, ISBN: 978-3-527-31663-2.

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