Principles of Occupational Toxicology 2 – Types of toxicity Tiina Santonen Paide 3.11.03

Types of Toxicity Acute toxicity Irritation and corrosivity Sensitization Repeated dose toxicity Genotoxicity Carcinogenicity Reproductive toxicity

Types of toxicity continues Mechanisms of toxicity: Direct local toxicity to tissues first in contact with the substance Systemic effects due to the absorption of the substance Mechanism may be relevant to all species or it may be species-specific Quite often with industrial chemicals the mechanism is unknown Does the mechanism of toxicity possess a threshold? (What is the shape of the dose-response curve like?)

Examples Sulphuric acid: strong acid, miscible in water corrosive, exposure to fumes may cause damage to the respiratory tract, and in repeated exposure in the respiratory tract cancers (laryngeal cancer) type and mechanism of toxicity: locally toxic at the first site of contact mechanism of cancer related to its destructive effect on respiratory tract epithelium threshold

Examples, con’t Cyanides Phthalates (e.g. Di(2-ethylhexyl) phthalate) toxic when absorbed into the body, mechanism well-known and apply to all species Phthalates (e.g. Di(2-ethylhexyl) phthalate) causes liver effects and liver tumours in rodents, however the mechanism of action (peroxisome proliferation) is known not to be relevant to humans

Dose-response D O S linear, E non-linear, no threshold threshold EFFECT

Dose-response relationship: lead decreased erythrocyte delta-ALAD activity increased zinc protoporphyrin anemia CNS effects decreased peripheral nerve conductivity Nervous paralysis, lead colics Adapted from Elinder C-G et al., Biologisk monitoring av metaller hos människa. Arbetsmiljöfonden, Uppsala, 1991

Information of the potential health hazards of chemicals is derived from... 1) Toxicological studies (in vivo, in vitro) 2) Case reports 3) Epidemiological studies

Toxicity testing Systematic toxicity testing started in 1960’s testing guidelines: OECD guidelines, EU guidelines for industrial chemicals GLP guidelines

Acute toxicity testing acute oral, dermal or inhalation toxicity provides information on acute health hazards likely to arise from acute exposure to the substance by the given route, and on the magnitude of acute toxicity of the substance usually these tests are made with rodents, dermal test quite often with rabbits LD50/LC50 values (dose level which is caused death to 50% of animals)

Acute toxicity testing, con’t principle of the acute LD50/LC50 tests: At least 3 groups of animals are exposed to different concentrations of the chemical, observations of effects and deaths are made, LD50/LC50 value is statistically derived value determined from the dose-response curve by using certain accepted extrapolation methods oral LD50 test is not used anymore, new substitutive tests are fixed dose test and acute oral class method which give an idea of the magnitude of the toxicity, but do not define the LD50 value

Irritation and corrosion Acute skin irritation/corrosion test (Draize test) usually albino rabbits are used, the test substance is applied to the skin of the rabbits and held under the semi-occlusive dressing usually for four hours. The degree of irritation is scored at specified intervals. NOT if the substance is a strong acid or a strong base (pH<2 or >11.5), or it has not caused skin irritation at maximal dosage in acute dermal toxicity test. Note: albino rabbits are usually more sensitive to the skin irritation/corrosion than humans

Irritation and corrosion con’t Alternative methods: Two in vitro skin corrosion –test included in EU test guidelines (rat skin TER assay and human skin model assay) Several promising in vitro skin irritation tests have been developed but have not yet been ready for systematic use in skin irritation testing

Irritation and corrosion con’t Acute eye irritation/corrosion test (Draize test) principle: the test substance is applied to the conjuctival sac of albino rabbit, the effects are observed and scored according to the severity of the effects. Repeated examinations at specified intervals NOT if the substance is a strong acid or a strong base (pH<2 or >11.5), or it is corrosive to the skin Note: albino rabbits are usually more sensitive to the eye irritation/corrosion than humans In vitro –methods for eye irritation testing has been developed, but have not yet been good enough to replace in vivo eye irritation test

Skin sensitization Traditional Guinea Pig models for skin sensitization guinea pig maximization test (GPMT) Buehler test New alternative: Local lymph node assay (LLNA) measures cellular proliferation in the lymph nodes draining the site of topical application of the substance more specific, objective and reproducible, gives more quantitative information

Repeated dose toxicity 28- or 90–day toxicity tests (usually oral or inhalation studies) provide information on health hazards likely to arise from repeated exposure by the given route of exposure throughout evaluation of every organ system interpretation of the results demands careful consideration

Special tests... e.g. neurotoxicity test evaluates potential neurotoxic effects of chemicals should be performed if there is a reason to believe that the substance may have effects on nervous system e.g. organophosphate pesticides

Genotoxicity the ability of the substance to induce genetic damage in cells In vitro tests: bacterial tests (e.g. Ames test); gene mutations mammalian cell gene mutation assays in vitro chromosomal aberration test

Genotoxicity con’t In vivo tests: e.g. bone marrow micronucleus test or chromosomal aberration test germ cell mutagenicity tests ability to cause genetic damage in germ cells

Carcinogenicity 2-year cancer bioassay usually rat or mouse are used, can be performed by any relevant route of exposure (usually oral or inhalation) should be performed if some indication that the substance could be carcinogenic laborous and costly, high number of animals needed

Reproductive toxicity 2-generation reproductive toxicity test evaluates the reproductive system as a whole over 2-generation, both male and female laborous and costly, high number of animals needed

examination of the offspring 21 days post partum killing and F0 male, treatment 10 wk F0 female, treatment 10 wk pairing T R E A M N birth of the offspring, external examination 21 days post partum unselected offspring killed, histopathology selection of F1 generation F1 animals paired littering, external examination of the offspring 21 days post partum killing and histopathology

Reproductive toxicity, con’t Developmental toxicity study (teratogenicity) treatment during the organogenesis, careful evaluation of fetal losses and malformations In interpretation attention is given to the severity of the effects and to the effects seen in fetuses without signs of maternal toxicity (Developmental neurotoxicity)

EU Classification and Labelling principles EU classification and labelling is very much based on the data obtained from toxicity tests

T+ Very toxic T Toxic Xn Harmful C Corrosive Xi Irritating F+ Extremely flammable F Flammable O Oxidizing E Explosive N Environmentally dangerous

Very toxic (T+) acutely very toxic chemicals labelling: e.g. oral LD50 is < 25 mg/kg or dermal LD50 <50 mg/kg LC50 is <0,25 mg/l for aerosols or <0,5 mg/l for gases or fumes labelling: R 26 (very toxic by inhalation) R 27 (very toxic in contact with skin) R 28 (very toxic if swallowed)

Toxic (T) Acutely toxic chemicals e.g. oral LD50 is bw 25 – 200 mg/kg, or dermal LD50 is bw 50 – 400 mg/kg LC50 is bw 0,25-1 mg/l for aerosols and 0,5-2 mg/l for gases or fumes labelling R23/24/25 (toxic by inhalation, in contact with skin or if swallowed) Cat 1 and 2 carcinogens (R45 or R49), mutagens (R46) and reproductive toxicants (R60, R61)

Harmful (Xn) e.g. acute toxicity: Oral LD50 between 200 –2000 mg/kg, or dermal LD50 bw 400 – 2000 mg/kg LC50 between 1-5 mg/l for aerosols and 2-20 mg/l for gases or fumes labelling R20/21/22 (harmful by inhalation, in contact with skin or if swallowed) if there is data showing that prolonged exposure can cause serious damage to health at the dose levels significantly lower than the dose levels used for acute toxicity classification (R48) ......continues in next slide....

Harmful (Xn), con’t Respiratory tract sensitizers (R42) Cat. 3 carcinogens (R 40, Limited evidence of a carcinogenic effect), cat. 3 mutagens (R 68, Possible risks of irreversible effects), and cat. 3 reproductive toxicants (R 62, Possible risk of impaired fertility or R 63, Possible risk of harm to the unborn child)

Irritants (Xi) and Corrosives (C) R-phrases: R36/R37/R38 and R41(risk of irreversible eye damage) skin sensitizers R43 Corrosive: R-phrases R34 or R 35 (causes burns or causes severe burns)

Classification and labelling Note: based on hazard, not on risk If there is a risk caused by these inherent characteristics of the chemical depends on the use and exposure potential In future: Global harmonization of Classification and Labelling