W507 – Dose response and toxicity testing

“In all things there is a poison, and there is nothing without a poison. It depends only upon the dose whether a poison is poison or not.” Paracelsus (1493 - 1541)

Dose response curves Dose response relationships describe the effect on an organism caused by differing levels of exposure (or dose) Dose levels are usually expressed in mg/kg body weight of the test animal for solids and mg/m3 or parts per million for aerosols/vapours These levels can be plotted on a graph against the response The dose response curve is a valuable tool to understand the levels at which substances begin to exert adverse effects and the degree of harm expected at various levels

Dose response curves Dose response curves can show a number of points including: The ‘no effect level’ where no effect occurs or no effect is detectable The threshold dose of the substance – the level at which the effect starts to occur The levels at which the effect occurs in a set percentage or all of the test animals

The dose-response curve threshold? It is possible to ‘overdose’ on large volumes of water A 10g dose of caffeine causes convulsions and vomiting (1 cup of coffee ~ 150 mg caffeine A fatal dose of salt is estimated to be ~ 250g

Dose response curves Particular points on dose response curves include: LD50 (Lethal dose, 50%) the dose that kills 50% of the test population LC50 (Lethal concentration, 50%) the concentration that kills 50% of the test population TD50 (Toxic dose, 50%) the dose that causes a particular effect in 50% of the test population TC50 (Toxic concentration, 50%) the concentration that causes a particular effect in 50% of the test population

The dose-response curve 100% LD50 = 50% of species exposed to dose die (Oral route) measured in mg/Kg LC50 = 50% of species exposed to concentration die (Inhalation route) measured in ppm or mg/m3 NOAEL – Highest dose at which there is No Observed Adverse Effect Level. Some dose response curves may not have a threshold, starting at zero. % mortality Dose threshold? 50%

No observed adverse effect level (NOAEL) Experimentally derived value – highest level/dose at which no adverse effect observed in the test species Lowest observed adverse effect level (LOAEL) Lowest dose/concentration that causes any observed effect Other terms include: LD/LCLo (Lowest dose/concentration at which death occurs) TD/TCLo (Lowest dose/concentration at which particular effect occurs) – effectively the same as LOAEL As values usually established in species other than humans, various safety or uncertainty factors are applied before this data is used in the establishment of workplace exposure standards

Threshold Dividing line between no-effect and effect levels of exposure It can be shown experimentally that thresholds exist, as for a given population low levels of exposure do not produce a detectable effect and as the dose increases the effect appears Thresholds vary between species Thresholds vary within species due to natural variability

Threshold Dose response curves plotted from data from experimental studies Curve drawn from a number of points, one from each exposure group More exposure groups – more accurate the shape of the graph Without infinite number of groups the precise shape of the curve cannot be known accurately For many substances the body can deal with the exposure without an effect occurring At the threshold the defence mechanism is overwhelmed for more susceptible individuals and the effect begins to appear Uncertainty on the shape of the curve is a particular concern at very low doses

Threshold Uncertainty on the shape of the curve at very low doses is a particular concern for carcinogens This has led to debate whether there is a threshold (no-effect) level for carcinogenic effects If no threshold the curve will go through the origin (zero dose) If there is a threshold the curve will cross the axis at some point greater than zero dose For carcinogens most experimental data has been derived at high dose levels – so extrapolation of the curve to very low levels is often difficult Low dose studies require too many animals to determine effects

Variability Within any group there are: Some susceptible individuals (hypersensitive) who are affected at low concentrations Some highly resistant individuals (hyposensitive) who are not affected at higher concentrations Vast majority of ‘average’ individuals It is important to recognise that some hypersensitive individuals may be in a work group and that they may suffer health effects at exposures below the recognised exposure standard Note: In animal testing studies the effects of variability are reduced as much as possible by using groups of test animals that are bred to be genetically very similar

Slope of the curve Another characteristic that can be examined is the slope of the curve Slope of the curve can vary widely between different substances Some substances show a steep curve (rise rapidly from threshold level to the ceiling level) Particular care needs to be taken as exposures just above threshold may produce effects in a large proportion of the group A relatively flat slope suggest the effect of an increase in dose is generally minimal and that there is much greater variation in the likelihood of the effect occurring in the whole exposed population

Slope of the curve

Toxicity testing To make judgements on risks and appropriate control measures information needs to be obtained on the hazardous properties of the substance This may be from studies of human exposures (epidemiological studies) – however, these often do not provide the full range of information required A range of other approaches have been developed which toxicologists use to investigate the toxicity of a substance Physico-chemical properties Similarities in chemical structure to other substances Usually more information is required and some experimental testing will be needed

Toxicity testing Toxicity testing on animals remains main approach Potentially controversial issue - progress has been made to replace animal tests, reduce numbers used and refine testing methods to reduce suffering Most toxicity tests use rodents (specially bred rats, mice, guinea pigs, rabbits) Animal testing usually carried out to established international guidelines to ensure they meet agreed standards and that results are acceptable to regulatory authorities Other approaches use cells or tissues in ‘culture’ (e.g. test tubes or Petri dishes) Known as in vitro (‘in glass’) methods

Toxicokinetic studies Undertaken to study how a substance is absorbed, distributed, metabolised and excreted from the body Studies involve administering a substance (often tagged with radioactive atom) to animals via one of the routes of entry Presence of substance (or metabolite) measured in various tissues over a period of up to a few weeks Considerable recent progress in computer aided modelling of toxicokinetics, particularly as a means of extrapolating from one species or route of exposure to another, and across a range of doses

Acute toxicity studies Aim is to provide information on what general toxicity might be seen if someone was exposed to a single, relatively high dose of a substance e.g. accidental poisoning Substance administered to groups of animals as single doses May be by inhalation, ingestion or dermal route Animals observed for up to two weeks after administration of dose for signs of toxicity After this period, organs of the animals examined for signs of damage as markers of toxic effects

Acute toxicity studies Historically these studies designed to derive LD50 or LC50 values These values are relatively crude measures of toxicity However, they are useful to make relative comparisons of acute toxicity of substances in order to rank them in terms of severity Have been used to derive hazard classifications Still find these values on Material Safety Data Sheets However, studies often overlooked more critical information e.g. toxic signs that might indicate how a substance exerts its effects Newer methods have been developed which use fewer animals and depend more on signs of toxicity, rather than just mortality These methods are becoming more common and are now part of regulatory schemes with established international guidelines

Acute toxicity studies Irritation studies Some substances can cause localised inflammation on contact with skin, eyes or respiratory tract In worst cases damage can be severe leading to corrosive destruction of the tissue Traditionally animals, usually rabbits, used for skin and eye irritation tests Significant efforts have been made to find alternative non- animal test methods for assessing potential for skin or eye irritation Internationally validated methods have been developed which use in vitro systems for skin irritation and corrosion

Acute toxicity studies Traditional irritation studies Skin irritation tests (Draize Tests) Substance applied to skin, covered for a few hours under a dressing which is then removed Skin assessed by looking for redness (erythema) and swelling (oedema) and a scoring system based on severity of reaction Used to provide a semi-objective classification of substances as irritants Eye irritation tests Substance applied inside lower eye lid Severity of response judged by scoring redness and swelling of conjunctiva and eyelids, opacity of the cornea and effects on iris over a period of up to 21 days

Sensitisation studies Skin sensitisation studies usually performed on guinea pigs A series of exposures applied to a part of the skin After a break from this induction phase (inducing the immune system to respond) another part of skin is exposed to a lower (non-irritant) dose of the substance If the substance has sensitising properties this shows as a skin reaction Reaction scored for severity – substance may be designated a skin sensitiser depending on proportion of test animals showing specified levels of response Newer approaches have been developed using mice and require less severe responses

Sensitisation studies Attempts have been made (usually using guinea pigs), to develop tests for predicting respiratory sensitisation potential However, no single validated method has been recognised For humans who show signs of skin sensitisation, ‘challenge’ tests can be performed under medical supervision to help diagnose the condition and identify the causative agent As for skin sensitisation, humans who show signs of occupational asthma can undergo ‘challenge’ tests to determine causative agents

Repeated dose toxicity studies Testing for effects of repeated exposure to a substance is most often carried out on rats, over varying periods of time 28 days – sub-acute testing 90 days – sub-chronic testing One or two years – chronic testing Exposure usually by oral route (less commonly by inhalation route) Three dose levels usually used Highest dose chosen to induce clear toxic effects Middle dose chosen to induce less effects Lowest dose chosen to induce no effects (Shorter term tests can act as a guide to select doses for longer term tests)

Repeated dose toxicity studies At end of exposure period a range of measurements undertaken on the animals together with an examination of body tissues and organs Range of tissues and organs examined increases with increasing length of exposure period Number of animals used is generally larger in the longer exposure period studies Main aims of these studies are to produce information on: Type of toxicity Potential target organs Dose-response relationships If possible, the No Observed Adverse Effect Level

Genotoxicity studies Wide range of tests Strategic step-wise approach usually adopted Initial in-vitro tests on bacterial cells to screen whether a substance may be genotoxic (Ames test) Ames test based on assumption that if substance is genotoxic to Salmonella bacteria used in test it may also be a carcinogen While some carcinogens do not give a positive Ames test (and vice- versa) the ease and low cost of the test make it valuable as a screening test Some tests also conducted in presence of liver extract to investigate possibility of genotoxic metabolites Other tests use yeast cells or fruit flies If all tests are negative, then usually the substance is not considered as possessing genotoxic potential

Genotoxicity studies If initial in-vitro tests show genotoxic potential further studies can be performed to determine if this potential could be expressed in animals Generally performed in mice One or two doses of the substance administered and bone marrow derived cells examined for evidence of genetic damage Positive results indicate potential to cause genetic damage and substance is potentially a carcinogen If genetic damage found in normal (somatic) cells further tests on germ cells (or offspring) undertaken to assess whether likely to induce heritable genetic damage

Reproductive and developmental toxicity studies Testing for a substance’s ability to cause toxicity to reproduction usually carried out on mice or rats Groups of male and female animals dosed with a range of doses Allowed to breed in pairs and compared with a control group Some of the offspring of these tests may be dosed and allowed to breed again (multi-generation studies) To investigate effects on developing offspring dosing may be carried out during period of pregnancy

Carcinogenicity studies Testing for carcinogenic properties is an extension of the chronic two-year repeated dose studies. Larger groups (50 male and female) of animals (usually mice or rats) receive two years exposure Control group also observed Highest dose usually chosen to cause a small toxic effect but not enough to cause excessive deaths At end of test (and whenever any animal dies) the animals are examined for any tumours and their type determined If significant increase in number of tumours compared to control group this may indicate the substance having carcinogenic properties in humans

Allergy assessment methods in humans There are a number of different assessment methods that are available for determining whether a person is allergic to a particular substance including: Lung function tests (spirometry) Challenge tests Skin prick tests Patch tests Serological tests (Blood IgE testing)

Lung function tests (spirometry) Undertaken to evaluate how well the lungs are working Assess conditions such as asthma, pulmonary fibrosis and chronic obstructive pulmonary diseases such as emphysema and chronic bronchitis Uses a spirometer to measure the amount (volume) and speed (flow rate) of air that can be inhaled or exhaled Results given as actual values or as percentage of ‘predicted values’ for given age, gender, height etc Results within about 20% of predicted values considered ‘normal’ One limitation – highly dependent on patient co-operation and effort

Lung function tests (spirometry) Common terms FVC – Forced vital capacity FEV1 – Forced expiratory volume in 1 second FEV1% - Ratio of FEV1 to FVC – in healthy adults this is usually about 75 – 80 % PEF – Peak expiratory flow

Challenge tests Spirometry can also be used as part of a challenge test Undertaken to determine whether sudden contraction of the bronchioles is as a result of exposure to a particular substance Can confirm specific substance causing the sensitisation Starting with small dose, the challenge involves increasing the dose of the substance in question Only performed under medical supervision

Skin prick allergy tests Usually first test recommended when allergy is suspected Simple, quick, inexpensive Gives information on all types of allergy including substances inhaled or ingested Suspected allergens mixed with liquid to form solution Up to 20 allergen solutions tested at a time by placing drop on marked position on forearm Top surface of skin pricked beneath each drop Skin observed for reaction – usually within a few minutes – reddening, itchy, swollen

Patch testing Skin prick testing introduces allergens into the body Used to test for allergies that do not necessarily occur on the skin Patch testing places substances on the surface of the skin (usually upper back) and aims to identify skin allergens Various test substances placed on the skin and covered for 48 hours At end of 48 hours, patch removed, skin examined and examined again after further 48 hours Irritant reactions are most prominent immediately after patch removed and fades over next day Allergic reactions take a few days to develop so is more prominent on day 5 than when the patch is first removed

Serological tests Involve analysis of blood serum for presence and quantity of specific IgE antibodies As quantitative measure of specific IgE in an individual For individuals taking antihistamine drugs For individuals with extensive skin disease such that prick tests are difficult to undertake However, invasive, relatively costly and results not available immediately Important that laboratories used take part in suitable external proficiency testing schemes