NITRATES AND NITRITES IN FOOD

 SURVEY OF NITRATES AND NITRITES IN FOOD AND BEVERAGES IN AUSTRALIA

Summary

Background

Nitrate and nitrite ions are ubiquitous in the environment and occur naturally in plant foods as a part of the nitrogen cycle. Nitrate and nitrite, as the sodium or potassium salts, have also been used as food additives in cured meats for many years primarily to prevent growth and toxin production of Clostridium botulinum.

Human exposure to nitrate and nitrite occurs mainly through the ingestion of fruit and vegetables. The consumption of fruit and vegetables is widely recommended due to the strong evidence of beneficial effects for health. However, dietary nitrate and nitrite have also raised some concerns because of implications for adverse effects including methaemoglobinaemia (which results in reduced oxygen transport in the blood) and possible increased cancer risk.

In order to estimate the Australian dietary exposure to nitrate and nitrite, and to determine whether there are any risks to human health at current dietary exposure levels, FSANZ has funded and coordinated surveys for both nitrate and nitrite in Australian foods and beverages. Food regulatory agencies in State and Territory governments collected the food samples in their region.

Key findings

  • The major sources of estimated nitrate dietary exposures across different population groups were vegetables (42-78%) and fruits (including juices) (11-30%). Highest concentrations of nitrate were generally found in leafy green vegetables, such as spinach, consistent with other international findings.
  • Vegetables (44-57%) and fruits (including juices) (20-38%) were also the major contributors to estimated dietary nitrite exposure across the population groups. Nitrite exposure from processed meats accounts for only a relatively small amount of total dietary nitrite exposure (5-7%).
  • Estimated Australian dietary nitrate and nitrite exposures are not considered to represent an appreciable health and safety risk.
  • However, the health benefits of fruit and vegetables are widely accepted, including strong evidence of a protective effect of certain vegetables, legumes and fruit against the development of a number of non-communicable chronic diseases, among them cancer and cardiovascular disease.

Abbreviations

ADI Acceptable Daily Intake
ATDS Australian Total Diet Study
bw Body weight
DIAMOND Dietary Modelling of Nutritional Data – FSANZ’s Dietary Modelling computer program
FAO Food and Agriculture Organization
FSANZ Food Standards Australia New Zealand
JECFA Joint FAO/WHO Expert Committee on Food Additives
KEKP Kids Eat Kids Play (2007 Australian Children’s Nutrition and Physical Activity Survey)
kg Kilograms
LOR Limit of Reporting
mg Milligram (one thousandth of a gram)
NMI The National Measurement Institute (NMI) (formerly the Australian Government Analytical Laboratory)
NNS National Nutrition Survey
NOAEL No observed adverse effect level
QHSS Queensland Health Clinical and Statewide Services Division
the Code The Australia New Zealand Food Standards Code
WHO World Health Organization

 

Note: A glossary of terms can be found in Appendix 1

Introduction

Nitrate and nitrite ions are ubiquitous in the environment and occur naturally in plant foods as a part of the nitrogen cycle. Nitrate levels may vary significantly in fruit and vegetables dependent on a number of biotic and abiotic factors. Conversely, nitrite levels are generally relatively low in fresh undamaged vegetables but may increase in some nitrate rich vegetables after harvesting, particularly if stored at room temperature (reviewed in Maynard et al, 1976).

Nitrate and nitrite, as the sodium or potassium salts, have also been used as food additives in cured meats for many years primarily to prevent growth and toxin production of Clostridium botulinum which causes the illness botulism (Davidson et al., 2002, Sofos and Raharjo, 1995). The addition of nitrite or nitrate improves the microbiological safety of these foods and extends their safe shelf life. This offers significant benefits to consumers in terms of the availability of a variety of different foods that are safe, convenient and cost effective. An alternative to sodium nitrite for production of cured meats has not been identified despite significant research effort (EFSA, 2003).

The safety of nitrate and nitrite has been comprehensively reviewed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Historically, there have been two main safety concerns around the presence of nitrate and nitrite in the diet. Those relate to the reaction of nitrite with haemoglobin to form methaemoglobin which can reduce oxygen transport in the blood, and a theoretical possibility of the potential for carcinogenicity through the formation of N-nitroso compounds in foods or in humans in vivo.

In order to estimate the Australian dietary exposure to nitrate and nitrite, FSANZ has funded and coordinated analytical surveys for both nitrate and nitrite. Food regulatory agencies in State and Territory governments collected the food samples in their region and provided these for analysis. These surveys have included:

  • An assessment of nitrate and nitrite concentrations in processed foods conducted as part of the 21st Australian Total Diet Study (ATDS) which also examined sulphites, benzoates and sorbates.
  • An assessment of nitrate and nitrite concentrations in fruit, vegetables and water as part of the 22nd ATDS which also estimated the dietary intake of the Australian population of the trace elements iodine, selenium, chromium, molybdenum and nickel.
  • A supplementary survey of selected fruit and vegetables conducted in April 2010.

Background

The diet constitutes an important source of human exposure to nitrate and nitrite either as natural constituents of plant foods or as intentional additives. Drinking water can also be an important potential source of nitrate (Gangolli et al, 1994).

Nitrate and nitrite levels in fruit and vegetables

Significant concentrations of nitrate are found naturally in various fruits and vegetables. It has long been established that these levels are dependent upon a number of factors including; the use of fertilisers, location and soil type, carbon dioxide concentrations (in greenhouse vegetables), seasonal light intensity and duration of light exposure and water availability (reviewed in Maynard et al, 1976).

Nitrate concentrations in vegetables may also vary up to orders of magnitude dependent on the vegetable species and the part of the plant sampled. High concentrations of nitrate tend to accumulate in the leaves, roots, petioles or stems of certain plants meaning that leafy vegetables including lettuce or spinach, and root crops such as beetroot, may accumulate high concentrations of nitrate. In contrast, levels of nitrate in vegetables such as carrots or onions are likely to be lower (EFSA 2008; Maynard et al, 1976).

Nitrite concentrations generally tend to be low in fresh undamaged vegetables, however levels can increase rapidly in certain nitrate rich vegetables, particularly if pureed and stored at room temperature. In addition to temperature, this increase is dependent upon nitrate reductase activity in the plant and the level of bacterial contamination (Chung et al, 2004; Ezeagu, 1996; Lin and Yen, 1980; Phillips et al, 1968).

Nitrate and nitrite in drinking water

The World Health Organisation (WHO) and Australian drinking water guideline levels are 50 mg/L for nitrate (as NO3) and 3 mg/L for nitrite (as NO2). The guideline values are established to protect young infants from methaemoglobin formation, however the guideline advises that water with a nitrate concentration of up to 100 mg-nitrate/L can be used by adults and children over 3 months of age without risk of significant health effects (NHMRC, 2004).

The WHO has also set a provisional guideline level for nitrite in drinking water of 0.2 mg/L for long term exposure (WHO, 2008).

In Australia, nitrate concentrations in major public supplies of drinking water are typically below 0.15 mg/L, however elevated nitrate concentrations (200-300 mg-nitrate/L) have been recorded in groundwater sourced for drinking in some rural areas. Nitrite is rapidly oxidised to nitrate in water and is rarely detected in well-oxygenated or chlorinated water (NHMRC, 2004).

 

Food additive permissions in Australia and New Zealand

Standard 1.3.1 of the Australia New Zealand Food Standards Code (the Code) permits the addition of nitrite and nitrate, in the form of sodium or potassium nitrite and nitrate, to a range of food products.

Nitrate is permitted to be added to slow dried cured meats and fermented uncooked processed comminuted meat products to a maximum level of 500 mg/kg and to cheese and cheese products at a maximum level of 50 mg/kg.

Nitrite is permitted to be added to commercially sterile canned cured meats to a maximum level of 50 mg/kg and to cured meats, dried meats, slow dried cured meats and processed comminuted meat poultry and game products to a maximum level of 125 mg/kg. Permissions for nitrate and nitrite in the Code are shown in Table 1.

Table 1. Food additive permissions for nitrate and nitrite in Australia and New Zealand

Product Additive Permitted level
Cheese and cheese products Nitrates (potassium and sodiumsalts) 50 mg/kg calculated as nitrate ion
Commercially sterile canned cured meat Nitrites (potassium and sodiumsalts) 50 mg/kg total of nitrates and nitrites, calculated as sodium nitrite
Dried meat Nitrites (potassium and sodiumsalts) 125 mg/kg total of nitrates and nitrites, calculated as sodium nitrite
Product Additive Permitted level
Additive Additive Permitted level
Permitted level Additive Permitted level
Slow dried cured meat Nitrites (potassium and sodiumsalts)Nitrates (potassium and sodiumsalts) 125 mg/kg total of nitrates and nitrites, calculated as sodium nitrite500 mg/kg total of nitrates and nitrites, calculated as sodium nitrite
Processed comminuted meat, poultry and game products Nitrites (potassium and sodiumsalts) 125 mg/kg total of nitrates and nitrites, calculated as sodium nitrite
Fermented, uncooked processed comminuted meat products Nitrates (potassium and sodiumsalts) 500 mg/kg total of nitrates andnitrites, calculated as sodium nitrite

 

Objectives of the survey

The objectives of this survey were to analyse levels of nitrate and nitrite in Australian food and beverages, and to determine whether estimated dietary exposure to nitrate and nitrite poses a risk to human health and safety.

Survey design and analytical method

Sample selection

21st ATDS

Foods sampled as part of the 21st ATDS represented mainly processed foods for which there are permissions to contain preservatives in the Code. Foods sampled included those that may be expected to show regional variation (regional foods) and those available nationwide and not expected to show regional variation (national foods). For each food, three samples were combined to give a composite sample that was analysed to measure the levels of nitrate and nitrite. A detailed description of food sampling conducted as part of the 21st ATDS can be found at http://www.foodstandards.gov.au/_srcfiles/21st%20ATD%20Study%20report-Aug051.pdf

22nd ATDS

The 22nd ATDS analysed nitrate and nitrite concentrations in a selection of fresh produce including fruit, vegetables and other food products such as beverages and some snack foods. Two composite samples, of three purchases each, were collected in three capital cities, making six composite samples for each national food. For regional foods two composite samples, consisting of three purchases each, were collected in five capital cities, making ten composite samples for each regional food. The collection period varied slightly for each State or Territory in order to stagger the arrival of samples at the analytical laboratory, as soon as practicable after purchase. All perishable samples were frozen prior to forwarding to the laboratory. The analytical laboratory prepared foods in accordance with detailed instructions. Perishable foods were prepared within 48 hours of arrival at the laboratory. Full details of sample selection as part of the 22nd ATDS can be found at http://www.foodstandards.gov.au/_srcfiles/ATDS.pdf.

2010 Survey of selected fruit and vegetables

Food items were collated into a sampling plan which included food preparation techniques consistent with how the food was prepared for analysis for the 21st and 22nd ATDS. Samples for testing were collected by food regulatory agencies in the Australian Capital Territory, Western Australia and Queensland from a variety of retailers during May 2010. Jurisdictions sampled three purchases of each food type. For each food type, the products available on retail shelves were reviewed and purchased. Where possible, two samples of the same product, each with different batch numbers/date markings were purchased to account for variation between batches. In this instance, the same products with different batch dates were composited together for analysis.

Sample preparation

The 52 foods selected according to the above sampling plans that were analysed for nitrate and nitrite are set out in Appendix 2, Table A1. Foods were collected and forwarded to the analytical laboratory as soon as practicable. All perishable samples were refrigerated or frozen prior to forwarding to the laboratory. All the foods examined in the study were prepared to a ‘table ready’ state before analysis (refer to Appendix 2, Table A2 for details on food preparation instructions). For example, potatoes were boiled and bacon was dry fried until cooked through.  A number of the foods surveyed in this study, such as ham and cheese, were available in a table ready form and required no further preparation.

Sample analysis

Analysis was conducted by Queensland Health and Scientific Services (QHSS) for food samples as part of the 21st ATDS, and by the National Measurement Institute (NMI) for foods sampled during the 22nd ATDS. Inter-laboratory checks were also conducted for certain fruit and vegetables as part of the 22nd ATDS.  Some inconsistencies were identified between laboratories. Therefore, some fruit and vegetables were resampled in 2010. Symbio Alliance conducted the nitrate and nitrite measurements in additional samples. All analyses were carried out in the food samples in accordance with accredited quality assurance procedures and the results were provided to FSANZ. The Limit of Reporting (LOR), which is the lowest concentration level at which the laboratory is confident in the quantitative results reported, ranged from 0.6 (liquid matrix) to 10 mg/kg for sodium nitrate (solid matrix) and 0.6 (liquid matrix) to 7.5 mg/kg (solid matrix) for sodium nitrite dependent upon laboratory method. Analytical methods are summarised in Table 2.

 

Table 2: Methods of Analysis for nitrate and nitrite

Laboratory Method Reference
 
QHSS FIA/Spectrophotometry QIS 12641 based on the method of Kirk and Sawyer in Pearson’s Composition and Analysis of foods
NMI Ion chromatography Based on method 4110B from APHA Standard method for the examination of waters. 20th Edition
SymBio Alliance Spectrophotometry NATA accredited method based on AOAC 973.31

The concentration of nitrate and nitrite can be expressed as a number of different units including mg/L, mg/kg, mg/L nitrate-nitrogen, mg/L nitrite-nitrogen, or also in terms of number of moles, and as the sodium salt. In this report, units are reported as the sodium salt (mg/kg) unless otherwise specified. Conversion factors between nitrate and nitrite and the sodium salts of nitrate and nitrite were based on the figures shown in Table 3. To convert NO3 to NaNO3 data were divided by 0.73 and to convert NO2 to NaNO2 data were divided by 0.67.

Table 3: Conversion of nitrate and nitrite ions to the sodium salt.

mM mg/L NO3 mg/L NO2 mg/L NaNO3 mg/L NaNO2
1 62 46 85 69

 

Estimating dietary exposures to sodium nitrate

A dietary exposure assessment (dietary modelling) is a tool used to estimate the exposure to (or intake of) agricultural and veterinary residues, contaminants, nutrients, food additives and other substances from the diet. To estimate dietary exposure to food chemicals, food consumption data is combined with food chemical concentration data. Food regulators have used dietary modelling techniques internationally for many years to determine if dietary exposures to specific food chemicals present an unacceptable risk to public health and safety.

To estimate the dietary exposures to sodium nitrite and sodium nitrate for each individual, the concentration of these chemicals in each analysed food was multiplied by the amount of food consumed and summed over all foods to determine the exposure to sodium nitrate and sodium nitrite from the whole diet (Equation 1).

Dietary Exposure = sodium nitrite or sodium nitrate concentration x food consumption amount

Equation 1: Dietary exposure calculation

In addition, approximately 5% of ingested nitrate is converted into nitrite in the saliva of humans (Appendix 6.2). This additional (endogenous) nitrite exposure also needs to be taken into account in the total dietary exposure assessment of nitrite. Therefore, 5% of the sodium nitrate concentration was added to the concentration of sodium nitrite for each food (Equation 2) before applying (Equation 1). This accounts for total nitrite exposure obtained through endogenous conversion of nitrate in the saliva and exogenous nitrite exposure in the diet to be calculated.

Total Sodium Nitrite Concentration (including endogenous formation)= sodium nitrite concentration in the food + (5% of sodium nitrate concentration in the food)

Equation 2: Total sodium nitrite concentration calculation

The dietary exposure assessments for all food chemicals (sodium nitrite, total sodium nitrite

The dietary exposure assessments for all food chemicals [sodium nitrate, sodium nitrite and total sodium nitrite (endogenous and exogenous sodium nitrite)], were conducted using a computer program known as DIAMOND (Dietary Modelling of Nutritional Data), which was designed to automate dietary exposure calculations. DIAMOND multiplied the allocated sodium nitrate, sodium nitrite and total sodium nitrite concentrations for each food consumed in the national nutrition surveys (NNS) with the amount of that food that each survey respondent consumed. This gave an estimation of each individual’s exposure to sodium nitrate, sodium nitrite and total sodium nitrite from each food. Once this had been completed for all of the foods, the total amount of sodium nitrate, sodium nitrite and total sodium nitrite consumed from all foods was summed for each individual. Population statistics (e.g. mean and 90th percentile exposures) for each age group were then derived from the individuals’ ranked intakes.

DIAMOND enables the dietary exposure assessments to be conducted using actual diets for males and females aged 2 years and above, as derived from national nutrition surveys. The dietary exposures to each chemical were calculated for each individual in the survey before mean dietary exposure results were derived for the specified age categories. Use of specific food consumption data greatly improves the reliability and accuracy of the dietary exposure estimates, and takes into account the different eating patterns of consumers.

Population groups assessed

Dietary exposures were estimated for:

  • infants aged 9 months
  • children aged 2-5 years
  • children aged 6-12 years
  • children aged 13-16 years
  • 17 years and above
  • females aged 16-44 years

Dietary exposure assessments were conducted for children because children generally have higher exposures due to their smaller body weight and they consume more food per kilogram of body weight compared to adults. A detailed description of food consumption data, model diet construction, food mapping and assumptions and limitations in dietary exposure assessment are at Appendices 4 to 4B.

Treatment of analytical values below the LOR

Nitrate and nitrite can be distributed in foods at very low concentrations and occur naturally in the environment. These amounts may make a substantial contribution to dietary exposures and should be accounted for. Therefore, it was not reasonable to assume they were not present in the food when the analytical results were less than the LOR. To allow for this uncertainty, the results for dietary exposures to sodium nitrate, sodium nitrite and total sodium nitrite were presented as a range, using the mean analytical concentration. The lower end of the range was calculated based on the assumption that results below the LOR were equal to zero (lower bound mean). A more conservative approach assumes the concentrations were present at half of the LOR and is indicated by an inner point in the range (middle bound mean). The upper end of the range, representing a very conservative ‘worst-case’ estimate, was calculated on the assumption that results below the LOR were equal to the LOR (upper bound mean). Where sodium nitrite or sodium nitrate concentrations were expected to be from intentionally added sources only, analytical results that were less than the LOR were assumed to be zero in all cases (e.g. sodium nitrite in cottage cheese).

Analytical results

Nitrate

The mean analytical concentrations for lower and upper bound concentrations for nitrate are shown in Appendix 3, Table A3. Upper bound mean nitrate concentrations (expressed as sodium nitrate) were highest in raw and fresh cooked spinach (2741-2963 mg/kg), canned beetroot (2009 mg/kg), fresh parsley (1957 mg/kg), raw celery (1527 mg/kg) and raw lettuce (1144 mg/kg). For banana, broccoli, cabbage, cucumber, potato crisps, pumpkin, salami, and strawberries concentrations were between 100 and 450 mg/kg. All other surveyed foods had nitrate concentrations of less than 100 mg/kg.

These results are relatively consistent with a comprehensive survey of nitrate concentrations in vegetables in Europe which examined 41,969 analytical results from 20 member states and Norway (EFSA, 2008). A large variation in median nitrate concentrations was observed ranging from around 1 mg/kg in peas to a high of 4,800 mg/kg for rucola (expressed as the nitrate ion). Median concentrations for cabbage, cauliflower and onions were 223 mg/kg, 122 mg/kg and 60 mg/kg, respectively. For cucumber and tomatoes, median reported concentrations were 156 and 26 mg/kg, respectively. High concentrations were typically reported in leafy vegetables including spinach (785 mg/kg), silverbeet (1,510 mg/kg) and mixed lettuce (1,878 mg/kg).

The results for nitrate are also consistent with those observed in a 2007 New Zealand survey. Mean concentrations of nitrate (expressed as sodium nitrate) were: cabbage (331 mg/kg), lettuce (1590 mg/kg), celery (1610 mg/kg), broccoli (133 mg/kg), spinach (990 mg/kg), beetroot, canned (763 mg/kg), potato (129 mg/kg), carrot (58 mg/kg) and pumpkin (67 mg/kg). Mean nitrate levels in bacon (36.5 mg/kg), ham (16.6 mg/kg) and luncheon sausage (30.9 mg/kg) were typically lower (Thomson et al, 2007). Some variation in results between surveys is expected because it is known that nitrate concentrations are influenced in particular by the season, methods of production and sunlight available. In addition, differences in survey methodology and reporting are likely to contribute to variations in reported nitrate concentrations for the same commodity between surveys and countries.

Nitrite

Upper bound mean nitrite concentrations (expressed as sodium nitrite) were generally highest in processed meats including bacon (27 mg/kg), frankfurts (30 mg/kg), ham (28 mg/kg), luncheon sausage (35 mg/kg), and strassbourg (35 mg/kg). The upper bound mean concentration of sodium nitrite was 38 mg/kg in spinach and 29 mg/kg in pumpkin. Other foods or beverages that reported upper bound mean concentrations of more than 10 mg/kg included beans, broccoli, cabbage, cucumber, grapes, parsley, peaches, peaches, pineapple and strawberry. White wine also contained nitrite at a concentration of above 10 mg/kg. All other foods and beverages contained nitrite at concentrations close to, or below the LOR. Individual results are shown in Appendix 3, Table A4.

Comparatively fewer data are available for nitrite concentration in surveys of foods and beverages internationally, however nitrite concentrations were generally comparable with concentrations in processed meats in surveys conducted in New Zealand and France (Thomson et al, 2007; Menard et al, 2008). Concentrations of nitrite were generally higher in fruit and vegetables in Australia than in the New Zealand survey (Thomson et al, 2007). Upper bound concentrations were more comparable with those reported in France (Menard et al, 2008). The storage temperature and length of storage of samples of some fruit and vegetables in the current survey prior to purchase was not known. Unfavourable conditions such as high storage temperature and long storage periods have previously been shown to increase nitrite levels in vegetables (Aworh et al. 1978, 1980 and Chung et al. 2004) and may have contributed to results in this study.

A 1996-1997 survey commissioned by Queensland Health found that of the 107 vegetable samples analysed, 18 samples had nitrite (expressed as sodium nitrite) levels of greater than 5 mg/kg. Nitrite concentrations of up to 10 mg/kg were seen in cabbage and celery; concentrations in lettuce ranged up 20 mg/kg and levels in silverbeet were up to 50 mg/kg (unpublished data). In a separate survey of nitrate and nitrite in Australian leafy vegetables, Parks et al., (2008) reported that fresh leafy vegetables available during a 6-month period on the Australian market can range in nitrate-N from 12 to 1400 mg/kg fresh weight and nitrite-nitrogen from 0 to 37.5 mg/kg. Meah et al., (1994) also reported a wide variation in nitrite (expressed as the ion) levels for lettuce (not detected (nd)-15 mg/kg), celery (nd-19 mg/kg), potatoes (nd-60 mg/kg) and beetroot (nd-71 mg/kg) in the United Kingdom. Nitrite concentrations in spinach, cabbages and tomatoes were below the limit of detection.  A recent survey of nitrite in vegetables available in Hong Kong found that nitrite concentrations were generally low (around 1 mg/kg) with higher levels reported in some cabbage (ca 3 mg/kg) and beetroot (ca 8 mg/kg).

 

Estimated dietary exposure to sodium nitrate and sodium nitrate

 

Sodium nitrate

The estimated mean and 90th percentile dietary exposures to sodium nitrate, in milligrams per day, for each age category are given in Table A9 in Appendix 5 and in Figure 1 below. For dietary exposures expressed in mg/kg bw/day, see Table A10 in Appendix 5. Across the three scenarios there was a minimal to no estimated range of mean and 90th percentile dietary sodium nitrate exposures, reflecting the low number of analytical samples at below the LOR (Figure 1). The estimated dietary exposure to sodium nitrate for the mean and 90th percentile increased with increasing age. Infants aged 9 months had the lowest estimated dietary exposure and people aged 17 years and above had the highest.

Figure 1: Range of mean and 90th percentile estimated dietary exposure to sodium nitrate, in milligrams per day_

_ lower end of the range represents where all <LOR analytical results have a concentration of zero; the upper end of the range represents where all <LOR analytical results have a concentration equal to the LOR.

 

Major contributing foods

As shown in Figure 2, the major sources of sodium nitrate dietary exposures across the different population groups were vegetables (42-78%) and fruits (including juices) (11-30%). Non-alcoholic beverages (excluding juices) were also a major contributing food group for infants aged 9 months.

More specific details regarding the major food group contributors to sodium nitrate are presented in Table A11 and A12 of Appendix 5. Lettuce (5-18%), stalk and stem vegetables (7-13%), starchy root vegetables (7-12%) and green leafy vegetables (cooked) (7-10%) were major contributors to dietary exposure to sodium nitrate for all age categories 2 years and above. Leafy vegetables and herbs (14%) and starchy root vegetables (8%) were the major contributing vegetables to sodium nitrate dietary exposures for infants aged 9 months.

For children aged 2-12 years, bananas, tropical fruits and figs (7-13%) were the major contributing foods to sodium nitrate exposure, followed by root vegetables (starchy) (11-12%). Bananas and plantains were the major contributing fruit to estimated dietary exposures for infants.

Based on the theoretical infant diet, bottled water (including plain mineral water and soda water) (17%) was the major contributor to sodium nitrate exposure for infants aged 9 months. Other non-alcoholic beverage food groups that were major contributors to sodium nitrate dietary exposures were infant formula (8%) and non-bottled water (7%).

 

Figure 2: Contributors to dietary exposure to sodium nitrate

Sodium Nitrite

 

The estimated mean and 90th percentile sodium nitrite dietary exposures for all age categories included in this survey , are presented in Table A13 (in milligrams per day (mg/day) and in Table A14 (in milligrams per kilogram body weight per day (mg/kg bw/day) of Appendix 5A. Generally, as age increased, the estimated mean and 90th percentile dietary exposures (in mg/day) increased. Infants aged 9 months had the lowest estimated dietary exposures and people aged 17 years and above had the highest (Figure 3).

 

Figure 3: Range of mean and 90th percentile estimated dietary exposure to sodium nitrite, in milligrams per day_

_ lower end of the range represents where all < LOR analytical results have a concentration of zero; the upper end of the range represents where all < LOR analytical results have a concentration equal to the LOR. The upper end of the mean range and the lower end of the 90th percentile range for some population groups overlap. This is represented by the green on the figure.

Note: the black line on each mean range and 90th percentile range represents where all < LOR analytical results have a concentration equal to half the LOR.

 

Major contributing foods

Fruits (including processed and juices), vegetables (including cooked) and processed meats and sausages were the major dietary sources of sodium nitrite exposure across all age categories examined (Figure 4), contributing 31-47%, 29-35%, 13-31% respectively.  Alcoholic beverages were also a major source of sodium nitrite intake for the population group aged 17 years and above (20%) and females aged 16-44 years (22%). More specific details regarding the major food group contributors to sodium nitrite intake are presented in Table A15 and A16 of Appendix 5A.

The fruits that had the highest contributions to estimated dietary sodium nitrite exposures for children aged 2-16 years were pineapple (9-11%), berries (6-12%) and bananas, tropical fruits and figs (<5-9%). For infants aged 9 months, the fruits that had the highest contribution were bananas and plantains (8%), berries and jams (8%), grapes (6%) and dried apricots, peel, cherries, ginger and fruit leathers (6%). For population groups aged 17 years and above, the major contributing fruits were fresh stone fruits and persimmon (10%) and pineapple (6-8%).

‘Pumpkin, squash and zucchini’ (12-13%) and ‘cucumbers, capsicums, chokos and chillies’ (6-8%) were the major contributing vegetables to sodium nitrite dietary exposures for children aged 2-16 years, the population aged 17 years and above and for females aged 16-44 years. Similarly, ‘pumpkin, squash, marrows and zucchini’ was the major vegetable contributor for infants aged 9 months (12% of estimated exposure).

Deli meats in whole pieces or cuts (except bacon) were a major source of sodium nitrite exposure for all age groups (8-15%). These results may be explained by the use of sodium nitrite as a preservative in cured meats. Bacon and pancetta was a major contributing food group for children aged 13-16 years only. No other deli meats (e.g. salami, frankfurts, strassburg, luncheon) were major contributing foods to estimated sodium nitrite dietary exposures for population groups aged 2 years and above. ‘Pork (except bacon) and deli meats (except frankfurts and poultry-based)’ (18%) and sausages and frankfurts (11%) were the major contributing processed meat foods for infants aged 9 months.

 

Figure 4: Contributors to dietary exposure to sodium nitrite

Total Sodium Nitrite

A proportion (approximately 5%) of ingested nitrate is converted into nitrite in the saliva of human beings. Therefore it is necessary to consider this endogenous contribution to total nitrite exposure in addition to nitrite exposure in the diet. The estimated mean and 90th percentile dietary exposures to total sodium nitrite, in milligrams per day, for each age category are given in Table A17 in Appendix 5B and in Figure 5 below. For dietary exposures expressed in mg/kg bw/day, see Table A18 in Appendix 5B. The estimated mean and 90th percentile for total sodium nitrite increased with age across the three scenarios.

Figure 5: Range of mean and 90th percentile estimated dietary exposure to total sodium nitrite, in milligrams per day_

_ lower end of the range represents where all < LOR analytical results have a concentration of zero; the upper end of the range represents where all < LOR analytical results have a concentration equal to the LOR. The upper end of the mean range and the lower end of the 90th percentile range for some population groups overlap. This is represented by the green on the figure.

Note: the black line on each mean range and 90th percentile range represents where all < LOR analytical results have a concentration equal to half the LOR.

 

Major contributing foods

As shown in Figure 6, the major contributors to total sodium nitrite dietary exposures across the different population groups were vegetables (44-57%) and fruits (including juices) (20-38%). Alcoholic beverages were also a major contributing food group for people aged 17 years and above and females aged between 16 and 44 years.

More specific details regarding the major food group contributors to total sodium nitrite are presented in Table A19 and A20 of Appendix 5B. Deli meats in whole pieces or cuts (except bacon) (5-7%), pumpkin, squash and zucchini (6-8%), starchy root vegetables (5-7%) and cucumbers, capsicums, chokos and chillies (5-6%) were major contributors to dietary exposure to total sodium nitrite for all age categories 2 years and above. In addition, bananas, tropical fruits and figs (6-11%) and berries (5-8%) were also major contributors for children aged 2-12 years.

Figure 6: Contributors to dietary exposure to total sodium nitrite

 

 

Health significance of survey results

Background

There is an extensive toxicological database for nitrate and nitrite. These studies were last reviewed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2002. JECFA established an Acceptable Daily Intake for nitrate and nitrite on the basis of studies in rats (Appendix 6.10). EFSA and the International Agency for Research on Cancer (IARC) have also evaluated potential health effects associated with nitrate and nitrite ingestion in food (EFSA 2008; 2010; Grosse, 2006).  These evaluations include an analysis of toxicological and epidemiological studies for nitrate and nitrite that have been published since the JECFA evaluation.  In this report, the pharmacokinetics and toxicology of nitrate and nitrite have been summarised to provide context to the consideration of possible health risks that may be associated with current estimated dietary exposure levels in the Australian population (Appendix 6). The assessment has considered the JECFA, IARC and EFSA reviews, together with other relevant data in humans.

Summary of nitrate and nitrite toxicity

Nitrate is generally regarded to be of relatively low toxicity. The toxicological sequelae of nitrate exposure are considered to be virtually entirely attributable to its conversion to nitrite. Nitrite reacts with haemoglobin (Hb) to form methaemoglobin (MetHb) in the blood which is the critical toxicological endpoint following nitrate and nitrite exposure. MetHb levels of up to 10% are typically not associated with clinical signs in humans. At higher levels, MetHb is associated with clinical signs including cyanosis, impaired aerobic respiration, metabolic acidosis, and in severe cases, death (Table 4). Levels of up to around 5% MetHb were not considered adverse by JECFA, or in a review of MetHb following acute exposure to MetHb inducing chemicals (Solecki, 2005; Speijers and Brandt, 2002).

 

 

Table 4: Clinical signs associated with elevated MetHb levels in blood (Wright et al, (1999)). 

% MetHb Clinical signs
<10 None
10-20 Cyanotic skin discolouration
20-30 Anxiety, headache, tachycardia
30-50 Fatigue, confusion, dizziness, tachypnea, increased tachycardia
50-70 Coma, seizures, arrhythmias, acidosis

 

Other toxicological endpoints are summarised in detail in Appendix 6. In addition to methaemoglobinaemia, the other main concern related to nitrate and nitrite exposure has been the potential for increased cancer risk. The available evidence does not support a conclusion that nitrate intake from the diet is associated with increased cancer risk. There was equivocal evidence of an increased incidence of squamous cell papilloma and carcinoma in the forestomach in female mice given sodium nitrite at 165 mg/kg bw/day. Nitrite was not carcinogenic in male mice or male and female rats. Epidemiological evidence that nitrite in the diet is associated with increased cancer risk in human beings is equivocal. It is known that nitrite and dietary amines can react to form N-nitroso compounds, but whether endogenous nitrosation takes place under actual food intake conditions in sufficient amounts to pose a risk to human health is uncertain.

 

Nitrate exposures associated with elevated blood MetHb levels in infants, children and adults

 

MetHb levels in blood of human adults were not increased at exposures of up to 15 mg/kg bw/day for 28 days (Table 5). In infants, aged 3-8 months, 16.5 – 21 mg/kg bw/day nitrate (ions) administered as nitrate-rich vegetables, did not induce elevated MetHb blood levels. Oral administration of bolus doses of 50-100 mg/kg bw/day nitrate were associated with MetHb levels of around 5-8% in the blood of infants. Detailed summaries of these studies are at Appendix 6.4.1.

 

Table 5: MetHb levels in adults and infants exposed to sodium nitrate or nitrate in food   

Nitrate exposure Dose(mg/kg bw) Study duration Age Number of subjects Percentage MetHb Reference
Sodium nitrate 10 Single dose Adult 8 No change Colbers et al, (1996)
Sodium nitrate 15 28 d Adult 10 0.1-0.6% Lambers et al, (2000)
Nitrate (ions) 50 2-18 d 11 d to 11 m 4 Maximum of 5.3% Cornblath and Hartmann, (1948)
Nitrate (ions) 100 6-9 d 2 d to 6 m 4 Maximum of 7.5% Cornblath and Hartmann, (1948)
Nitrate (ions) 16.5 -21 7 d 3.5 – 8 m 7 0.8 (0.2-3.4) Kubler, (1958)

 

Blood MetHb levels following exposure to nitrate in drinking water have been investigated in infants and children (Table 6 and Appendix 6.4.2). The weight of evidence supports that exposure of infants and children to drinking water containing concentrations of up to 100 mg/L nitrate is not associated with increased MetHb levels. Assuming a high water intake of 150 mL/kg bw/day, this is equivalent to about 15 mg/kg bw/day nitrate, consistent with observations from controlled exposure studies.

 

Table 6: MetHb levels in infants and children exposed to nitrate in drinking water

Nitrate (ion)Concentration(mg/L)  Dose*(mg/kg bw/day) Age Number of subjects Percentage MetHb Reference
0 0 90-180 d 89 0.8 Simon et al., (1964)
50-100 11 90-180 d 38 0.8
>100 15 90-180 d 25 0.7
5  <1 >91 d 556 0.97 Shuval et al., (1972)
50-90 10 >91 d 1426 0.99
<43 3 1-8 37 0.98 Craun et al., (1981)
95-482 18 1-8 62 1.13
<44 7 ≤ 9 y 234 1.4-1.8 Diskalenko et al., (1968)
792 119 ≤ 9 y 126 2.1-3.3
898 135 ≤ 9 y 208 3.1-7.1
8.8 1 12-14 y 10 0.8 Subbotin et al., (1961)
101 15 12-14 y 11 5.3
  • Cited in Craun et al., (1981) * Unless water intake was detailed in the study, an intake of 150 mL/kg bw/day was assumed

 

Nitrite exposure associated with elevated blood MetHb levels in infants and adults

 

Approximately 6.3% of ingested nitrate on a molar basis, or 5% by mass, of ingested nitrate is converted to nitrite in the saliva of humans (reviewed in Appendix 6.2). In many cases, endogenous nitrate reduction by saliva is the primary source of dietary nitrite exposure in humans, because dietary nitrate exposure in the diet far exceeds that of nitrite. Therefore, nitrate exposure studies can be used to support an estimated nitrite exposure that is not associated with elevated MetHb levels in blood. The equivalent sodium nitrite exposure at 15 mg/kg bw/day sodium nitrate is approximately 0.75 mg/kg bw/day sodium nitrite. This exposure to nitrite did not cause an increase in MetHb levels in infants, children or adults following administration of nitrate in controlled experimental studies, or in drinking water. Studies in infants support that MetHb levels are not elevated below exposures of up to 0.75 mg/kg sodium nitrite. Oral doses of 0.3 and 1.2 mg/kg bw/day nitrite administered to healthy infants aged 1-3 months for 10 days did not increase MetHb levels. At higher doses of 3.7 and 5.2 mg/kg bw/day MetHb concentrations increased to about 3-4%, and were maintained at that level for the 10 day experimental period (Toussaint and Selenka, 1970). In adult volunteers, bolus oral doses of sodium nitrite of approximately 2.4 mg/kg bw caused maximum MetHb levels of around 3-4% (Hunault et al, 2009). However, MetHb levels following exposure in the diet over the course of the day would be expected to be lower due to the short blood half-life of nitrite and MetHb. Overall, the available data support that exposure of up to 0.75 mg/kg bw/day sodium nitrite is not associated with elevated MetHb levels in humans.

Options for establishing an Acceptable Daily Intake or Acute Reference Dose

 

Available human data were not sufficient to support the establishment of an Acceptable Daily Intake (ADI), or an Acute Reference Dose due to confounding factors in nitrate and nitrite exposure studies. These included bacterial contamination, the concurrent presence of nitrate and nitrite in water, and limited exposure data in drinking water studies. Laboratory animal studies were not considered entirely suitable for establishment of a Reference Health Standard because quantitative differences exist in the conversion of nitrate to nitrite in the oral cavity (Speijers and van den Brandt, 2002; Walker, 1990; 1996) and species differences are also evident in MetHb formation and reduction rates in blood (See Appendix 6.3).

 

Risk Characterisation

 

Estimated mean dietary exposure to sodium nitrate ranged from 1.0 to 2.1 mg/kg bw/day for all population groups. At the 90th percentile, estimated dietary exposure ranged from 2.1 to 3.5 mg/kg bw/day. For nitrite, estimated mean dietary exposure ranged from 0.15 to 0.36 mg/kg bw/day, and exposure at the 90th percentile ranged from 0.3 to 0.6 mg/kg bw/day for all population groups. Exposures for nitrate and nitrite at the 90th percentile are below levels that were not associated with elevated MetHb in blood of adults, children or infants in experimental and drinking water studies. Therefore, estimated dietary exposure to sodium nitrate and sodium nitrite in Australian food is not considered to represent an appreciable human health and safety risk.

 

Vegetables (42-78%) and fruits (including juices) (11-30%) were the major sources of sodium nitrate dietary exposures across the different population groups. For sodium nitrite, the major contributors to total dietary exposures across the different population groups were also vegetables (44-57%) and fruits (including juices) (20-38%). Deli meats (except bacon) represented only around 5-7% of total dietary exposure to sodium nitrite. As such, the risks associated with nitrate and nitrite exposure also need to be considered in terms of the benefits of consumption of fruit and vegetables (See below). Overall, it is considered that because the estimated exposures to nitrate and nitrite are unlikely to result in any appreciable health risks, the strong evidence of health benefits from fruit and vegetable consumption outweigh the risks associated with nitrate and nitrite exposure.

 

Beneficial Effects of Fruit and Vegetables.

 

The Dietary Guidelines for Australian Adults[1] recommend eating plenty of vegetables, legumes and fruits. Regularly including a variety of vegetables, legumes and fruits in the diet will provide a wide range of vitamins, minerals, dietary fibres and beneficial, non-nutrient phytochemicals found in plant foods for very few kilojoules.  Vegetables include green leafy varieties, red and yellow and starchy vegetables. Fruits include those high in vitamin C and those high in vitamin A (and its analogues).

 

The Dietary Guidelines concluded that ‘there is strong evidence of a protective effect of certain vegetables, legumes and fruit against the development of a number of non-communicable chronic diseases, among them cancer, cardiovascular disease, type 2 diabetes, hypertension, and cataract and macular degeneration of the eye. This may, in part, be mediated through phytochemicals. Adults are encouraged to consume on average at least two helpings of fruit and five of vegetables each day, selected from a wide variety of types and colours and served cooked or raw, as appropriate.’

The Dietary Guidelines for Children and Adolescents in Australia[2] reference the Australian Guide to Healthy Eating which recognises the importance of fruits and vegetables in a healthy diet for all sections of the population.  It recommends “consumption of between one and two servings of fruit and two to four of vegetables each day for children aged 4–7 years; one to two servings of fruit and three to five of vegetables each day for children aged 8–11 years; and three to four servings of fruit and four to nine of vegetables each day for adolescents (12–18 years).”

 

Risk Management Considerations

Data from this survey indicated that estimated levels of nitrite dietary exposure levels are below the threshold for elevated MetHb levels in blood of infants, children and adults. As the current estimated dietary exposures to sodium nitrate and sodium nitrite are not considered to represent an appreciable human health and safety risk neither a regulatory nor a non-regulatory approach to risk management is considered necessary.

 

Conclusion

The large majority of estimated dietary nitrate and nitrite exposure occurred through the ingestion of fruit and vegetables. Exposure to nitrate and nitrite through uses as a food additive represented only a relatively small proportion of dietary exposure. Current estimated Australian dietary nitrate and nitrite exposures are not considered to represent an appreciable health and safety risk. Any health risks that may be associated with ingestion of nitrate and nitrite in the diet, are outweighed by the strong evidence of health benefits of consumption of fresh fruit and vegetables as part of a balanced diet.

 

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[1] National Health and Medical Research Council. Dietary Guidelines for Australian Adults. Canberra 2003.

 

[2] National Health and Medical Research Council. Dietary Guidelines for Children and adolescents in Australia incorporating the infant feeding guidelines for health workers. Canberra 2003.

 

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