“RISK ASESSMENT OF CHEMICALSGENERAL CONCEPTS”5797‐Food toxicologyDEGREE IN PHARMACYYear 2013‐14 – First semesterProfessors: Eugenio Vilanova and Jorge EstévezDepartment: Applied Biology

Unidad de Toxicología y Seguridad Química

RISK ASESSMENT OF CHEMICALSGENERAL CONCEPTS

Eugenio Vilanova GisbertUniversidad Miguel Hernández De Elche

Unidad de Toxicología. Instituto Bioingeniería

http://tox.umh.es

Biological systems interact with thousands of substances in your environment:

Exogenous substances

Exogenous substances

Once absorbed some haveENDOGENOUS BILOGICAL FUNCTIONS(Nutrients, vitamins and essential elements)

Others without endogenous biological function in our body, we call them:

XENOBIOTICS

SOME numbers:

Genes that encode proteins: prokaryotes: 2,000 insects: 7,000 higher animals: around 50,000 different enzymatic reactions in the cell: around 5000

Endogenous molecules Deifferent proteins in a cell: around 5,000 monomers of biopolymers and metabolic: around 5,000

EXOGENOUS MOLECULES with endogenous function:

Essential nutrients: 31 molecules 9 amino acids, 10 trace minerals, 12 vitamins)

Probably essential: 11 molecules

Food components: some 10,000 (?) (considering only the monomers of biopolymers)

XENOBIOTICS Xenobiotics of natural origin: ????? Several hundred thousands

Industrial Xenobiotics: European register: >100,000 substancesregular interaction: 10,000 substances

Biological systems have mechanisms to deal with the interaction with thousands of external molecules:

•destruction of foreign macromolecules (digestion and immune system)

•Some are used for endogenous functions (nutrients, vitamins, oligo elements)

•Conversion to endogenous molecules (intermediary metabolism)

•Elimination of Xenobiotics (xenobiotic metabolism) BUT...

CHEMICALS

MECHANISM OF DETOXICATION(ADME)

ELIMINATIONDISPOSAL

NERVOUS SYSTEM

MECHANISM OF DETOXICATION

ELIMINATION

CHEMICALS

NERVOUS SYSTEM

Biological systems have mechanisms to deal with the interaction with thousands of external molecules:

Interaction with xenobiotics are part of the normal physiology

•Elimination of Xenobiotics (xenobiotic metabolism)

BUT...

SOME Xenobiotics at low doses, in certain circumstances, can cause desired changes as a remedy of diseases:

THERAPEUTIC OR PROPHYLACTIC EFFECTS

in these circumstances we call:

MEDICINES, PHARMACEUTICAL DRUGS

The XENOBITICS or their metabolites at doses greater than the capacity of detoxication and disposal can cause :

adverse effects to the health of the individual or ecosystem:

TOXIC EFFECTS.On these circumstances we call them:

TOXIC SUBSTANCES • TOXICANTS • TOXINS • POISONS …

SOME Xenobiotics in certain circumstances can selectively kill a way of life "while" respecting others: biocidal effects

In these circumstances we call: •BIOCIDES (general concept or specific regulatory concept)•PESTICIDES (insecticides, herbicides, fungicides, rodenticides,...)•ANTIBIOTICS •DISINFECTANT •PLANT PROTECTORS

BUT ...

ALL BIOACTIVE SUBSTANCE

REPRESENTS A HEALTH RISK TO NON-TARGET SPECIES

From the classic (archaic?) concept of study of the poison up

to

The current concept of • CHEMICAL SAFETY • risk assessment • risk prevention

based on the modern science of Xenobiotics:

molecular mechanisms of toxicity and biotransformation

TOXICOLOGY

Conceptual analogy with Pharmacology but it is dealing with pharmaceutical drugs used for therapy

¿Métodos para hacer estos ensayos

¿Why we need regulation internationally accepted ?

To:

guaranty safety and confidence of the citizens(right to be safe and filling safe)

guaranty free circulation of goods

For that, harmonizing:

Testing methods Criteria of evaluation Mutual acceptance

SOME BASIC PRINCIPLES: Priority: to guaranty safety of citizens The limits must not compromise other priority public health measures The proposal of a safety limit is a scientific process. Establishing a legal limit is a decision affected by other factors of technical viability and social sensitivity.

SOME CRITERIA for limits (1) SAFETY (1): based on a threshold. (2) (3) (4) (5) (6)

TOXIC EFFECT WITH THRESHOLD

For most toxic effects (not all):

are believed to have a “threshold”:dose below that no toxic effect is caused

A threshold can be established

NOAEL: “No observed adverse effect level”The highest tested dose with no effect

L o g (D O S IS )

EFFE

CT

TOXIC EFFECT WITH THRESHOLD

NOAEL

EVALUATION TOXICITY

Acute toxicity. Determinacion DL50.

Toxicidad aguda por via oral, dérmica y por inhalación Irritación cutánea y, en caso necesario. ocular. Se hará si no es corrosivo. Sensibilidad cutánea.

SHORT TERM (28 DAYS) Toxicidad oral a dosis repetitivas (estudio de 28 días). No si hay estudio a 90 días Otras vías (inhalación, dérmica, según proceda).

SUBCRONIC TOXICITY. Estudio via oral durante 90 días. Otras vías si procede.

CRONIC TOXICITY (2 years) Toxicidad oral a largo plazo (2 AÑOS)

Carcinogenicidad (2 años)

GENOTOXICITY. Mutagenicidad invitro

Genotoxicidad in vivo..

REPRODUCTION TOXICITY. Teratogenicidad. Reproduccion a multiples generaciones Fertilidad y desarrollo.

METABOLISM Absorción, distribución y excreción, tras la administración tanto oral como dérmica.

Metabolismo.

ESTUDIOS ADICIONALES Neurotoxicidad. Neurotoxicidad retardada en gallinas adultas. Metabolismo en vegetales (para agroquímicos) Efectos tóxicos de metabolitos procedentes de vegetales. Estudios de mecanismos para clarificar los efectos detectados. Efectos tóxicos sobre el ganado y los animales domésticos. HUMAN DATA Datos existentes de exposición humana epidemiológicos Estudios copn voluntarios Métodos de diagnóstico y tratamiento. RESUMEN de toxicología en los mamíferos y CONCLUSIONES. Resumen de toxicología en los mamíferos y conclusiones.

Propuesta de NOAEL, IDA, AOAEL y otros parámetros. Evaluación global de los datos toxicológicos.

ESTABLishing the lowest relevant NOAEL Estudy NOAEL Subacute dermal (28 dias) 110 mg/Kg/day Subchronic dermal (90 days) in dogs 35 mg/Kg/day Subchronic oral 90 days in rats 50 mg/Kg/day Subchronic oral 1 year dogs 25 mg/Kg/day Chronic-carcinogenicity oral (2 years) in rat (systemic effects)

10 mg/Kg/day

Reproduction 3 generation oral in rats (systemic maternal effects)

25 mg/Kg/day

Teratogenicity in rabbit (10 days, GD 6-16) (systemic maternal effects)

50 mg/Kg/day

… AND OTHER STUDIES AS REQUIRED FOR

REGISTRATION)

Lowest relevant NOAEL: "The NOAEL is established as 10 mg/Kg/day on the basis of decrease in the body weight gain observed at the next higher tested dose in a 2 year oral study in rat

10 mg/Kg/day

ACCEPTABLE DAILY INTAKE (ADI)

A DOSE WE CAN CONSIDER THAT HUMAN POPULATION CAN BE EXPOSED FOR LIFE WITHOUT RISK

NOAEL is usually obtained from animal experimentation We need extrapolation to humanWe need to consider the interindividual variability in the

sensitivity

We need a safety factor for

NOAEL (SAFETY FACTOR) ADI

Equivalent concepts:Reference dose (RfD), Acceptable exposure level (AEL), Tolerable daily intake, (Other)

SAFERTY FACTOROther names: “uncertainty factor “(UF)

“assessment factor” (AF)

From Cassaret y Doull, Principals in Toxicology, 2005

INTERSPECIES DIFFERENCES

INTER INDIVIDUAL HUMAN DIFFERENCES

KINETICKINETICKINETICDINAMIC DINAMIC

SAFETY FACTOR(Uncertainty factor, UF)(Assessment factor, AF)

10 x extrapolation frm animal tohuman

10 x for human interindividual variability

Usually UF= 100

Possible additional factors:

• 3 x when NOAEL is from subchronic study indeed of chronic• N x if other uncertainties are observed (2x, 3x, or 10x).

Then UF may be 200x, 300x, 1000x

Proposal of a limit in water (substance with threshold) We need: To know the toxicological properties and to establish:

1. Estimate a ADI from the NOAEL and a safety factor for an adult

2. Establish the standard water consumption: (2 Litres /day in adults of 60-70 Kg bw) (1.5 L/d children of 30 Kg bw) (1 L/d infants 10 Kg bw)

3. Allocation in water: proportion of the ADI we accept to be exposed via drinking water (usually <10% of the ADI)

MAC

LIMIT OR ALLOWABLE MAXIMUM CONCENTRATION IN WATER: CONCENTRATION THAT WOULD PROVIDE 10% OF THE MAXIMUM DAILY INTAKE IN 2 LITERS OF WATER

MAC = ADI x (%contribution from water) Daily intake

For example in drinking water, it is assumed: Estimated consumption: 2 liters / person / day Contribution: 10% of the intake is due to the drinking water. It is assumed that the food is also contaminated and may provide 90% of the maximum permitted intake

MAC = ADI x 10 mg/L100 x 2L

NOAEL (mg/Kg/day) x 70 (60) Kg body weight/person x 10/100 Contribution (mg/Litre)

UF (10-100-1000) x 2 Litre /day

NOAEL x 70 (60)(mg/Litre)

UF x 2 x 10

EXAMPLE: A PESTICIDE for post-harvest thiabendazol, NOAEL =10 mg /Kg/day UF=100. ADI= NOAEL/UF =10/100 = 0,1 mg/Kg/day For adult of 60 Kg bw = 0.1x60= 6 mg/day

Recommended limit (under tox criteria): <10% ADI: 6*10/100= 0.6 mg Assuming 2L consumption: =0,6/2 = 0,3 mg/L = 300 ug/L

EXAMPLE: A PESTICIDE for post-harvest thiabendazol, NOAEL =10 mg /Kg/day UF=100. One experts suggested adding an additional safety factor of 3 or 10 due to some uncertainty in the carcinogenicity study but it was not agreed by most other experts. In that worse case: F=300 or 1000 ADI= 10/1000 = 0,01 mg/Kg/day

0,6 mg/day (adult 60 Kg) Recommended limit (under tox criteria): <10% ADI: 0,6*10/100= 0.06 mg =0,06mg/2L = 0,03 mg/L = 30 ug/L Then the conclusion is between Worse case : 30 ug/L Stardard criteria: 300 ug/L

SOME CRITERIA for limits in water (1) SAFETY (1). Based on a threshold. Establishing:

· Safety dose (no effect NOAEL) · safety factor (AF, UF) · acceptable daily intake (IDA) =NOAEL/AF) ·Accepted allocation of water (10% and 2 L daily water consumption in adult 60Kg)

(2) (3) (4) (5) (6)

Most of recommended guide values of WHO Most of water quality parameter in European Directive

Are based in this criteria:

WHAT IS SAID IN THE EUROPEAN DIRECTIVE?

Our estimation is 300 g/Litro (by toxicological criteria under treshold) The regulation is 3,000 time lower

OBVIOUSLY OTHER CRITERIA IS USED FOR PESTICIDES

All pesticides has the same máximum limit: 0,1 g/Litre

examples of guide values by WHO

Guideline value (μg/litre) aldicarb 10 aldrin/dieldrin 0.03 atrazine 2 bentazone 300 carbofuran 7 chlordane 0.2 chlorotoluron 30 cyanazine 0.6 DDT 2propanilo 20 methoxychlor 20 lindane 2 permethrina 20 DICHLORPROP 100 2,4-DB 90 FENOPROP 9 Simazina 2

WHO guides

Values per individual substance (Toxicology criteria) Values in the range of units to hundreds g/L (except 3 not used with 0,03 g/L)

European Directive All pesticides the same limit

0,1 g/L (based in analític criteria)

PESTICIDE IN EUROPE Very simple: No function in water, no needed in water It is possible to avoid by good agricultural practice Consequently: European citizens DO NOT want pesticides in drinking

water. (Criteria based in SOCIAL SENSITIVITY) How controlling concentration = CERO? Legal limit: Based on a limit of detection/quantification

(Technical criteria based on analytical chemistry)

METHODS FOR ANALYTICAL CONTROL The methosd should be able to: Detect and measuring <10 % de legal limit With a error <10%

For a limit of 0,1 ug/L (plaguicidas) The analytical method with error <0,001 ug/L (1ppt) (LD<0,01; LQ<0,02 ug/L)

SOME CRITERIA for limits in water (1) SAFETY (1). Based on a threshold.

(2) SEGURIDAD-2. (3) ANALYTICAL CRITERIA. Based on limit of quantification (4) SOCIOECONOMICAL (1): Social Sensitivity (5) (6)

special cases of Carcinogens (by genotoxic mechanism)

Scientifically consensus: NO THRESHOLD

Lower dose less probability BUT no cero, any dose has a probability Acceptable risk?

OMS acepta riesgo de 1 / 100.000 EUROPA acepta riesgo de 1 / 1.000.000

Risk 1/10,000 to 1/100,000 is considered of concern Risk higher that 1/10,000 unacceptable

SOME CRITERIA for limits in water (1) SAFETY (1). Based on a threshold. (2) SAFETY(2). Probabilístic

carcinogens by genotoxic mechanism (mutagenic) Excess of Risk <1/100,000

(3) ANALYTICAL CRITERIA. Based on limit of quantification (4) SOCIOECONOMICAL (1): Social Sensitivity (5) (6)

ACRILAMIDE, BENCENE and other Level regulated EU and WHO guide with probability of excess of risk <1/100.000 ARSENIC

10 μg/L (as WHO) (probability of excess on skin cancer 6/10.000) (unacceptable?)

No lethal cancer, The lung cancer Risk is lower than 1/million Severe problem in India-Bangla Desh Problems in Mexico and Chile In the future should be revised, BUT Technical-economical difficulties to be reduced

SOME CRITERIA for limits in water (1) SAFETY (1). Based on a threshold. (2) SAFETY(2). Probabilístic – carcinogens) (3) ANALYTICAL CRITERIA. Based on limit of quantification (4) SOCIOECONOMICAL (1): Social Sensitivity (5) SOCIOECONOMICAL (1): technical and economical viability (6)

HALOMETHANES (HALOFORMS) and OTHER HALOGENATED

Formed by reaction organic material and chlorine (CHLORINATION) Due to potential carcinogenicity level should be convenient to reduce Legal limit considered higher than convenient BUT We cannot eliminate chlorination Future strategy for decreasing organic material

CRITERIUM: Other priority measures for public health should not be compromised (WE NEED GURANTY OF MICROBIOLOGICAL HYGIENE IN WATER) (We need chlorination)

SOME CRITERIA for limits in water (1) SAFETY (1). Based on a threshold. (2) SAFETY(2). Probabilístic-carcinogens (3) ANALYTICAL CRITERIA. Based on limit of quantification (4) SOCIOECONOMICAL (1): Social Sensitivity (5) SOCIOECONOMICAL (1): technical and economical viability (6) COMPATIBLE WITH OTHER PUBLIC HEALTH MEASURES (as chlorination for disinfection)

RISK MANAGEMENT AND COMMUNICATION. What to do if the level detected is > 0,1 g/Litro?

(case A) level higher to level of risk (case B) level higher than the legal but no health risk Escenarios:

i) Tap water in a city/tawn of a. <1000 inhabitanys b. Between 1000 and 100,000 inhabitants c. 100,000 -1 million d. Magapoli of more that 1 million

ii) Bottle water iii) Wáter in a food industry

New chemicals EC Directive 67/548/EEC

Data requirement Directivae92/32/EEC Principles

(Criteria for evaluation) Directive 93/67/EEC

Technical Guidance Documents TGD, EC, 1993

A basic concept is that it should be based on the evaluation of data that are required and must be submitted by the “Notifier” (producer/importer that aims to put it on the European market for a given application)

Not in the European Inventoy of Existing Substance (EINECS) by Sept de 1981

Existing chemicals EC Reg. Consejo 793/93

Risk assessment principles EC Reg 1488/94

TGDs TGDs for EC Reg 1488/94

TGDs package for harmonization of the evaluation of News and Existing Chemicals TGDs EC, 1996

Those in the European Inventory (EINECS) by Sept de 1981 (n=100.106 substances)

Sustancias con normativas específicasNombre Nombre en ingles Referencia

Directiva de Plaguicidas Directive of Pesticides (orPPP Directive)

EC Directiva 91/414/EEC

Medicamentosveterinarios

Veterinary drugs EC Regulacion 2377/90

Aditivo alimentarios Feed additives EC Directiva 70/524/EEC

Aditivos de alimentos Food additives 89/107/EEC

Cosméticos Cometics SCP/803/90

Materiales de embalaje Packaging materials EC Directiva CSIPM/1025

Biocidas Biocides EC Directiva 98/8/EEC

TGDs en preparación

The concept of "EXISTING" and "NEW" Europe

It is applied in various fields (any chemical, pesticides, biocides, cosmetics, food)

Different security requirement would be applied temporarily

NEW substances:inmediate application of the new regulation

EXISTING substances:A calendar for adaptation to new regulationTRANSIENT PERIOD

Critical date

EXISTING BEFORE CRITICAL DATE NEWSAuthorization needs

PREVIOUSLY

to demonstrate safety and efficacy

The authorization to continue marketing remains.

Industry is required to demonstrate their safety and effectiveness to continue commercialization

It is only deactivated if AFTER the evaluated a risk is deduced

Limit of NotificationDate Directive or

Regulation

FIELD Fecha crítica Directiva o norma europea

Chemicals 1967 y 1981 Directive 92/32/CEE (mod Dir 67/548) (Dir 2001/58 28 mod)

Pesticides (agricuture)

1991 91/414/CCE

Biocides 2000 98/3/CE

Cosmetics 1976 y 2003 Dir 2003/15/CE (7ª modif Dir 76/768/CEE)

Foods 1997 Regulation (CE) 258/97 1829/2003 y 641/2004

The concept of "EXISTING" and "NEW" Europe

It is applied in various fields

REgulation REACH

Registration of CHemicals Evaluation

Autorization

REACH

1 Protection of human health and the environment 2. Competitivity of the chemical industry 3. Avoid fragmentation of the market 4. Increase transparency 5. International integration 6. Promotion of non-animal tests. 7. Accordance with international obligations of the EU

Objetives REACH

Table 1.1 The proposed schedule for the registration of 30,100existing substances (6)

Number ofsubstances

Volume (tonnesper annum)

Deadline forregistration

2600 >1000 end of 20052900 100-1000 end of 20084600 10-100 end of 201220,000 1-10 end of 2012

REACH

Asignación de responsabilidad a la industria

Cambio drástico de planteamientoLos ensayos costará >2.100 M€

Assignment of responsibility to industry.Drastic change of approach Testing industry cost > 2,100 M€

Important change

REACH

Toxicity in animal modelsand human data

No observed effect adverse effect level(NOAEL)

Acceptable daily intake

(IDA = NOAEL/UF)

Margen de Seguridad IDA/(Ingesta diaria) (MOS) (<1 hay riesgo) (>1 no riesgo)

Source and route of exposure

Contribution to daily intake

Daily exposure

EXPOSURE ASSESSMENT EFFECT ASSESSMENT

RISK CHARACTERIZATION

RISK ASSESSMENT TO HUMANS

Extrapolation(Uncertainty factor UF)

(Evaluationn factor)

Base set minus:45 (partly)6b,c,d,e7a-d8

Base set minus:4 (partly)5 (partly)6d, 1genotoxtest7a,b,c8

Base set plus:Reproductive toxicitySubchronic/chronic toxicityExtended genotoxicityToxicokineticsChronic ecotoxicityTerrestrial ecotoxicityExtended fatetests

Othertestsconsiderednecessary fora fullrisk assessment

1. Identitydata,methodsofanalysis

2. Quantity, functions, applications3. Precautionary measures,

emergency measures4. Physical properties5. Chemical properties6. Toxicological properties:

a. acutetoxicity(2 routes)b. skin/eye irritationc.sensitizationd.subacute toxicitye.genotoxicity(2 tests)

7. Ecotoxicological propertiesa. acutetoxicity(algae, fish,

Daphnia)b.inhibition bacteriac.ready biodegradabilityd.hydrolysis

8. Methods renderingthe substanceharmless

Base set minus:45 (partly)6b,c,d,e7a-d8

Base set minus:4 (partly)5 (partly)6d, 1genotoxtest7a,b,c8

Base set plus:Reproductive toxicitySubchronic/chronic toxicityExtended genotoxicityToxicokineticsChronic ecotoxicityTerrestrial ecotoxicityExtended fatetests

Othertestsconsiderednecessary fora fullrisk assessment

1. Identitydata,methodsofanalysis

2. Quantity, functions, applications3. Precautionary measures,

emergency measures4. Physical properties5. Chemical properties6. Toxicological properties:

a. acutetoxicity(2 routes)b. skin/eye irritationc.sensitizationd.subacute toxicitye.genotoxicity(2 tests)

7. Ecotoxicological propertiesa. acutetoxicity(algae, fish,

Daphnia)b.inhibition bacteriac.ready biodegradabilityd.hydrolysis

8. Methods renderingthe substanceharmless

The need for development of alternative methods is highlighted by dividing the research needs in two sections:

• Development of Alternative Methods

• Other priorities of research

Research and Validation

REACH

Supported regulation on animal protections. Animal should be replaced as possible

Lago Maiore. Ispra. ECVAM, ECB

http://tox.umh.es

http://remanet.net

RED ESPAÑOLA DE METODOS ALTERNATIVOS A LA EXPERIMENTACION ANIMAL

EFFECT ASSESSMENTToxicity in vitro and animal models

Toxicological properties.HAZARD IDENTIFICATION

Acceptable daily intake

(ADI/IDA = NOAEL/UF)

Extrapolation(Uncertainty/safety/factor, UF)

No effect level (NOAEL)

Dose effect relationship

CLASSSIFICATION and LABELING

Directive 67/548Reg. 1272/2008

RESTRICTIONS(independently of risk

ESTABLISHING CONCENTRATION LIMITS