Toxicology Assessment cont. (Lectures 19-20)

how are TTC values calculated

--613 chemicals with 2,941 NOAELs and good supporting toxicological data are assigned to a class based on their structures (broad range of industrial, food, environmental, agricultural, pharmaceuticals and consumer product chemicals) --the most conservative NOAEL selected, based on the most sensitive species, sex and endpoint selected --TTC value calculated for each of the 3 Cramer classes (5th percentile NOAEL calculated for each class, 100 fold uncertainty factor used)

challenges to toxicology

-->100,000 industrial chemicals, although most used in very small quantities --in recent decades Europe has tested some 200-300 new chemicals /year --toxicologists do not have the appropriate tools: high-throughput methods, or alternatives to animal testing

prenatal reproductive toxicity assay (>10 and >100 tonnes): what information does it give

--OECD 414: a prenatal developmental toxicity study --information on the effects of repeated oral exposure to a substance during pregnancy --maternal and fetal health is assessed

evaluation under REACH involves...

--a requirement for experimental animal studies --an important ethical principle of animal use in research is that alternatives to live animals should be used whenever possible --this principle is explicitly stated as part of REACH and similar new US legislation --3Rs

carcinogenicity testing: how are tests conducted

--conducted over the greater period of the animal's life (2 years for rats) --3 doses: maximum tolerated dose plus 2 lower doses (also, a vehicle control) --50 males and 50 females per group --diet formulation very important --over 2 years, body weight, food and water consumption, haematology and clinical biochemistry are measured --possibly an interim kill at 12 months (gross necropsy and histopathology) --same endpoints as subchronic studies (interim and terminal tests)

when is TTC not used

--for substances for which EU requires submission of toxicity data --when sufficient data are available for a risk assessment, these data should be used

short term toxicity test endpoints

--functional observations: on the 4th exposure week, sensory reactivity to stimuli of different types, grip strength, motor activity --body weight and food/water consumption --haematology --optional end points; urinalysis (protein in urine = kidney damage), oestrus cycle, T3, T4, TSH levels --gross necropsy (full, detailed, all animals), with organ weights

what does carcinogenicity testing allow scientists to do

--identify target organs --tumor dose-response relationships --identify NOAEL --extrapolation of carcinogenic effects to low dose human exposure levels --provision of data to test hypotheses regarding mode of action

subchronic testing: what information does it provide

--information on the possible health hazards likely to arise from repeated exposure over a prolonged period of time covering post-weaning maturation and growth well into adulthood --information on the major toxic effects, indicate target organs and the possibility of accumulation --an estimate of NOAEL --exposure which can be used in selecting dose levels for chronic studies and for establishing safety criteria for human exposure

non-mammalian models

--invertebrates, nematodes (Caenorhabitis elegans), hydra, insects (Drosophila) --vertebrates: fish (Danio rerio)

extended one-generation reproductive toxicity study (OECD 443)

--males and females mater after 5 weeks to produce F1 generation --F1 generation in utero from 7-9 weeks, weaning from 9-12 weeks --after weaning, animals divided between 3 cohorts; females are sacrificed for gross necropsy and histopathology cohort 1: 20 males and 20 females, reproductive toxicity assessed cohort 2: 10 males and 20 females, neurotoxicity assessed cohort 3: 10 males and 20 females, immunotoxicity assessed

replacement disadvantages

--models are dependent on pre-existing (correct?) information --in a live organism, all variables in physiology and pathology are not known --research on new biological processes must utilize a living organism at some point

reduction: methods

--performing pilot or sighting studies to determine some of the potential problems before numerous animals are used --designing a study to utilise animals as their own controls --gathering maximum amount of information from each animals --ensuring statistical power --minimising variables such as disease, stress, diet, genetics, etc that may effect results --performing appropriate literature searches and consulting with colleagues to ensure that experiments are not repeated --using appropriate species of animal so that useful data is collected

read-across, TTC, toxico-geomics, systems toxicology and structure activity relationships

--prediction from existing data on toxicity of chemicals. --extrapolation to chemicals of unknown toxicity based on physical and chemical similarities. --goal is not to eliminate in vitro or in vivo testing, but to make testing more focused

target organ toxicity can be assessed through...

--primary cell cultures (ex. blend up liver, release hepatocytes that act sort of like liver for 5-7 days) --established cell lines (HeLa) --cell culture models (pulmonary cells, liver cells, blood cells (erythrocytes, granulocytes, platelets), neuronal cell cultures) --organ culture (perfused heart, brain slices, perfused kidney/renal slices/tubule cells, thymic culture for immunotoxicity)

mammal mutagenicity tests: what kinds of cells are used

--primary cells difficult to culture with poor cloning ability --established cell lines: Chinese hamster ovary and mouse lymphoma cells --these cells have a limited xenobiotic metabolism --abnormal; usually show aneuploidy

in vitro eye irritation assay

--reconstituted human corneal epithelium (RhCE) --in vitro RhCE models are made from human-derived cells, which are cultured on specially designed cell culture inserts --these cells form a multi-layered structure which closely models the corneal epithelium --test materials are applied topically, just as they are in vivo --the viability of RhCE is assessed using the MTT assay (see how many cells are alive vs. dead)

Russel and Birch's "3Rs" (1959)

--replacement --reduction --refinement

replacement

--replacing "higher" with "lower" animals (microorganisms, eggs, reptiles, amphibians) --inanimate models for dissection to teach anatomy of animal, mechanical or computer models, audiovisual aids, or in vitro modeling

mammal mutagenicity tests

--resistance to toxic purines, 8-azaguanine, 6-thioguanine (HGPRT) --resistance to toxic pyrimidines (thymidine kinase, TK) --sister chromatid exchange assay

TTC values are established by

--using chemical structures to grouping experimental toxicity data from animal bioassays --applying a probabilistic methodology to define a threshold such that there is a low risk of an adverse effect at exposures below that threshold

replacement advantages

--utilizing pre-existing knowledge for teaching --applying known principles to new systems to look for similarities --using less expensive animals/models to screen large numbers of agents for toxicity or mutagenicity

determining route, structure and risk

--volatile chemicals showing increased risk via inhalation route --reactive chemicals (epoxides) equally dangerous --aromatic hydrocarbons much more dangerous by the oral route

when is TTC used

--when chemical structure of substance is known and where human oral exposure can be estimated to be relatively low --when there is limited chemical-specific toxicity data --for substances with or without structural alerts for genotoxicity and for cancer and non-cancer endpoints

5 classifications of carcinogens based on carcinogenicity testing

1) carcinogenic to humans 2) likely to be carcinogenic to humans: carcinogenic in animal models, knowledge of mechanism 3) suggestive evidence of carcinogenic potential: not all data can be integrated, carcinogenic in an animal model 4) inadequate information to assess carcinogenic potential 5) not likely to be carcinogenic to humans: data suggests that it is not carcinogenic

>1000 tonnes of chemical per year tests (2)

1) carcinogenicity testing 2) hazard identification for carcinogens

>10 tonnes of chemical per year tests (5)

1) in vivo/in vitro eye irritation assays (Draize, RhCE) 2) mammalian mutagenicity test 3) HPRT assays 4) short term toxicity testing (28 days) 5) reproductive toxicity assay

>100 tonnes of chemical per year tests (2)

1) subchronic testing (90 day test) 2) reproductive toxicity assay (prenatal and one-generation)

reproductive toxicity assay

10 males and 12 females per group 3 doses and 1 control --dose all animals before breeding, so there is opportunity for damage --repeated dosing of animals for 77 days, repeated measurements of body weight, food consumption, oestrus cycle --males sacrificed earlier: gross necropsy and histopathology performed --pups: litter size, sex, and abnormalities noted --pregnant rats and non pregnant rats: gross necropsy and histopathology at 77 days --understand teratogenicity

prenatal reproductive toxicity assay (>10 and >100 tonnes): how is the study carried out

20 females per group 3 doses + control over 5 weeks... weeks 1-2: mating weeks 3-5: repeated dosing terminal: foetus analyzed for sex, weight, and abnormalities, pregnant rats sacrificed one day before expected delivery for gross necropsy and histopathology over the weeks, body weight, food consumption, morbidity, and mortality are assessed

short term toxicity testing

28 day test --determination of oral toxicity using repeated doses, after initial information on toxicity has been obtained by acute toxicity testing --one species, male and female exposed by appropriate route at 3 dose levels, 5 animals per group (may be an issue determining dose response curves with 3 different dose levels) --USUALLY oral route, appropriate route determined by likely route of human exposure

subchronic testing

90 days one species, male and female exposed by appropriate route, 10 animals per group and 4 groups repeated doses

mutagens damage...

DNA (heritable changes)

how many generations can be tested in reproductive toxicity tests

F1, F2, F3.... some toxicants have been shown to have adverse effects generations down the line

mammal mutagenicity tests: HPRT assays

HPRT (hypoxanthine phosphoribosyl transferase) metabolizes 6-thioguanine to a toxicant (makes a protoxicant toxic) --mutations in HPRT gene causes resistance to the drug --chinese hamster ovary cells (CHO) have a single copy of HPRT (haploid) --CHO cells treated with 6-thioguanine die --cells that have been exposed to a mutagen may have a mutated HPRT and so become 6-TG resistant, forming a colony --cells in image are stained with crystal violet to see resistant colonies

TTC decision tree

This modified TTC approach includes two extra steps: the first asks whether there are chemical structures associated with genotoxicity in the chemical in question. The second asks whether the chemical in question is an organophosphate. However, the logic is: what is the chemical's structure and does it resemble a known toxicant, if so, what is the estimated intake and does that intake exceed a threshold determined from the NOAELs of known, similar toxicants? These "simple" questions lead to two outcomes: there is either a low risk of toxicity being observed at the estimated exposure level, or further toxicity testing is required to properly determine the risk. Notes: Low risk of toxicity in the case of genotoxins is at the level of cancer risk less than 1 in 106. Estimated intake assumes body weight of 60 kg.

advantages and disadvantages of in vivo testing

advantages: clearly defined genetic constitution, controlled exposure, detailed necropsy examination disadvantages: metabolic differences, mode of action

advantages and disadvantages of in vitro

advantages: reduced animal suffering so fewer ethical concerns, faster, cheaper, experiments more specific disadvantages: reductionist and so relevance to organism may be unclear

three Cramer classes

based on chemical structures with different TTC values for each class

mutagens can be...

carcinogens

most toxicity tests are currently on...

cells from experimental animals, some human (occupational exposure)

carcinogenic risk does not necessarily correlate with...

class --a known carcinogen present at very low levels possesses less risk than a likely carcinogen present at high levels

alternatives to skin and eye tests

eye: corioallantoic membrane (chickens) skin: cytotoxicity (mouse fibroblast), keratinizing and stratifying (mouse teratoma)

in 2007, US National Academy of Sciences called for a major shift in toxicology to

focus on in vitro studies to minimize suffering and cost of tests

TTC values describe

generic human chronic exposure thresholds

elements of haematology

haematocrit, haemoglobin, erythrocyte count, reticulocytes, total and differential leucocyte count, platelet count, blood clotting

why is it important that CHO cells are haploid for HPRT

if diploid, would need 2 mutational events to show mutation

what kind of information does short term toxicity testing provide

information on the possible health hazards likely to arise from repeated exposure over a relatively limited period time, including effects on the NS, immune, and endocrine systems

what does an AOP do (carcinogens)

integrates data on the effects of a toxicant at a molecular, cellular and organism level

subchronic testing: what parameters are assessed

interim tests: appearance, eyes, food, water, bodyweight, haematology, blood chemistry, urinalysis terminal tests: necropsy, tissue weights, examination, histopathology

histopathology tests

look for evidence of toxic effects

refinement

maximize data from animals

glyphosphate

may be carcinogenic? different organizations decide on this based on data integration mechanism data (2019) shows that glyphosphate is a carcinogen, but maybe not at low doses mice given MHC gene to make them more susceptible to mylomas --glyphosphate upregulates gene production in B-cells; see high incidence of myloma

in vitro mutagenicity tests

mutagenicity tests identify toxicants with the potential to cause genetic damage in germ and somatic cells --prokaryotes (S. typhimutium) --eukaryotes (like chinese hamster ovary cells)

reduction

reduction of animal suffering --identify pain and distress and prevent/relieve it --set earliest possible experimental endpoint --adequate training prior to performing procedure --ensure procedures to be performed are reasonable for that species --use appropriate analgesics and anaesthetics for harmful procedures --perform appropriate post-surgical care including thermoregulation and fluid balance

the cost of toxicology (REACH)

reproductive toxicology is the most costly, and uses the most animals

organ on chips

shows evolution of in vitro cell culture --cells can be manipulated so they stick to each other instead of to a piece of plastic, acts more like the organ

describe Cramer class I

simple chemical structures that suggest low oral toxicity. For example, normal constituents of the body (excluding hormones); acyclic aliphatic hydrocarbons; common carbohydrates

describe Cramer class III

structures do not permit a strong initial presumption of safety or may even suggest significant toxicity. For example, structures that contain elements other than C, H, O, N or S certain benzene derivatives; aliphatic substances with more than 3 types of functional groups

describe Cramer class II

structures that are less innocuous than Class I substances, but the structures do not suggest toxicity. For example, common components of food; substances with no functional groups other than alcohol, aldehyde, side-chain ketone, acid, ester

in vivo eye irritation assay: Draize test

tested to assess prospect of permanent blindness; specifically called the Draize test --performed in rabbits --administration of toxicant into the conjunctival sac and graded after 24 hours --most criticized on grounds of animal welfare --however, continues due to regulatory requirements for assessing visual impairment

as the amount of chemical used per year increases...

tests become more sophisticated and involved

carcinogenicity testing: what is it

the identification of the carcinogenic properties of a chemical, resulting in an... --increased incidence of neoplasms --increased proportion of malignant neoplasms (same amount of neoplasms as expected, but more are malignant than expected) --a reduction in the time to appearance of neoplasms

when to use linear/nonlinear extrapolation

the mode of action is used to decide whether linear or nonlinear extrapolation is used --linear extrapolation if mode of action is by direct DNA reactivity or when available evidence is not sufficient to support a nonlinear extrapolation --nonlinear extrapolation if there is sufficient evidence to support a non-genotoxic mode of action

TtC approach

threshold of toxicological concern --pragmatic screening and prioritization tool for use in food safety assessment

structure activity relationships

what structural features of the molecule contribute to, or detract from, the desired biological activity of the molecule of interest

thymus

where T cells are educated