Survey of 3-Monochloropropane-1,2-Diol (3-MCPD) in Soy Sauce and Related Products (Number 14/01)

Monday 18 June 2001

Food Survey Information Sheet

Key Facts

• 3-monochloropropane-1,2-diol (3-MCPD) is a chemical contaminant which is known to occur at low levels in many foods and food ingredients.
• 3-MCPD is known to cause cancer in animals, but recent studies have shown that it does not directly damage genetic material. Expert committees are currently evaluating data on 3-MCPD to establish a safe level of consumption.
• This survey repeats an earlier survey of 3-MCPD in soy sauce and similar products published in 1999. It was undertaken to measure the progress by industry in reducing levels of 3-MCPD.
• In March 2001, the European Commission adopted a regulatory limit of 0.02mg/kg based on 40 per cent dry matter content for 3-MCPD in soy sauce and hydrolysed vegetable protein (HVP), to come into force in April 2002.
• 22 per cent of the 100 samples of soy sauce and related products analysed in this survey contained levels of 3-MCPD above the European Union (EU) limit.
• Consumers are advised to avoid products identified in this survey as containing 3-MCPD levels above the agreed EU limit.
• The Agency is working with Local Authorities to ensure that products identified in this survey as containing unacceptable levels of 3-MCPD are not available in the UK.
• The Agency will conduct a further survey of 3-MCPD levels in soy sauces shortly. This will target those products found to have unacceptable levels of 3-MCPD to assess the progress made by manufacturers in reducing levels.

Summary
A survey of 3-MCPD in a range of soy sauces and related products available in the UK has been completed. A similar survey was reported in 1999.1 100 samples of soy sauce and related products were purchased by Ventress Technical Ltd. from retail outlets in the UK. 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 for 3-MCPD. This survey assessed the levels of 3-MCPD in soy sauces currently available in the UK against the EU limit of 0.02 mg/kg, based on 40 per cent dry matter content which will come into force in April 2002.2

In this survey, 22 per cent of samples analysed contained levels of 3-MCPD equal to or above the EU limit compared with 32 per cent of the samples analysed in the 1999 survey. The highest level of 3-MCPD was 93.1 mg/kg.

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-HVP,3 which is produced by treating proteins from vegetables, such as soya, with hydrochloric acid. In acid-HVP, components of fats and oils in the starting materials are chlorinated at high temperature to form chloropropanols.

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".4 3-MCPD has since been found to occur at low levels in many foods and food ingredients,5-8 though the origin and formation of 3-MCPD in these is not yet fully understood. In line with the SCF's opinion, the FAC recommended in 1996 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.9

An earlier survey of 3-MCPD in soy sauce and similar products1 found significant levels of 3-MPCD in some products and the companies concerned were contacted. They were urged to take action to reduce the levels of 3-MCPD in line with the FAC's advice.

In October 2000, the Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM), considered two new toxicological studies and has advised that 3-MCPD need no longer be considered as mutagenic in vivo; nevertheless, it is still carcinogenic in animals.10 The FAC considered the COM's new opinion at its meeting in October 2000 and advised 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."11

The toxicological data and the statements and conclusions of the COM,10 and the UK Committee on Carcinogenicity of Chemicals in Food, Consumer Products and the Environment (COC)12 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 ultimately lead to an estimate of the amount of 3-MCPD which can be consumed daily over a lifetime without appreciable harm to health (Tolerable Daily Intake, TDI) being set.

This survey was commissioned to monitor changes in levels of 3-MCPD in soy sauce and related products since the previous survey. During the course of the survey, the European Commission adopted a regulatory limit of 0.02 mg/kg for 3-MCPD in soy sauce and HVP12 based on a 40 per cent dry matter content. The levels of 3-MCPD in these sauces have been assessed against this new EU level, with the limit of quantification in this survey being set at 0.01 mg/kg, This regulation was adopted in March 2001 and is due to come into force in April 2002, although the level in the regulation may be revised before coming into force, following the new advice from the SCF.

In line with the guidelines for planning, conducting and reporting surveys,13 manufacturers and distributors of products found to contain levels of 3-MCPD, at or above 0.02 mg/kg were sent details of their individual results. They were invited to submit comments of up to 200 words. These are reported in Annex 1.

3-MCPD has been shown to be a precursor of 1,3-dichloropropanol (1,3-DCP) formation and 1,3-DCP has been shown to occur in soy sauces.14 In light of this information and the continuing toxicological evaluation of this substance, the concentrations of 1,3-DCP were measured in the samples collected for this survey. Further to their consideration of 3-MCPD, the COM and COC have recently considered 1,3-DCP.15,16 The results of the analyses will be assessed and interpreted against the COM and COC advice and published concurrently in Food Survey Information Sheet 15/01.

Sampling
100 samples of soy sauces and related products were purchased from retail outlets in four areas of the UK; London, Leeds, Manchester and Cambridge during August 2000. Of these samples 67 were soy sauces and the remainder were various other related sauces e.g. mushroom soy, oyster and teriyaki. The details of each sample were recorded on receipt and descriptions are reported in Table 1. The absence of a particular brand from this table means only that the product was not included in the survey. 34 of the products sampled had been analysed in the 1999 survey so that a comparison could be made. Products found in the 1999 survey to have levels of 3-MCPD above 0.01 mg/kg are listed in Table 2. The sample plan aimed to include as many of the products tested in the previous survey as possible. The remaining samples were selected at random to reflect the wide range of products available and included own brands as well as proprietary brands.

Methodology
Analyses were carried out using a validated and accredited gas chromatographic method (GC-MS) for the determination of 3-MCPD17 with a limit of quantification of 0.01 mg/kg. The method uses deuterated 3-MCPD (d5-3-MCPD) as an internal standard. This is added to a known amount of sample followed by saline solution and the mixture is blended to achieve homogeneity. After sonication, the contents of a diatomaceous earth refill pack are added and mixed thoroughly. This mixture is transferred to a glass chromatography column and the non-polar components are eluted using a mixture of hexane and diethyl ether. The 3-MCPD is eluted using diethyl ether and the sample extract is concentrated under rotary evaporation to a small volume. A portion of this extract is subsequently derivatised using N-heptafluoro-butyrylimidazole (HFBI). The derivatised sample is analysed by gas chromatography-mass spectrometry (GC-MS).

Quantification was based on a calibration series of 3-MCPD standard solutions, equivalent to a range of concentrations 0.006 to 1.88 mg/kg 3-MCPD, measured with each batch. Samples that were found to contain 3-MCPD at levels higher than 1.88 mg/kg were diluted and re-analysed. The samples were analysed in batches. Each batch included a reagent blank and at least one spiked sample fortified at a level of 0.010 mg/kg. All results were corrected for recovery.

Analytical results have a variability known as the measurement uncertainty (details are given in Annex 2). 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 + y mg/kg. The measurement uncertainty was assessed to be + 0.004 mg/kg at a level of 0.01 mg/kg 3-MCPD. The analytical data showed that the analyses met the quality assurance criteria set (details are given in Annex 3).

Results
The results of the survey are summarised in Table 3 and Figure 1. A full list of results, including details of the brand names for each product is given in Table 1. 22 of the 100 (22 per cent) samples contained levels of 3-MCPD above the EU limit, these are listed in Table 4. Of the 67 samples of soy sauce, 12 (18 per cent) contained levels of 3-MCPD above the EU limit, and of the 33 samples of other similar products (such as oyster and teriyaki sauce), 7 (21 per cent) contained levels above the EU limit. The highest level of 3-MCPD was 93.1 mg/kg, in a soya bean sauce.

Interpretation
This survey was commissioned to monitor changes in levels of 3-MCPD since the previous survey. During the course of the survey, the European Commission proposed a regulatory limit of 0.02 mg/kg for 3-MCPD in soy sauce and HVP, based on 40 per cent dry matter content, which was agreed by Member States in December 2000. This regulation was adopted in March 2001 and is due to be implemented in April 2002.12 The levels of 3-MCPD in the sauces in this survey have been assessed against this limit.

In the 1999 survey,1 40 samples were analysed and the reporting limit was 0.01 mg/kg based on advice from the FAC.8 In the 1999 survey, 35 per cent of samples contained levels of 3-MCPD greater than the EU limit while in the current survey 22 per cent of samples contained 3-MCPD levels above the EU limit. Figure 2 and Table 5 show the percentage of total samples analysed in 1999 and 2000 that fall into ranges of levels of 3-MCPD. The current survey included 34 samples of products also analysed in the 1999 survey. Of these, 27 products contained levels less than the EU limit for 3-MCPD in both surveys.

A significant number of products sampled in this survey (22 per cent) still contained 3-MCPD concentrations above the EU limit. 3-MCPD was found in the range of sauces analysed and no correlation was observed between 3-MCPD levels and country of origin.

Possible sources of 3-MCPD in soy sauces are:

• The addition of acid-HVP,
• Acid hydrolysis of some or all of the soya bean/wheat and
• The toasting of the wheat component.

However, it is not possible to explore any link between method of manufacture and level of chloropropanols without further verifiable information from the producers.

Conclusion
22 per cent of samples analysed in this survey contained levels of 3-MCPD above the EU limit. The results show a decrease in frequency of contamination compared with the 1999 survey. However the current survey includes several brands of soy sauce not analysed in 1999 and some of these brands contained very high levels of 3-MCPD. The results show that sauces with unacceptable levels of 3-MCPD are still available in the UK. Indeed, the highest level found in this survey was higher than that found in the previous survey.

Those retailers responsible for selling soy sauces identified as containing high levels of 3-MCPD were asked to remove any remaining stock from the shelves. Details of all products found to have 3-MCPD levels greater than or equal to 0.05 mg/kg have been forwarded to the European Commission's Rapid Alert System. (The level of 0.05 mg/kg allows for the variation in the solid content of the products analysed). This information will then be forwarded to all Member States who may use this to arrange product recalls. The Agency is working with the Local Authorities Co-ordinating Body on Food & Trading Standards (LACOTS) to take this forward in the UK. Enforcement Officers will visit retailers whose products were shown to have high levels and inform them that a legal limit for 3-MCPD will be in force from April 2002. Retailers will also be advised to stock only those products where there is clear evidence that they have been tested for 3-MCPD and that they are within acceptable safety limits. This course of action supplements our dialogue with UK distributors of these products. Consumers are advised to avoid products identified in this survey as containing 3-MCPD levels above the agreed EU limit.

The Agency will conduct a further survey of 3-MCPD levels in soy sauces shortly to assess the progress made by industry in reducing levels of 3-MCPD in soy sauce and related products in both retail and catering establishments. Those products found to have unacceptable levels of 3-MCPD in this survey will be targeted in the follow up survey.

Summary of Units
Milligram (mg): one thousandth of a gram
Kilogram (kg): one thousand grams
Milligrams per kilogram (mg/kg)

1. Survey of 3-Monochloropropane-1,2-diol (3-MCPD) in soy sauce and similar products. Food Surveillance Information Sheet no. 187,1999.
2. Commission Regulation (EC) No 466/2001. Setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Communities. 8th March 2001.
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. 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.
5. Collier, P. D. Cromie, D. D. O. and Davies, A. P. (1991). Mechanism of formation of chloropropanols present in protein hydrolysates. Journal of the American Oil Chemists Society. 68, 785-790.
6. Survey of 3-Monochloropropane-1,2-diol (3-MCPD) in Selected Foods. Food Surveillance Information Sheet no.12/01, 2001.
7. Survey of 3-Monochloropropane-1,2-diol (3-MCPD) in Food Ingredients. Food Surveillance Information Sheet no.11/01, 2001.
8. Survey of 3-Monochloropropane-1,2-diol (3-MCPD) in acid-hydrolysed vegetable protein. Food Surveillance Information Sheet no. 181, 1999.
9. Food Advisory Committee. (1996). Update on chloropropanols present in protein hydrolysates, Press Release 13/96.
10. Crews C., Le Brun G., Hough P., Harvey D. and Brereton P. Chlorinated propanols and levulinic acid in soy sauces. Czech Journal of Food Sciences, Vol.18, (2000), 276-277.
11. Brereton, P et al. (2001). Determination of 3-Chloro-1,2-Propanediol in foods and food ingredients by gas chromatography with mass spectrometric detection: collaborative study. Journal of AOAC International. 84 (2), 455-465.

Further Information Further information on this survey can be obtained from: Mrs Sue Johns Contaminants Division Room 707c, Aviation House 125 Kingsway London WC2B 6NH Tel: +44 (0) 20 7276 8712 Fax number: 020 7276 8717 Email: sue.johns@foodstandards.gsi.gov.uk The full report of this survey is held in the Elsie Widdowson Library at Aviation House, 125 Kingsway, London WC2B 6NH Tel: +44 (0) 20 7276 8181. 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: Library and Information Service Food Standards Agency Room 115B, Aviation House 125 Kingsway London WC2B 6NH Tel: +44 (0) 207 276 8187 Email: library&info@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 Floor 5, Aviation House 125 Kingsway London WC2B 6NH Email: john.caseley@foodstandards.gsi.gov.uk

Annex1 - COMMENTS
From Lee Kum Kee
"Following the Agency's UK survey in September 1999 about various brands of Chinese sauces and subsequent to its own reviews, Lee Kum Kee (LKK) has succeeded in eliminating any traceable level of 3-MCPD in its products produced from October 1999. LKK implemented new production techniques, product formulations and raw material sourcing to ensure that all products manufactured thereafter, while meeting or exceeding consumer expectations for quality, are also subject to rigorous testing procedures to meet or exceed stringent regulatory standards.

Working closely with internationally accredited laboratories, LKK has become the only Hong Kong food company with its own laboratory that has received Hong Kong Laboratory Accreditation Service's international accreditation for testing against 3-MCPD in raw materials and finished products.

Consensus within the scientific community about 3-MCPD effects has not been reached, and there appears to be no definitive reason to take any additional actions currently. Nevertheless, LKK is concerned enough about this issue (including the varying levels of 3-MCPD in many other food and beverage products) that it remains committed and open to constant review.

LKK takes its responsibility seriously and will continue to do whatever necessary to protect consumer trust and the long heritage of its global brand."

From Manning Impex Ltd.
"We work with our supplier and their manufacturers to meet EU regulations with regard to food safety and 3-MCPD. Our manufacturer gives surety of their Good Manufacturing Practices (GMP) certification. Furthermore, the EU mission is currently visiting our supplier's manufacturing plant to validate their Hazard Analysis and Critical Control Point (HACCP) plan and certify compliance with GMP and Sanitation Standards Operating Procedures (SSOP). We have every confidence in the systems they have in place to maintain the highest standards.

After we informed our suppliers of the EU standards for 3-mcpd, in Sept 1999, they took immediate action to obtain the Certificates of Analysis (COA) from their manufacturers to ensure that all their raw materials are below recommended levels. The results of independent analysis utilising the UK CSL methodology, confirms that the raw ingredients contain 3-MCPD at levels below 10ppb (less than 0.01mg/kg). In essence, the level of 3-mcpd in our product is undetectable. Moreover, our supplier will be conducting further independent testing to validate the COAs.

Our supplier and ourselves are aware and fully responsive to the health considerations of 3-MCPD. This undertaking therefore commits us to always taking the necessary precautions to maintain higher standards than those set by the EU."

From S. O. P. Edibles Ltd.
"I would like to inform you that we are working closely with our supplier in China regarding this matter, and they have been given a copy of your methodology to ensure that the tests they carry out are in line with the Food Standards Agency."

From Chadha Oriental Foods Ltd.
"We would like to confirm that since the initial survey was undertaken by MAFF in August 1999, we have reviewed our supplier base and discontinued imports of products where levels of 3-MCPD were in excess of recommendations. Additionally, stock that was held by us was returned to suppliers.

The product that you referred to in your letter 'Golden Mountain' Seasoning sauce is now a discontinued product line. As such, any samples that were found on sale within the trade precedes this review."

From S W Trading Ltd
Pearl River Bridge - Soy Sauces
We became aware of 3-MCPD in September 1999 whilst we were negotiating the 'sole agency' agreement with PRB, which we obtained in October 1999. From that time we have instigated procedures to ensure that our customers receive only genuine PRB products which all contain:

• clearly marked labelling stating: Imported by S. W. Trading, London Best Before end ______, Batch Nos.
• and analyse weekly by Batch Numbers to ensure that 3-MCPD is either not detected or certainly less than 10ug/kg which is now always the case.

This has been achieved by regular contact with MAFF at the outset, the Foods Standard Agency and daily contact with our manufacturers.

However, it has become apparent that random sampling by our Authorities has not produced the same results as our own analysis. There could be various reasons for this problem, but our investigations have uncovered two main reasons.

Firstly is that, unfortunately, there are counterfeit products on the market which are certainly not imported by us as our domestic sauce is 600ml and secondly we observe from the reports that the samples analysed were either not imported by us or imported before the 3-MCPD 1999 food alert was received

Annex 2 - 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."¹ 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
For this survey the measurement uncertainty was calculated from analyses of an in-house reference material containing 0.023 mg/kg and randomly selected survey samples fortified with 0.010 mg/kg 3-MCPD. The measurement uncertainty was calculated using a standard coverage factor of 2, equivalent to a confidence of approximately 95 percent. It was made up of a precision element (standard uncertainty 0.0018 mg/kg) and a bias element (relative standard uncertainty 7.3 percent). The assessment was targeted at samples containing 3-MCPD at levels close to the reporting limit and is therefore less applicable to samples containing 3-MCPD considerably in excess of 0.020 mg/kg.

Results
Measurement uncertainty for 3-MCPD was assessed to be ¿0.004 mg/kg at a level of 0.010 mg/kg 3-MCPD.

References

1. Eurachem/CITAC Guide on Quantifying Uncertainty in Analytical Measurement, second edition, 2000

Annex 3 - ANALYTICAL QUALITY ASSURANCE
3-MCPD
The following quality assurance criteria were set:

• The calibration line is linear and should have a correlation coefficient (r2) of at least 0.99.
• The sample peak area ratios fall within the calibration range.
• The modification for dilution has been calculated correctly.
• The 3-MCPD peak retention times are within ¿ 0.3 minutes of those in the standards.
• A minimum library fit of 800 is achieved for each spectrum.
• Each analytical batch should contain the following:
  1. A reagent blank.
  2. A spike recovery sample or a quality control sample of known 3-MCPD concentration.
• The reagent blank should contain < 0.005 mg/kg 3-MCPD. Spike recoveries should fall within 50 - 130 percent and the quality control sample should be within 2 standard deviations of the true value.
• If sample concentration exceeds the linear range either less sample mass should be taken (e.g. for soy sauce take 1g instead of 8g) or the sample should be diluted and the sample mass remains the same.

Results

• The correlation coefficients of all calibration standard solutions were higher than 0.99.
• Standard solutions run at the end of each batch all had a peak area within ¿15 percent of that standard run at the beginning of the batch.
• No reagent blank samples showed a response equivalent to more than 0.005 mg/kg 3-MCPD.
• All results for the soy sauce reference material all fell within 2 standard deviations of the characterised value.
• The internal standard peak area for all the calibration standards exceeded 50,000 units.
• The peak area ratios of all samples fell within the range of the calibration standards, in some cases after dilution and re-extraction of the sample.
• The mass spectra of the sample 3-MCPD peaks all exceeded a library fit of 800 against an authentic standard.
• The retention times of the sample 3-MCPD peaks were all within ¿0.2 minutes of those of a standard

Table 1 See right hand navigator bar.

Table 2 See right hand navigator bar.

Table 3 Summary of 3-MCPD Levels in the Current Survey

Range of levels of 3-MCPD (mg/kg)	Number of samples
<0.01	69
0.01-0.19	6
0.02-1.00	8
1.00-10.00	6
10.00-50.00	9
>50	2

Table 4 See right hand navigator bar.

Table 5 Comparison of the Results of the 1999 and the Current Survey (2000)

	Percentage of total number of samples in the survey
Range of levels of 3-MCPD (mg/kg)	survey 1999	survey 2000
<0.01	52.5	69
0.01-0.19	12.5	6
0.02-0.05	0	3
0.05-1	12.5	5
1.00-5.00	10	5
5.00-10.00	5	1
10.00-20.00	2.5	6
20-50	5	3
>50	0	2

Figure 1 See right hand navigator bar.
Figure 2 See right hand navigator bar.