Survey of 3-Monochloropropane-1,2-diol (3-MCPD) in Selected Food Groups (Number 12/01)

Monday 19 February 2001

FSA Surveillance Information Sheet

Introduction
The results of two surveys commissioned by the FSA into levels of 3-MCPD found in food and food ingredients were published on 1 February. The majority of samples in both surveys did not contain quantifiable levels of 3-MCPD. Where 3-MPCD was detected it was not at levels that would give cause for concern and there is no need for consumers to change their diet. Recent toxicological studies are currently being evaluated and the FSA will inform consumers if the advice changes.

3-MCPD is a chemical contaminant that can form during food processing and scientific studies have shown that it can cause cancer in animals.

Until recently, EU and UK expert committees considered 3-MPCD to be genotoxic and the Food Advisory Committee (FAC) had advised that levels should be 'undetectable.' UK expert committees have concluded in recent studies that 3-MCPD is not genotoxic and, as a result, the FAC have now advised that levels be reduced to the lowest technologically possible in all foods.

For the food survey, 300 retail samples were analysed, including cereal-based products, soups, meat and dairy products. Of these, 70 per cent did not contain quantifiable levels. The highest levels found were low (0.13mg per kg) ¿ in cream crackers. The food groups identified as most likely to contain 3-MCPD include biscuits, bread and cooked/cured fish and meat.

Also analysed were 63 food ingredients, including caramels, meat/yeast/malt extracts and breadcrumbs. There were no quantifiable levels of 3-MCPD in 78 per cent of these samples. The highest level measured was 0.49 mg/kg. This is higher than the levels in the food survey, but generally these ingredients constitute less than 2 per cent of the final food product. Even for ingredients such as malt flour and breadcrumbs, which could constitute up to 10 per cent, it is still not a cause for concern at the levels found in this survey.

To generate further information that will help reduce 3-MCPD levels, the Agency is funding a comprehensive project on the origin and formation of 3-MCPD. A further survey on soy sauces is also currently being carried out and the results will be available in the next few months.

Key Facts

• 3-monochloropropane-1,2-diol (3-MCPD) is one of a group of chemical contaminants known as chloropropanols. It occurs at low levels in many foods and food ingredients as a result of processing or storage conditions.
• 3-MCPD is a known carcinogen in animals, but recent studies suggest that it is not genotoxic in-vivo. Further evaluation by expert committees may permit a limit of exposure to be set.
• 300 retail food samples were analysed in this survey from five main food groups thought most likely to contain 3-MCPD, including cereal products, soups, meat products and dairy produce.
• 70 per cent of the foods analysed in this survey did not contain quantifiable levels of 3-MCPD.
• The survey identified that the food groups most likely to contain 3-MCPD include baked goods, bread and cooked/cured meat/fish.
• At this stage, there is no need for consumers to change their diets as a result of these findings. Action is being taken to generate more information to further reduce exposure.

Purpose
When this work was commissioned in 1998, expert committees both in the UK and Europe advised that 3-MCPD was a genotoxic carcinogen and as such should be undetectable in foods. 3-MCPD was originally identified as a contaminant of the savoury ingredient acid-hydrolysed vegetable protein (acid-HVP) and subsequently in soy sauce. This further survey was set up as a rapid response to the perceived urgent food safety issue at that time. The food groups chosen for investigation were those which available information, including the HVP and soy sauce studies, indicated were the most likely to contain 3-MCPD. The survey was also designed to identify which food groups were most likely to be major contributors of 3-MCPD in the diet. A relatively few samples from each food group were taken for analysis to allow the survey to cover the widest possible range of foods. Hence, no significance can be attached to the presence or absence of any particular brand as samples were taken at random in each of the food groups considered. It should therefore be recognised that the survey results primarily indicate those food groups where further work is needed to reduce levels of 3-MCPD. Based on new toxicological evidence, UK experts now consider 3-MCPD remains a potential carcinogen, but not genotoxic in-vivo. Therefore, the Agency's Food Advisory Committee now advises the food industry to reduce levels of 3-MCPD in foods to the lowest levels technologically achievable.

Summary
A survey of 3-monochloropropane-1,2-diol (3-MCPD) in a range of foods available in the UK has been completed. The analysis of the samples was conducted by the Central Science Laboratory (CSL) using a validated method of analysis with a limit of quantification of 0.01 mg/kg. The survey was initially commissioned to assess the progress made by manufacturers in addressing the Food Advisory Committee's (FAC) 1996 recommendation1 that 3-MCPD should not be found in any food or ingredients using a validated method of analysis capable of measuring down to 0.01 mg/kg. Whilst the survey was in progress the FAC modified its advice, as a result of data submitted by industry, and recommended that "industry should continue to take all steps necessary to ensure that 3-MCPD is undetectable (i.e. less than 10 ppb) in foods and, where technologically feasible, in food ingredients as well."2

The survey was mainly undertaken to identify the food groups which might provide a significant contribution to exposure from the diet. 3-MCPD was not quantified in 70 per cent (211/300) of food samples. Three of the food sub-groups tested, breakfast cereals, soups and confectionery, had no quantifiable levels of 3-MCPD in the samples analysed. Foods with quantifiable levels of 3-MCPD included breads, savoury crackers, toasted biscuits, burgers and salamis.

Foods which required cooking before consumption were prepared according to the packet instructions where available or in the absence of such advice by following typical practice before analysis. If these foods were found to contain 3-MCPD, uncooked portions which had previously been set aside were then analysed. In all the cases where 3-MCPD was detected in cooked foods it was also present in the uncooked food. However, the data were insufficient to say whether cooking contributed towards the formation of 3-MCPD in food.

The FAC have considered the results of the survey and issued a statement as set out below.

Background
3-MCPD is one of a group of chemical contaminants known as chloropropanols. 3-MCPD was originally identified as a contaminant of the savoury ingredient acid-hydrolysed vegetable protein (acid-HVP),3 which is produced using hydrochloric acid. In acid-HVP, components of fats and oils in the starting materials are chlorinated at high temperature to form chloropropanols. 3-MCPD has since been found to occur at low levels in many foods and food ingredients,4-7 though the origin and formation of 3-MCPD in these is not yet fully understood. In addition to the research being sponsored by industry, the Agency also intends to address this problem and has recently commissioned a comprehensive research project that is to start shortly.

In 1994, following a review of the available data, the EC Scientific Committee for Food (SCF) concluded that "3-MCPD should be regarded as a genotoxic carcinogen."8 In line with the SCF's opinion, the FAC recommended in 19961 that "within 18 months, the food industry should take all the steps necessary to ensure that 3-MCPD cannot be found in any food or ingredients, regardless of the method of manufacture, when using a validated method of analysis capable of measuring down to 10ppb, the lowest level of detection currently achievable." Industry has endeavoured to meet the Committee's advice through individual and concerted effort and investment.

In 1999, following earlier surveys of 3-MCPD in the savoury food ingredient acid-HVP,5 soy sauce and similar products,6 and supported by information from the industry, the FAC recognised the difficulty of attaining such low levels in food ingredients. They subsequently revised their advice and recommended2 that industry continue to take all steps possible to ensure that 3-MCPD is undetectable in foods and, where technologically feasible, in food ingredients as well.

In October 2000, the Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM) considered 3-MCPD in light of two new toxicological studies, with the following outcome: "The Committee concluded that both the rat bone-marrow micronucleus test and the rat liver UDS test had been carried out to an acceptable standard and were negative. Thus the additional information recommended by the COM as being necessary to provide adequate reassurance that the mutagenic activity seen in-vitro was not expressed in-vivo had now been provided.

The Committee agreed that the major urinary metabolite beta-chlorolactic acid in rats was formed by oxidation of 3-MCPD and that the two new mutagenicity studies supported the view that reactive metabolites if formed did not produce genotoxicity in-vivo. The Committee concluded that 3-MCPD can be regarded as having no significant genotoxic potential in-vivo."9

The FAC considered the COM's new opinion at its meeting in October 2000 and issued the following advice:
"Previous advice from the Committee on Mutagenicity (COM) and the Committee on Carcinogenicity (COC) concluded that 3-MPCD has shown mutagenic potential in-vitro and carcinogenicity in the rat, but that it was not possible to draw firm conclusions on the mechanism of carcinogenicity. New toxicological studies have been reviewed by the COM who have revised their advice and stated that 3-MCPD can be regarded as having no significant genotoxic potential in-vivo. Nevertheless it is still a carcinogen in animals. The data on 3-MCPD is to be reviewed by the COC and by international committees and it is anticipated that this will lead to the establishment of a tolerable daily intake (TDI). Until these considerations have been completed, the Committee advises industry that they should continue to take all steps necessary to reduce concentrations of 3-MCPD in foods and food ingredients to the lowest technologically achievable."10 Following the deliberations of the COM, the Committee on Carcinogenicity (COC) re-considered its advice and issued a comprehensive statement in December 2000. It concluded that:
"3-MCPD was unlikely to present a carcinogenic risk to man, provided the exposure was 1000 times lower than the no observed effect level (NOEL) of 1.1 mg/kg body weight/day for tumourigenicity."11

The data, statements and conclusions of the COM and COC will be reviewed by the SCF and by the Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Committee on Food Additives (JECFA) in 2001. It is anticipated that this will lead to a Tolerable Daily Intake being set by the end of 2001.

The survey described here was conducted to assess the levels of 3-MCPD in several different food groups which had previously been shown to contain 3-MCPD, foods likely to contain 3-MCPD formed during processing and foods containing compounds which are potential 3-MCPD precursors. All the samples analysed were obtained from retail outlets. The Food Standards Agency has also conducted a survey of the levels of 3-MCPD in food ingredients; this is to be reported concurrently with these results.

The process of planning, conducting and preparing to report this survey has been a consultative one with industry. As a result, the Food and Drink Federation Retailer 3-MCPD Liaison Committee have issued the following statement:
"The food industry, embracing both food manufacturing and retailing interests, has undertaken a substantial programme of work flowing from the FAC's recommendation in October 1996. The industry has been at the forefront of activities to reduce levels of 3-MCPD in manufactured ingredients and foods. Research has been encouraged and technically supported, extensive analytical surveys commissioned and best practice shared.

As a result, much new data have been generated enabling the various sectors of industry involved in this collective activity to reduce consumer exposure in line with the advice of the FAC. To date achievements include:

Improved knowledge of the occurrence of 3-MCPD in foods and ingredients.
Improved knowledge of the conditions which influence the production of 3-MCPD.
Reducing levels of 3-MCPD in foods and ingredients; e.g. HVP, soy sauce.
Introduction of alternative food contact materials to reduce migration of 3-MCPD into foods such as tea and salami.

It is acknowledged that, potentially, more could be achieved when the mechanism of how 3-MCPD occurs in ingredients and foods is fully understood. Nevertheless, in the short term there is no identifiable technical solution for certain products.

Industry's commitment to the objectives set out above, and its desire to work closely and collaboratively with Government, illustrate the priority it accords to food safety and protection of the consumer. The results of the FSA survey usefully complement the studies carried out by industry, have confirmed the low incidence and levels found currently and will assist in directing the continuing endeavours of industry and Government to minimise consumer exposure to 3-MCPD."

In line with the guidelines for planning and reporting surveys, manufacturers whose products were found to contain quantifiable levels of 3-MCPD, i.e. at or above 0.01 mg/kg, were sent details of their individual results and invited to submit comments of up to 200 words. Annex 1.

Sampling
300 samples from 5 food groups, namely; cereal products, dairy produce, meat and fish products, soups and miscellaneous foods, were purchased from representative retail outlets in the York area, between November 1999 and May 2000. A full list of these and their brand names were placed on the Agency's website.12 Descriptions of analysed samples are reported in Table 8. The absence of a particular brand from this table means only that the product was not included in the survey. The foods studied included food types shown previously to contain 3-MCPD (e.g. salami, cheese and malt extracts), foods likely to contain 3-MCPD formed during processing (pre-cooked meals) and foods containing compounds which are potential 3-MCPD precursors. A limited study of the effect of domestic cooking on levels of 3-MCPD has indicated that this too may be a contributory factor.13 Hence some samples, e.g. burgers and bacon, which are cooked prior to consumption were analysed before and after cooking.

Samples of 500 g minimum weight were purchased. Where more than one packet was required samples having the same batch code and/or use-by date were purchased. All samples were stored frozen, cool or dry as appropriate. Samples for any necessary future analysis were retained as required by the current guidelines for planning and reporting surveys.14 Foods which did not require cooking were divided into three portions, with one being used for analysis and the other portions being retained. For products which required cooking at least three packets were purchased with one pack being cooked as instructed and an unopened pack being retained for any required subsequent re-analysis. Cooked foods were prepared according to the packet instructions. Where 3-MCPD was detected in the cooked sample, the third packet was analysed uncooked.

Methodology
Analyses were carried out using a validated and accredited gas chromatographic method (GC-MS) for the determination of 3-MCPD15 with a limit of quantification of 0.010 mg/kg. This limit was the lowest common level at which 3-MCPD could be quantified in all the food matrices tested in this survey. It was based on the limits of detection measured in the collaborative trial of this method.15 An internal standard of deuterated 3-MCPD (d5-3-MCPD) was added to a known amount of sample followed by saline solution and the mixture blended to a homogenous consistency.

After sonication or centrifugation, depending on sample type, the sample was thoroughly mixed with diatomaceous earth. This mixture was transferred to a glass chromatography column and the non-polar components eluted using diethyl ether. The sample extract was concentrated under rotary evaporation to a small volume. A portion of the extract was then derivatised using n-heptafluorobutyrlimidazole. The derivatised sample was analysed by GC-MS with selected ion monitoring of 3-MCPD, m/z 253, 275, 289, 291, 453 and d5-3-MCPD, m/z 257, 456.

Samples were analysed in batches comprising up to 17 samples, a reagent blank, a soy sauce reference material and a matrix reference material appropriate to each food group. Details of this quality assurance procedure are given in Annex 2.

The measurement uncertainty of the reported results was estimated as described in Annex 3.

Results
Table 1 is a summary of the results; it classifies the samples in their constituent food groups and gives details of the maximum levels of 3-MCPD reported in each sub-group. The full data are presented in Tables 2-7, where reference is also made to the applicable measurement uncertainty. Table 8 contains all the details of the 300 samples purchased as part of the survey.

3-MCPD was not reported in 211 of the 300 (70 per cent) and was quantified in 89 of the 300 (30 per cent) food samples using a method with a limit of quantification of 0.010 mg/kg. The limit of quantification being the level at which the analytical methodology has confidence in the result, it is always greater than the limit of detection. For any analytical method each result reported is the best estimate for that sample, it is always qualified by the measurement uncertainty, e.g. x mg/kg plus or minus y mg/kg. (See Annex 2)

49 of the 106 (46 per cent) samples classified in the cereal products food group (Table 2) had quantifiable levels, though none of these were breakfast cereals. The highest level of 3-MCPD observed in the survey was in a sample of cream crackers. 3-MCPD was quantified in 4 of the 35 (11 per cent) samples of dairy products (Table 3), 26 of the 63 (41 per cent) samples of meat products (Table 4) and in 10 of the 62 (16 per cent) samples of miscellaneous foods (Table 5). None of the 34 soup samples contained 3-MCPD at quantifiable levels (Table 6).

Of the samples which required cooking before consumption and which were thus cooked before analysis only the chargrilled burgers, two bacon samples and a flame grilled chicken sample contained quantifiable 3-MCPD. Analysis of uncooked samples of these foods showed that 3-MCPD was also present at quantifiable concentrations in the uncooked food. None of the crumpets, soups, sauces or pizzas which were cooked prior to analysis were found to contain quantifiable 3-MCPD (Table 7).

The FAC considered the results of this work in the light the latest toxicological advice and made the following statement:
"The Committee welcomes the fact that 70 per cent of samples did not contain quantifiable levels of 3-MCPD. The Committee commends industry's efforts to deal with this contaminant and urges them to continue to work with the Food Standards Agency to identify the origin of the contamination in food groups where quantifiable levels of 3-MCPD were found, thus enabling concentrations to be further reduced to the lowest technologically achievable in all foods. The situation should be reviewed once the SCF and JECFA have considered whether it is possible to set a Tolerable Daily Intake for this contaminant."

Interpretation
The origin and formation of 3-MCPD is not yet fully understood. The Agency intends to address this problem by commissioning a comprehensive research project, which is to start shortly. The results from this survey will help to identify food groups and processes that will need to be considered as part of this work.

The objective of this survey was to identify those food groups where further work is needed to reduce levels of 3-MCPD. These results indicate that the ingredients and/or processing conditions of most savoury crackers and toasted biscuits often lead to the formation of 3-MCPD. A smaller proportion of other wheatflour based products such as biscuits, cakes, pies and breads were also found to contain 3-MCPD. However breakfast cereals, many of which contained wheat or wheat products and some of which may have been toasted during processing, did not contain 3-MCPD.

3-MCPD was quantified in samples of smoked salmon and anchovies; in contrast none of the battered or breaded fish products analysed contained quantifiable 3-MCPD. Processed fish require further investigation. The components and/or processing of cooked/cured meats, chargrilled burgers and salamis also appear to enhance the formation of 3-MCPD. These foods also warrant further investigation.

Where foods were found to contain 3-MCPD after cooking, uncooked samples were also analysed. 3-MCPD was also quantified in all these uncooked samples and the cooking process did not have a clear effect on the formation of 3-MCPD.

3-MCPD was not found in foods such as soups and sauces in which it was thought likely to be detected due to the presence of savoury ingredients, such as HVP. This may be explained by manufacturers moving away from the use of acid-HVP to enzyme-HVP as an ingredient; 3-MCPD is not formed in the production of the latter.

Following the recent considerations of the COM and COC, it has been suggested that a dietary exposure assessment might be undertaken. Until the TDI is available it is not possible to interpret or assess any estimation of dietary intake. The NOEL figure identified by the COC merely takes into account the carcinogenicity and does not include any other toxic effects that might be produced by 3-MCPD. In June 2001, JECFA will evaluate the full toxicological implications of 3-MCPD. The results of this survey will be supplied to JECFA to assist in their deliberations which it is anticipated will result in the setting of a tolerable daily intake (TDI) for 3-MCPD; it would be premature to anticipate their advice.

In addition the amount of published work investigating levels of 3-MCPD in foods to date is minimal; hence the data available are extremely limited. For example, some of the food groups in the survey had less than ten samples taken, which prohibited the estimation of batch-to-batch and brand-to-brand variation. It would be inappropriate to take these few results as anything more than indicative of the possible trends in 3-MCPD occurrence. Before an assessment of dietary intake can be carried out a more comprehensive data set needs to be available. To this end, a European Scientific Co-operation Task (SCOOP) has been set up, with the UK as joint co-ordinator, which will collect and collate information on the levels of 3-MCPD in foods. This is due to be completed early in 2002.

Furthermore, the limited data currently available suggest that some forms of domestic cooking can result in an increase in the levels of 3-MCPD observed in food.13 The Agency has recently commissioned a short research project which will investigate this further. Again, a comprehensive data set is necessary before a robust dietary intake estimation can be made.

It is anticipated that the coincidence of the setting of a TDI, the completion of the SCOOP task and data from the research project on domestic cooking will enable a robust and informed intake assessment to take place at that time.

Conclusions
Of the 300 food samples analysed 70 per cent had no quantifiable levels of 3-MCPD. However, the results also confirm that 3-MCPD may be present in a wide range of foods although the mechanism of its formation is not fully understood. The Agency has commissioned a comprehensive research project, due to start in April 2001, which will address this issue. Results indicate that the occurrence of 3-MCPD is erratic within the food groups in which it has been found and further investigation is needed. 3-MCPD was not quantified in some of the foods in which it has previously been found; this confirms the efforts industry has made to reduce the levels of 3-MCPD in these foodstuffs in line with the FAC recommendation. The Food Standards Agency and industry continue to work towards a reduction in levels of 3-MCPD in food.

Summary of Units
Milligram (mg): one thousandth of a gram
Kilogram (kg): one thousand grams
Milligrams per kilogram (mg/kg)
ppb: parts per billion
m/z: mass/charge

1. Food Advisory Committee, Update on chloropropanols present in protein hydrolysates, Press Release 13/96, 1996.
2. Food Advisory Committee, Recent developments on 3-monochloropropane-1,2-diol (3-MCPD) in food and food ingredients, Press Release 5/99, 1999.
3. Velisek, J. D., Davidek, J., Kubelka, V., Janicek, G., Svobodova, Z and Simicova, Z. New Chlorine Containing Organic Compounds in Protein Hydrolysates. Journal of Agricultural and Food Chemistry, 1980, 28, 1142-44.
4. Collier, P. D., Cromie, D. D. O. and Davies, A. P. Mechanism of formation of chloropropanols present in protein hydrolysates. Journal of the American Oil Chemists Society, 1991, 68, 785-790.
5. Survey of 3-monochloropropane-1,2-diol (3-MCPD) in acid-hydrolysed vegetable protein.
6. Survey of 3-monochloropropane-1,2-diol (3-MCPD) in soy sauce and similar products.
7. Macarthur, R., Crews, C., Davies, A., Brereton, P. and Harvey, D. 3-monochloropropane-1,2-diol (3-MCPD) in soy sauce and similar products available from retail outlets in the UK. Food Additives and Contaminants 2000, 17(11) 903-906.
8. European Commission Opinion on 3-monochloropropanediol (3-MCPD), expressed on 16 December 1994. Reports of the Scientific Committee for Food. Food Science and Techniques, Thirty-sixth Series, 1995, 31-33.
9. The effects of cooking on the level of 3-monochloropropane-1,2-diol (3-MCPD) in foods. CSL Report FD 98/60.
10. Determination of 3-monochloropropane-1,2-diol in food and food ingredients using mass spectrometric detection: Collaborative trial. CSL Report FD 97/95

Further Information:

Mrs Sue Johns Food Standards Agency Contaminants Division PO Box 31037 Room 210, Ergon House 17 Smith Square London SW1P 3WG Tel: +44 (0) 20 7238 6772 Fax: +44 (0) 20 7238 5331 E-mail: sue.johns@foodstandards.gsi.gov.uk

A full report of this survey is held in the Library at Nobel House, 17 Smith Square, London, SW1P 3JR; Tel: +44 (0) 20 7238 6575. If you would like to consult a copy, please contact the Library giving at least 24 hours notice or, alternatively, copies can be obtained from the Library; a charge will be made to cover photocopying and postage. Further copies of this Information Sheet can be obtained from: Food Standards Agency Information Centre PO Box 31037 Room 303b, Ergon House 17 Smith Square London SW1P 3WG Tel: + 44 (0) 20 7238 6223 Fax: + 44 (0) 20 7238 6330 Email: information.centre@foodstandards.gsi.gov.uk

Copies of the statements from the Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment and the Committee on Carcinogenicity of Chemicals in Food, Consumer Products and the Environment the can be obtained from: Khandu Mistry Room 692D Skipton House 80 London Road London SE1 6LH Tel: +44 (0) 20 7972 5020 Fax: +44 (0) 20 7972 5156 Email: Khandu.Mistry@doh.gsi.gov.uk

Further information on the work of the Food Advisory Committee (FAC) can be obtained from: Mr John Caseley FAC Secretariat PO Box 31037 Room 224, Ergon House 17 Smith Square London SW1P 3WG Tel: + 44 (0) 20 7238 6267 Fax: + 44 (0) 20 7238 6263 Email: john.caseley@foodstandards.gsi.gov.uk

Annex 1 COMMENTS The Biscuit, Cake, Chocolate and Confectionery Alliance have issued the following statement on behalf of their members: "The cake and biscuit industry has taken very seriously the recommendation from the Food Advisory Committee that industry should take all steps necessary to reduce 3-MCPD in foods to the lowest levels technologically achievable. Through their trade association, the Biscuit Cake Chocolate & Confectionery Alliance, cake and biscuit manufacturers have maintained a three year screening programme to measure and understand occurrence of 3-MCPD in the industry's products. The findings, like those of the present FSA survey, indicate that occurrence of 3-MCPD is erratic and the factors governing its production are complex. In order to understand better how 3-MCPD is formed, and therefore how it can be controlled, the Alliance commissioned research at Campden & Chorleywood Food Research Association. This showed that baking temperature and dough types may influence 3-MCPD formation. In the short term there is no technical solution, as altering the dough type would produce an unpalatable product. However the Alliance will commission further research next year following a meeting of an international expert committee which is due to consider 3-MCPD. The Alliance has given FSA copies of its earlier research findings as they became available and will continue to work with FSA to further our understanding and control of this substance." Statement from Pogen concerning 3- MCPD in foods: "Pogen is seriously concerned over the results shown in the report, and we have therefore listed a number of steps, which we will take in order to reduce the level of 3-MCPD in our products to the lowest technological achievable level. First of all we need to increase our knowledge about 3-MCPD. Further we need to contact our fat supplier for discussions on fat alternatives, and finally we need to review our recipes and production process in order to see which improvements can be made." Statement from Thomas the Baker: "On receiving the result of analysis of our product for 3-MCPD, Thomas the Baker has instigated a series of investigations in an effort to reduce levels down to the lowest technologically achievable, in line with current advice from the Agency's Food Advisory Committee. We have contacted each individual supplier of the ingredients used to make our wholemeal bread and requested up to date specifications, as well as any further useful information. In the light of these comments we will be looking closely at the baking cycle of our wholemeal breads to see if this has an impact on the presence of 3-MCPD. Within a smaller purely retail business such as our own, we do not have the facilities or resources to establish the exact routes or sources of 3-MCPD in food. We shall watch with interest for the outcome of the research that industry federations and the Agency are currently carrying out into the origins and formation of 3-MCPD." Statement from John West: "Anchovies are prepared using traditional salting techniques. Salting is done in 2 stages - immersion in brine to rapidly diffuse salt into the flesh preventing spoilage, followed by storage in barrels with dry salt under pressure which gives the anchovies their characteristic flavour and texture, and reduces the water activity to circa 0.75 to prevent bacterial growth. No other ingredients are added, nor are the canned anchovies heat processed. Because anchovies met some of the criteria considered important in the formation of 3-MCPDs, we have undertaken a series of analyses which have indicated sporadic and variable levels of 3 MCPDs from 10 ppb to over 50 ppb. The causes for higher levels are not fully understood, but are likely to be driven by variations in fat content and the dynamics of ripening as affected by pressure, storage time and temperature. It is not, therefore, currently possible to identify and avoid conditions that favour 3 MCPD formation. Anchovies are a traditional foodstuff and therefore this is an issue common to the industry. Contribution to the diet is extremely low so the concomitant intake of 3 MCPDs is insignificant."

Annex 2 ANALYTICAL QUALITY ASSURANCE The following quality assurance criteria were set: - Reagent blanks should not show a 3-MCPD response equivalent to or greater than 0.05 mg/kg. - The values for the cross matrix reference material should fall within 0.025 mg/kg plus or minus 0.006 the value determined by in-house homogeneity testing. - The values for the salami reference material should fall within 0.022 mg/kg plus or minus 0.002 the value determined by in-house homogeneity testing. - The values for the cheese alternative reference material should fall within 0.028 mg/kg plus or minus 0.004 the value determined by in-house homogeneity testing. - The values for the breadcrumbs alternative reference material should fall within 0.030 mg/kg plus or minus 0.004 the value determined by inter-laboratory collaborative trial. - The values for the soup reference material should fall within 0.035 mg/kg plus or minus 0.003 the value determined by in-house homogeneity testing. - The values for the aqueous reference material should fall within 0.010 mg/kg plus or minus 0.003 the value determined by in-house homogeneity testing. Results exceeding 2 standard deviations but lower than 3 standard deviations were regarded as having reached the warning level. In this instance the rules governing Shewart control charts were adopted. Where the quality control criteria were not adequately met the analytical batch was repeated. The results show that the analyses met the quality criteria set such that: - None of the reagent blank samples contained 3-MCPD at or above 0.005 mg/kg. - All determinations of 3-MCPD in the soy sauce reference material fell within plus or minus 2 s.d. of the homogeneity tested mean. - All determinations of 3-MCPD in the cheese alternative class reference material fell within plus or minus 2 s.d. of the homogeneity tested mean. - One determination for of 3-MCPD in the cheese bread reference material gave a value outside the warning limit (i.e. greater than 2 s.d.) of the control chart but subsequent values for the reference material were within the required limits. This was in compliance with the rules governing Shewhart control charts and the batch was accepted. - One determination for of 3-MCPD in the salami class reference material gave a value outside the warning limit (i.e. greater than 2 s.d.) of the control chart but subsequent values for the reference material were within the required limits. This was in compliance with the rules governing Shewhart control charts and the batch was accepted. fell within plus or minus 2 s.d. of the homogeneity tested mean. - All determinations of 3-MCPD in the soup class reference material fell within plus or minus 2 s.d. of the homogeneity mean. - All determinations of 3-MCPD in the aqueous reference material fell within plus or minus 2 s.d. of the homogeneity mean.

Annex 3
MEASUREMENT UNCERTAINTY
Introduction
All analytical results have a variability known as the measurement uncertainty. The Eurachem/CITAC interpretation of the definition of measurement uncertainty is "the range of values that the analyst believes could be reasonably attributed to the measurand." Thus, the values for 'measurement uncertainty' given in this report represent the range around the best estimate of the concentration of 3-MCPD within which the true value for the concentration of 3-MCPD is believed to lie.

Method It has been stated that results from a collaborative trial carried out to validate a published method are a recognised source of data to support an uncertainty estimate. The data from such trials typically include estimates of reproducibility standard deviation, SR, for several levels of response, a linear estimate of the dependence of SR on the level of response, and may include an estimate of bias based on CRM studies. How this response can be used depends on the factors taken into account when the study was carried out. The measurement system used in this survey has been shown to be in analytical control (Annex 2). The collaborative trial results have therefore been used to provide an estimate of the measurement uncertainty associated with the reported results. One of the principal objectives of the collaborative trial was to allow an estimate of the measurement uncertainty/reliability on the results to be obtained. The measurement uncertainty may be calculated from the critical differences calculated from the trial results, where it has been demonstrated that the measurement system is in control. These values equate to the range around the measured value in which the true value will fall. For example, in the case of breadcrumbs used in the trial, an analyst obtaining a result of 0.030 mg/kg knows that the "true value" will be in the range 0.030 mg/kg plus or minus MU. Thus, the true value is in the range 0.030 plus or minus 0.012 mg/kg . For survey matrices found to often contain 3-MCPD but not included in the collaborative trial (chargrilled burgers, savoury crackers and doughnuts) measurement uncertainty was estimated from data obtained by in-house testing. Five unspiked and five spiked (0.01 mg/kg 3-MCPD) samples of each matrix were analysed in duplicate on five days. The standard uncertainty was calculated from estimates of the in-house reproducibility and bias (recovery). A coverage factor of 2 was used, giving a confidence of approximately 95 per cent.

Results The estimates of the measurement uncertainty obtained for each material, by collaborative trial or in-house testing, are given below. It has also been shown that a generalised equation for SR values at concentrations less than 0.12 mg/kg is 22 per cent of the concentration value

Test Material	Mean value (mg/kg)	Sr	SR	HoR	MU plus or minus (mg/kg)
HVP	0.029	2	4	0.47	0.008b
Malt Extract	0.055	3	7	0.54	0.014b
Soup Powder	0.043	4	8	0.73	0.016b
Breadcrumbs	0.030	3	6	0.77	0.012b
Danish Salami	0.016	2	6	1.30	0.012b
Cheese Alternative	0.043	5	10	0.87	0.020b
Chargrilled Burgers	0.040	2	5a		0.011c
Savoury Crackers	0.014	0.4	1a		0.002c
Doughnuts	0.020	1	2a		0.005c

^a: 'within-laboratory reproducibility' ^b: measurement uncertainty based on collaborative trial results ^c: measurement uncertainty based on in-house testing Where: Sr is the standard deviation of the repeatability [ this is a measure of the between-laboratory variability expressed as a standard deviation on individual results obtained in the trial ] SR is the standard deviation of the reproducibility this is a measure of the between-laboratory variability expressed as a standard deviation on individual results obtained in the trial HoR is the ratio of the actual between-laboratory precision to the predicted between-laboratory precision value. [ this is a measure of the acceptability of the precision of the method. Ratios of less than 2 are found for methods being considered "satisfactory" with respect to their precision characteristics ] MU is an estimate of the measurement uncertainty for each sample derived from the trial results. It equates to the plus or minus range within which the true value for the concentration of the analyte is believed to lie with a confidence of approximately 95 per cent.

References
1. Eurachem/CITAC Guide on Quantifying Uncertainty in Analytical Measurement, second edition, 2000.
2. Thompson, M. and Wood, R. (eds), Guidelines on Internal Quality Control in Analytical Chemistry Laboratories, Pure Applied Chemistry, 1995, 67, 649-666.
3. Determination of 3-monochloropropane-1,2-diol in food and food ingredients using mass spectrometric detection: Collaborative trial. CSL Report FD 97/75.

Tables All tables (1-8) are available below.