Historial perspective

- Phase 1: hunting, fishing and gathering. Knowledge was present on mainly acute toxic properties of plants and mushrooms. But less knowledge was present of chronological toxicity effects.

- Phase 2: Agricultural development.New plants were eaten, due to new cooking (heating) strategies new plants were allowed to

eat. Storage and transport introduced new toxic compounds (mycotoxins).

- Phase3: Industrial development.Distance between food and consumer increases. New preservation methods (canning) and additives were produced to facilitate this greater distance.In 1919 the Warenwet was found. Next to Dutch laws, European laws are at work as well, such as general food law and veterinary medical products.

Organisations involved in risk assessments:

European level:- European Commission

USA:- Food and Drug Administration (FDA)

Worldwide:- World Health Organisation (WHO) Expert Committee on Food Additives- Food and agriculture organisation of the united nations (FAO)- Codex Alimentarius Commission

Advising bodies:- Joint FAO/WHO Meeting on pesticides residues JMPR- Joint Expert Committie on Food Additives (JECFA) - European Food Safety Authority (EFSA)

Ways of exposure to hazards in the modern food chain:- Industrial emission- Veterinary drugs- Pesticides - Natural toxins- Fycotoxins- Additives- Packaging materials- Heating of products

Risk assessment

Hazard identification- Chemical properties- Structural similarities- Uses/sources- Epidemiological and experimental data- Critical effect- Target organ

Hazard quantification- Dose-response curve- NOAEL and Benchmark dose- Extrapolation from experimental animals to humans- ADI, TDI, VSD (virtual safe dose = the dose would cause one extra case in 1 million humans due to lifelong exposure).

Exposure assessment- Levels in food/drinking water- Consumer questionnaires/production volume data- Estimated daily intake- Which population is to be considered?

Risk characterisation- Compare the ADI, TDI, VSD with EDI- Determine margin of safety or margin of exposure

Risk management- Politics, culture, society

Hazard vs Risk- Hazard: The possibility of doing harm- Risk: The probability of doing harm

The hazard remains the same, if a lion of next to u, outside a car or behind a cage. In a cage, the lion is a hazard, not a risk. If someone releases the lion, the hazard is transformed into a risk.

Hazard are described in qualitative and quantitative terms.Qualitative:- Type of effectsNeurotoxicity, hepatoxicity, nephrotoxicity, carcinogenicity (induction of cancer), mutagenicity (induction of mutations), teratogenicity (development disorders), sensitization (allergies), dermal, reproductive or endocrine disruptive effects. - Mode of actionInhibition of specific enzymes, specific reactions (DNA binding), competition with hormones, vitamins or metabolites.- Target organLiver, brain, lungs, hearts etc.

Acute toxicity: short exposure, high doses or concentrationsChronic toxicity: long exposure, low doses

Quantitative:- Dose-response relationship- Time-dose-response relationship

These response curves are obtained via fixed protocols in animals studies. Animals studies have disadvantages: - Ethical- Expensive- Time consuming- Relevance of effect observed in animals at high dose levels to the human situation

Alternatives to animal testing:- Structure activity relationships (SAR)- In silico (computer models)

- In vitro research with animal/human cells

Despite these alternatives, dose response curve still often obtained with animal experiments. There is a small shift to human cell based testing. Translation of in vitro data to in vivo data is problematic.

A compound might have different target organs or different effects at different dose levels.

The lowest dose levels are most important.

The NOAEL or LOAEL is used to calculate ADI or TDI. A safety factor of 100 or more is then used. Divided by 10 for interspecies differences, additionally divided by 10 for inter individual differences. Next to these 2 safety factors, other safety factors can be used, based on quality of data or type of exposure used.

ADI is for avoidable chemicals, such as additives and pesticides. TDI is for unavoidable contaminants, such as dioxins, mycotoxins and heavy metals.

If ADI or TDI is higher than the EDI than its okay.Occasionally more exposure to chemicals than described in the ADI or TDI is not bad, as these guidelines are made for lifelong exposure.

Disadvantages of NOAEL: - NOAEL is dependent on sample size. More animals -> lower NOAEL. So, better experimental designs produce lower NOAELs. - NOAEL is limited to experimental dose. - NOAELs differ across studies. - NOAELs use only one experimental data point, whilst the full dose-response information as a whole is not used.

Bench Mark Dose (BMD) approach- Bench mark response (BMR) = 5-10% increase effect over background level.- Bench mark dose = the dose causing the BMR- BMDL = lower confidence limit of the BMD

ADI/TDI = BMDL * 1/10 * 1/10 * other safety/uncertainty factors

Advantages of BMD:- Not dependent of setup of experiment- Makes use of all toxicity data- BMDL has on average the same level of protection as NOAEL- Same uncertainty/safety factors can be used.

EPA software can calculate the BMD(L).

ADI concept is not applicable for genotoxic or carcinotoxic compounds. - If avoidable: zero tolerance- If unavoidable: ALARA (as low as reasonably achievable). VSD (virtual safe dose) is the dose that gives 1 in 10^6 extra cancer risk upon life time exposure. - Margin of exposure (MOE). = BMDL10 / EDI

A MOE of 10.000 (risk of 10.000 fold lower than the dose that induces 10% cancer response in animals (BMDL10)) is considered the amount which separates the high and low priority risks.

Food supplements

This is a supplement to the diet which provides nutrients that may be missing or not consumed sufficiently in the diet. The definition of a food supplement varies between countries. Some countries describe them as drugs, some as natural health products.

When food supplements are considered as food, the manufacturer is responsible for safety. There are 2 categories within the EU:- Vitamins and minerals (regulated)- Plant food supplements (not regulated, only some species or compounds not allowed).

Vitamins and minerals: There are directives (richtlijnen) that describe that vitamin and minerals supplements are capsules, tablet, sachets in order to supplement a normal diet in a safe and appropriately labelled way.

In these supplements only compounds may be present with are listen in Annex I. Substances listed in Annex 2 may be used singly or in combination from which vitamins and minerals may be manufactured. EFSA regulates inclusion of vitamins and minerals based on scientific dossiers.

In Annex 1 tolerable upper intake levels may be used, which reflect the maximum level of total chronic daily intake of a nutrient. In Annex 2 safety evaluations are not made based on the nutrient itself but focus on the source of the nutrient.

Safety evaluation:- Nutrients are essential for human well-being within a certain range of intakes- There is a long history of safe consumption- Some nutrients may be consumed chronically at levels of significantly above those obtained from endogenous nutrients in foods without reported adverse effects. - Data on adverse effects in humans may help reduce uncertainty factors- Protection is present against nutrients through homeostatic regulation, such as excretion or absorption. - A tolerable upper intake level is present. This is the maximum level of total chronic daily intake of a nutrient which is judged to be unlikely to produce a risk of adverse health effects.

For example vitamin A. UL = 3000 ug RE/day

Plant food supplements (PFS)Safety of PFS is generally not assessed before entering the market.

Concerns of PFS:- Overconsumption- Naturel is assumed to be safe- Availability of harmful PFS through the internet- Often no requirement of safety of PFS

Plant food supplements may be toxic because:- Naturally present toxic ingredients- Harmless ingredients may be replaced with a toxic alternative- Adulteration (illegal compounds present) (deliberately)- Contamination of harmful compounds (accident or negligence)

Compounds 2L:

Naturally present compounds:- Pyrolizidine alkaloids (PA) : Symphytum officinalis: Hepatotoxicity (obstruction of small veins in liver and cancer in animals).

- Alkenylbenzenes : Present in PFS containing oil of basil, fennel, nutmeg etc: Genotoxicity and cause liver tumors in rodents at high dose levels.

Replacement of harmless ingredient with toxic alternative:- Anisatin: Star Anise (Illicium verum anise): Epileptic abnormalities

- Aristolochic acids (Aristolochia fanchi): cardiovascular damage, kidney damage, cancer of kidneys and urinary tract.

Adulteration- Sibutramine: increased of adverse cardiovascular events

- Sildenafil: increased risk on skincancer

Contaminants- Polycyclic aromatic hydrocarbons (PAH, bijvoorbeeld benzo[a] pyrene): unintentionally added from environment. Several PAH are genotoxic and carcinogenic.

EFSA uses 2 tiers to assess PFSTier 1 is for compounds of which the following is known and assessed to pose low risk for adverse effects:- Nature of botanical preparation or the botanical- Its intended use and levels of use- Whether the botanical preparation has a long term history of use showing no adverse effects.

Tier 2: For botanical compounds lacking history of no adverse effects. An ADI or TDA should be established, as Threshold of toxicological concern (TTC) or MOE (margin of exposure) can be used for genotoxic carcinogenic ingredients.

Labelling of PFSs:- Indication of nature of nutrients or substances- Recommended daily intake and warning of the risks to health if this is exceeded- Is not substitute for varied diet- Not a medicinal product- Should be out of reach of young children

- Most not contain a statement that it can cure prevent or treat a disease.- Any statement that a varied diet cannot provide appropriate quantities. - Any misleading claims -> elimination from the market

Health claims are assessed by the EFSA. Thus claims are regulated, but safety is not, as this is not judged before entering the market. Natural toxins, adulteration, contamination or replacement of ingredients are treats for adverse effects.

Natural toxins

Phytotoxins (plants)Some medicines are made from bioactive compounds produced by plants. The amount of bioactive compounds varies with portion and age of the plants, climatic and genetic conditions.

Anisatine in Japanese star anise: GABA inhibits neurotransmitter receptors and causes epileptic attacks.

Bioactive compounds are often found attached to sugar in plants. Aglycone is released by enzymes, which has potential to decrease bone loss in post-menopausal woman.

Toxiclogical concern

Improper preparation- Anti-Nutritional factors (ANFs) - Trypsin inhibitors (interfere with protein-digesting enzymes) - Lectins (interfere with absorption nutrients intestines)These are present in seeds and reduced by cooking (heat-labile).

- Solanine is present in flowers and potatoes. Solanine inhibits acetylcholinesterase, preventing breakdown of the neurotransmitter acetylcholine. This leads to irritation and injury of GI tract, abdominal cramps, vomiting, diarrhea, trembling, weakness and unconsciousness.

Famine foodCyanogenic glycoside: HCN inhibits cytochrome c oxidase (complex IV of OXPHOS) ultimately causing the paralytic disease Konzo. HCN detoxification leads to thiocyanate, which inhibits iodide uptake in the thyroid and may lead to swelling of the neck.

Propper preparation can reduce HCN exposure. HCN, which is a cyanogenic glycoside, is heat stable. Chopping, soaking and sometimes fermenting is necessary to remove HCN.

Toxic amino acidsLathyrogens: active compound is ODAP which causes muscle weakness, stiffness and paralysis of leg muscles. (neurotoxicity) due to overstimulation of the glutamate receptor (NDMA). BAPN: causes weak connective tissue due to inhibition of formation of cross connections between collagen and elastin.

SupplementsGlucosinolates: Isothiocyanates, are claimed to have anti-cancer properties but might be genotoxic themselves. Alkenylbenzenes: genotoxic and carinogenic.

Sensitive indivudalsImmune reactions should be mentioned on the label. Fava glycosides: Vicine and convicine. They produce low levels of gluthathione (GSH) by leading to low levels of G6PD, leading to low levels of protection againt oxidative stress, which affects red blood cells.

Malaria also leads to G6PD deficiency.

Herbal teasPyrrolizidine alkaloids -> liver necrosis.

Concluding remarks:Phytotoxins are Generally recognised as safe (GRAS).

Natural toxins in animals

Includes:- Bacterial toxins- Fycotoxins- Animals toxins

Bacterial toxins- Clostridium botulinum inhibits release of acetylcholine by presynaptic membrane of motor endplates. Leading to weakness of muscles by production of botulinum toxin which can be fatal. Honey is often cause of botulism in children, sodium nitrate inhibits growth of clostridium botulinum.

Fycotoxins (Marine algae)- Dinoflagellates are a group of flagellate eukaryotic organisms which accumulate in the food chain in particular shellfish.

1 Paralytic shellfish poison (PSP)- Mechanism: blockage Na-influx- Symptons: Nausea, vomiting, diarrhoea, numbness, tingling lips, coordination probelsm, confusion, paralysis, respiratory distress and death. - Saxitoxin (typical PSP toxin)

2 Neurotoxic shellfish posion (NSP)- Mechanism: activating sodium channel through enhancement of Na-permeability. - Symptoms: Nausea, vomiting, diarrhoea, numbness, tingling lips, coordination problems, confusionBut NO paralysis, respiratory distress or death. - Brevetoxin (typical NSP toxin)

3 Ciagatura fish posion (CFP)- Mechanism: Enhancement of Na permeability activating sodium channels- Symptoms: Nausea, vomiting, diarrhoea, numbness, tingling lips, coordination problems and confusion. But NO paralysis, respiratory distress or death. - Ciguatoxin (typical CFP toxin)

4 Amnesic shellfish poison (ASP)- Mechanism: Overstimulaion of glutamate neurons -> neurotoxicity. High intracellular calcium leading to neuronal cell death and lesions in areas of the brain where glutaminergic pathways are concentrated. - Domoic acid (typical ASP toxin)

5 Diarrhetic shellfish poison (DSP)- Mechanism: inhibitor of ser/thr protein phosphatases -> hyperphosphorylation of proteins, including ion channels in the intestinal epithelia, resulting in impaired water balance and loss of fluids. - Okadaic acids (typical DSP toxin)

Detection of shellfish poisons- Mouse bioassay -> injection of suspected sample -> endpoint mortality- In vitro alternative required -> E.g. electrical activity neurons

Fish toxinsTetrodotoxin: neurotoxin in organs such as intestine, ovaria, liver. Paralysis begins 10-45 minutes after ingestion. Death can occur within 6-24 hours. Mechanism: it blocks sodium channels.

Scombrotoxins: Bacterial contaminated histidine rich (scombroid) fish: tuna, mackerel, bonito, sardines. Histidine becomes histamine, which leads to an allergic reaction. Such as redness, swelling, heat, pain and loss of function (inflammation).

Concluding remarksMany natural toxins in animals are a result of contamination such as bacteria or algae. Adequate control of products prevent adverse effects in population. Detection methods are required for quality control of products.

Food additives

1) To maintain product consistency2) To improve or maintain nutritional value3) To maintain pleasure & wholesomeness 4) To regulate pH5) To enhance flavour or change colour

Direct additive:- Substance added for specific purpose

Indirect additive:- Becomes part of food unintentionally though predictably (packaging).

Evaluation and re-evaluation is continuously done. European Commission and EFSA authorise additives in Europe. In America the Food and drug administration (FDA) does this. For the whole world joint FAO/WHO Expert Committee on Food Additives (JECFA) does this.

EU directive (richtlijn)- Regulation of E-numbers, only additives on this list may be used.

Evidence must be prodivded that the additive:- Performs as intended- Will not cause adverse health effects- The use does not mislead the consumer at expected levels of human consumption

ADI’s are used for additives, though not all additives have ADI.Genotoxic carcinogens are not accepted, never as food additive. Genotoxic: not allowed; genotoxic carcinogen; not allowed, carcinogen; allowed below ADI

There are four groups of additives:- Additives with an ADI defined- Additive with a temporary ADI (insufficient toxicological information; therefore an extra safety factor is used). - Additives with ADI not specified; based on tox data and use foreseen: MOS is adequate.- Additives with no limit due to safe history of use. Due to Incredibly high ADIs or incredibly low EDIs.

Considerations- Children: lower body weight, daily intake must be lower, at increased risk for artificial sweeteners. - Risk benefit balance: risk of use considered lower than the risk expected upon their absence. For example: Nitrate may form nitrosamine, which can cause stomach cancer. However, nitrate is added to red meat to prevent infection with Clostridium, and prevent production of botulinum toxin.- Carcinogenicity of additives: aspartame ‘krebs cyclus’ cancer? No, TCA cyclus. ADI of aspartame is not applicable to PKU patients (unable to degrade Phe, resulting in high plasma levels of Phe resulting in brain damage).

Carcinogenic additives are classified. Group 1: carcinogenic to man -> forbidden

Group 2A: Probably carcinogenic -> forbiddenGroup 2B: Possibly carcinogenic -> more researchGroup 3: Compound cannot be classified because of insufficient data -> more researchGroup 4: Proven to be non-carcinogenic for humans

Concluding remarks- Food additives best regulated chemicals- EDI far below ADI and is considered safe- Increasing consumer criticism- Possible adverse health effects related to: suspected insufficiently investigated carcinogenic risk, immunotoxicity, allergenicity and hyperactivity.

Food additives allergy and hyperactivity

Positive points:- Inhibit bacterial growth- Prevention negative change of smell or taste- Maintenance of aroma and color- Enhance anti-oxidant status

Negative points:- Enhance cancer risk? (I don’t think so)- Allergy or intolerance?

Food allergy or intolerance:- Unwanted physiological reaction based on immunological mechanisms.Type 1 = hypersensitivity: Mast cells release histamine and cytokines when allergen is encounteredType 4 = Delayed hypersensitivity: Macrophages present allergens to T-cells, cells release interleukins which stimulate the immune system and cause inflammation. The first encounter creates memory for the second encounter (faster response).

Intolerance is not an allergy. Lactose intolerance = deficiency of lactase.

Food additives may contain compounds which can initiate allergic reactions. Types of allergy or hypersensitivity caused:- Skin: eczema itching, oeema- GI disorders: vomiting, intestinal cramps, diarrhoea- Respiratory disorders: asthma, rhinitis - Other like: migraine, hyperactivity, lack of muscle coordination

Fun facts:Hyperkinesis (hyperactivity) may be caused by ADS or ADHD. There are AZO dyes and non-AZO-dyes. There are lots of dyes which may cause allergy and hypersensitivity. AZO dyes are considered to be more likely to increase hyperactivity of children.

Since 2010: Foods containing azo dyes require warning; may have an adverse effect on activity and attention in children. More research is needed. Urticaria (netelroos) and asthma patients seem to be more sensitive.

Underlying mechanisms:- Mostly unknown- Immunological mechanisms: Type 1 or 4. - Non-immunological mechanisms (ADS/ADHD)- Direct injury of stomach mucous- Interference with prostaglandin synthesis due to the compound acetyl salicylic acid (ASA) (mechanism of aspirin) may also cause hypersensitivity reactions. Whilst ASA inhibits prostaglandin synthesis, thereby inhibits PGH2 which leads to less leukotrienes.

Biotransformation

Bioavailability depends on 5 steps:- Liberation and dissolution in GI tract- Transport across intestinal membrane- Blood circulation and distribution- Metabolism or biotransformation- Excretion

Biotransformation processes are described in physiologically based kinetic models

- Hydrophilic compounds: Excrete in urine or bile - Polar compounds: Conjugation -> Hydrophilic compound -> Excretion- Lipophilic compounds: Modification -> polar ->Conjugation -> hydrophilic -> excretion- Highly lipophilic compounds: Accumulate in fat tissue- Reactive compounds: Via xenobiotics or via Phase I or II biotransformation -> covalent binding to tissue macromolecules.

Phase 1 (P450) Reaction: Oxidation, reduction and hydrolysisProduct: Polar metabolite with functional OH, NH2, SH, COOHCofactor: NADPH

Phase 2 Enzyme: Epoxide hydrolase, glutathione S-transferase, glucuronosytransferase, sulfotransferase

Product: Hydrophilic metabolitesConjugation with: Water, glutathione, glucuronic acid, sulfate

Biotransformation enzymes are predominantly present in liver. But also in GI tract, lungs, skin and kidneys. Bioactivation in specific organ may explain target organ effect. So, biotransformation/activation is important for detoxification but also bioactivation. Expression of biotransformation enzymes differ with age, genetics and lifestyle.

Measurement of biotransformation is done based on microsomes, cytosol, S9, rat sample, human samples and velocity of reaction rates.

Based on kinetic aspects, humans are expected to be less sensitive than rats. With ages the effectiveness of enzymes vary a lot. There are also genetic specific differences; poor and fast metabolizers. Lifestyle, such as smoking, medicine use and vegetables and fruits consumption also play a role.

Lastly, difference in gut microbiota may explain differences in uptake of glycoside conjugates.

The abundance of P450 enzymes differ with age. Some isoenzymes of P450 differ with gender. Isoenzymes of CYP1A2 and P450 differ with lifestyle, for example smoking, grapefruit juice or bbq meat but also with genetic variation.

Natural carcinogens and cancer modulating factors

Diet hugely impacts cancer, looking a migration studies Japan -> USA. Fat intake and breast cancer.

There are non-genotoxic and genotoxic compounds.

Non-genotoxic:- Oxidative stress- Immunosuppression- Increasing proliferation (epigenetic)

Genotoxic (DNA damage)- DNA adducts- Chromosome damage (breakage & fusion)

Genotoxic carcinogens: NO safe exposure levelMOE < 10.000 priority risk management

Chemical carcinogens in food

Chemical carcinogens can also be formed endogenously such as nitrite + amine -> nitrosamines.85% of nitrosamines are carcinogenic.

Chemical carcinogens can also be formed due to heating of food. Pyrolysis of fat, polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene. Benzo[a]pyrene can be formed due to smoking, grilling, barbecuing or burning foods. PhIP is one of the most formed heterocyclicamines, it is carcinogenic. Also Maillard reactions: sugar-amino acids (acrylamide).

Genotoxicity and carcinogenicity studiesGenotixicity can be tested in vitro via the Ames, TK or CAT test. If one or more is positive, two in vivo tests should be conducted.

In vitro:Ames test: Salmonella Thyphimurium cannot form colonies due to absence of external amino acid source, unless reversion of the mutation restores biosynthesis of the amino acid.

Mammalian gene mutation test (TK). TK+/- (non mutated) die, whilst TK-/- survive. So mutation is needed to survive.

Chromosome aberration test (CAT). Chinese hamster ovary cells or human lymphocytes. Arrest cells in metaphase, then after exposure to copound examine structural changes in chromosomes.

In vivo:Micronucleus test: Mice or rats exposed to compound, then microscopic analysis of bone marrow.

DNA repair test: detection of repair of DNA damage liver cells of rats.

If genotoxic: no carcinogenicity testing necessary.If genotoxic and non-avoidable: carcinogenicity studies still needed.

Standard carcinogenicity study4 groups, 50 male and female rats or mice, 1 control 3 dose groups. Check for tumours.

Extrapolation to humans often difficult:- No epidemiological data available to support correlation- High doses in animal studies vs low doses in human studies- Specific differences between rodents and humans- Pure compound vs exposure in a complex food matrix.

Chemical anti-carcinogen in food- Reduce effects of carcinogens by modulation of biotransformation enzymes- Reduce inflammation- Reduce oxidative stress- Reduce proliferation

However, this effect is mainly found in in vitro studies.

Concluding remarks- Several natural (genotoxic) carcinogens present in food- Deliberate addition is not done- Removal unavoidable chemicals not possible- MOE is then used- Difficulties assessing risk in humans- Anti-carcinogen effect mainly shown in in vitro studies

Metals in food

Metal: a material that is hard, shiny, good electrical and thermal conductivity.Metalloid: a chemical element with properties of a mixture of metals and non-metals.Non-metal: a chemical element that mostly lacks metallic attributesEssential metal: part of enzymes involved in metabolic processes.

Chelator: an electron donating compounds which are capable of binding to toxic metal ions to forms structures which are easily excreted from the body. Its properties are an amino, thiol and carboxy group.

Examples:- Methallothionein- Ethylene-diamino-tetra-cetic-acid-Dimercaptopropanol

Bioavailability of metals depends on:- Solubility of salts- Valency of ions (Fe2+ versus fe3+)- Presence of other ions (competition of uptake, more Cd uptake with Ca deficiency). - Ability to dissolve in fats- Food in GI tract.

Fe2+ can enter and leave behind the intestinal cell. Heem iron can enter but only leave as Fe2+. After leaving the cell Fe2+ is converted to Fe3+.

Methylmercury- Is released in air by industrial processes.- Converted to MeHg by microorganism, which is more bioavailable and toxic. - Fish accumulate MeHg in fat.- Accumulation causes neuro(developmental) toxicant

Methylmurcury overactivates NMDA receptor, which leads to increased intracellular calcium and cell death and ROS. Symptoms are mainly expressed as neurotoxicity

Lead- Exposure via food, water, air, dust, soil.

- Inhibits heme synthesis leading to anemia, initiates demyeliation which leads to neurotoxicity.- Mechanism is via ROS and disturbance of calciumhomeostasis - Absorption 4 fold higher in children. - Accumulates in soft tissue and bones. In bones the half-life increased drastically.

Cadmium- Cigarette smoke, environment, food- Toxicity: nephrotoxicity (proximal tubule dysfunction) and osteoporosis. When metallothionein binding saturates severe kidney damage occurs. - Mechanism: ROS, kidney damage via calcium loss and bone loss. - Subgroup such as smokers, children, vegetarians and people living in cadmium rich areas may exceed TWI 2-fold.

ArsenicGenomic instability due to decrease of global DNA methylation leading to cancer risk, especially skin cancer.

Concluding remarks:Exposure is such that adverse health effects in humans cannot be concluded.

Veterinary drugs

Antibacterial compounds, anthelminitics, coccidiostats and others.

Risk for consumer: residues in meat, eggs and milk. Many adverse effects: a few are reproductive, immune and teratogenicity toxicity.

MRL = Maximum residue level in animal products, for ADI are established. If levels are higher than the MRL, a withdrawal period is made:This ensures that levels are lower than the MRL

Antibiotic resistance:- Fear that large scale use of antibacterial agents will cause resistance- Drugs no longer useful for treating patients- Increased resistance in farmers, but less at organic farms.

Useage of drugs and hormones for growth promotion is prohibited.

Forazolidone- Widely used in pigs and chickens- Mutagenic and carcinogenic so no NOAEL. - NO residues of forazolidone and its metabolites. - But mouse study proven opposite, so no risk taken and banned.

B-agonist leads to more meat, less fat. But prohibited as this affects the human heart.

Persistant organic pollutants

- POPs accumulate in the food chain, like DDT, PCBs, Dioxins.They mostly have larges MOEs. Dioxins are mainly chronicly toxic, but are used by asssin attempts for its acute toxicity.

Neem powerpoint door.

Combination toxicology

Kinetics: ATME: absorption, transport, metabolism and eliminationDynamics: Target organ & receptor

Prediction of plasma levels: integration of in vitro data in kinetic model

Complex mixture > 10 compounds

Simple compound < 10 compounds

Simple similar action:- No interaction, similar mode of action

Simple dissimilar action:- No interaction, different mode of action

Interactions:potentiation, antagonism

Response addition: Summation of effect A and B (independent joint action)Dose addition: Summation of doses A and B (simple joint addition)

Cumulative risk assessment for multiple compounds in food

Similar action: Compounds act in the same way, but differ in potencies.

Dissimilar action: Compounds act independently from each other, through different mode of actions that do not affect each other

Interactions: A combined effect may be strong or weaker than expected based on dose addition.

Oxidants

Oxidants influence:- Food quality- Shell life- Taste- Smell- Vitamin contents

Reactive oxygen species:- Superoxide anion radical- Hydroxyl radical- Hydrogen peroxide- Singlet oxygen

Reactive species derived from ROS:- Lipid (peroxide) radicals

Reactive nitrogen species- Nitric oxide - Peroxynitric acid

Sources of ROS1- Decoupling one-electron transport chains2- Enzymatic reactions3- Redox cycling (paraquat)4- Inflammation neutrophils/macrophages5- From other ROS (Haber-Weiss reaction)

Damage produced by ROS:- Oxidation proteins/enzymes -> loss of function- Oxidation DNA -> mutagenicity and carcinogenicity - Oxidation unsaturated fatty acids -> lipid peroxidation (disturbs membrane structure)

Antioxidants:- Vitamin C- Vitamin E- Carotenoids- GSH (glutathione)- Ubiquinone- Polyphenols

Antioxidants enzymes:- Superoxide dismutase (SOD) - Catalase- Glutathione peroxidase

Natural anti-oxidants:- Carotenoids- Vitamin E

- Vitamin C- Polyphenols like flavonoids- Peptides (glutathione)- Conjugated linoleic acid- Coenzyme Q (ubiquine)

These can donate a e- or H+ or form a stable AOX radical. Claims can be made on antioxidant presence in foods, but are often not proven.

Antioxidants

Beta carotene reduces risk on lung cancer, cardiovascular disease etc. Carotene deficient diets increase risk on lung cancer. 4-5 times increase in plasma levels causes adverse effect.

Beta-carotene supplementation may increase risk on long cancer, possibly due to reactive oxidative beta carotene metabolites. Or due to disturbing of retinal signalling and thereby activating oncogenes and down regulating tumour suppressor genes.

Haber-Weiss reaction

ROS and RNS may have signalling function to promote health effects of physical exercise in humans.

Sint janskruid: verminderde werking P450 en bepaalde medicijnen. Ongewenste zwangersschap.

Other polyphenols:- Quercetin: health claims; inhibition of cancer, ageing, cardiovascular disease and photosensitivity diease. At high levels more indicdence of tumors. The possible mechanism of protection is induction of EpRE mediated cancer preventive genes by the flavonoid quinones.

Flavourings

Exposure is low, flavours have a long history of safe use. The FDA made a first GRAS list. Flavours are no food additivesEFSA makes regulations for flavours.

Three types of flavours are recognised in the EU:- Flavour substances which are chemically identified- Flavour preparations from plant or animal origen (Natural flavour complexes)- Thermal process flavours or smoke flavours.

Flavours are obtained synthetically to ensure purity and identity, although also found naturally (1700 substances). 350 substances are also synthetically produced but are not found in nature.

Artificial flavouringsThese are almost always structurally related to naturally occurring flavouring substances.

Natural flavour complexesExtracts, oils, fruit juices, animal or vegetable material.

Thermal process flavouringsProduced by heating together substances, containing NH2 and a reduced sugar, similar to changes that happened when food is heated. Are safer, as heterocyclic amines are removed.

Smoke flavouringsExtract from smoke, that give the same flavour. Are safer than traditionally smoking process, because harmful compounds are removed as much as possible.

Safety evaluation:

In vitro:Ames test: Salmonella Thyphimurium cannot form colonies due to absence of external amino acid source, unless reversion of the mutation restores biosynthesis of the amino acid.

Mammalian gene mutation test (TK). TK+/- (non mutated) die, whilst TK-/- survive. So mutation is needed to survive.

Chromosome aberration test (CAT). Chinese hamster ovary cells or human lymphocytes. Arrest cells in metaphase, then after exposure to copound examine structural changes in chromosomes.

Flavours are grouped in 3 classes:Class 1: flavours have simple chemical structure, suggesting low oral toxicityClass 2: flavours have less innocent structures, but are not suggestive of toxicityClass 3: may suggest toxicity, or have structural features that permit no strong initial presumption of safety.

The Threshold of toxicological concern (TTC) is used. Class 1 – 1800ug/dayClass 2 – 540 ug/dayClass 3 – 90 ug/day

Diacetyl: flavour giving buttery flavour. Inhalation is dangerous, however the safety evaluaton is only for oral exposure. Couramin is not genotixic, but is carcinogenic. So a thresholded mode of action. Alkenylbezenes is genotixic, restricted in the EU.

Nanotoxicology

There are nano-formulated food additives. Current specifications on nano forms of these additives are re-evaluated by the EFSA. Nano-particles are better for bio-availability. Food packes have improved mechanical, barrier and antimicrobial properties.

Nanoparticles tend to stay close to each other.

First point of entry provides opportunity for local effects;- Lungs- GI tract- Skin

Then the target organ for systemic effects.

Uptake:- Transcellular through normal intestinal enterocytes.- Paracellular (between enterocytes through tight junctions). - Peyers patches lymphatic tissue containing M cells. These M cells take up, process and present the nano-particles to the immune system.

Uptake of nano-particles depend on:- Particle size- Surface charge- Attachment of ligands- Coating with surfactants

Surface charge:- Positively charged particles become entrapped in negatively charged mucus.- Negatively charged diffuse across the mucus layer. Smaller sized particles pass through the mucus layer faster.

To study translocation organs on a chip are used.

Possible health hazard:- ROS production.

- Frustrated pahocytosis: when particles are too large or cannot be degraded.

- fibrosis and risk on Gi carcinomas.

- systematic effect: particles found in several organs and can be transported via nerves.Interleukines may be important for systematic effect (inflammation effect of fibrosis in GI tract).

Safety assessment:- Precautionary principle: new technologies should not be allowed when they imply risks for men or environment. Even if this has not been scientifically proven yet. - Proportionality principle: measures taken should be based on risk-benefit evaluation.

So, currently:- They are a hazard but we don’t know if they are a risk.

Pesticides

Are against: insects, fungi, weeds and other biological causes.

Acceptable daily intake vs acute reference dose

There is also an acceptable operator exposure level (OAEL).

Maximum residue limit. - Reflect the minimum quantities of pesticides to achieve sufficient effectivity.- This is also below the ADI- Which means that the MRL are below the ADI, in most cases well below, so exceedance of the MRL does not mean the ADI is exceeded. - However is the MRL is exceeded, the product will not be placed on the market.

Exceedance can result from:- Unauthorised pesticides- Pesticides not authorised for a specific crop- Higher application rate or shorter pre-harvest intervals- Insufficient washing out

Risk management actions are taken by withdrawing food or lowering MRLs.

Additional effects of different pesticides can be considered if pesticides target similar metabolic pathways -> cocktail effects.

Organophates:- Inhibit acetylcholinesterase - Leads to overstimulation of postsynaptic receptors.

Pesticides that exhibit a similar toxicological effect can produce joint, toxicity effects:Cumulative risk assessment:

Total residues of the index chemical= Sum of residues (mg) * (potency factor)