Teil 3

Question 1

What are the 6 main principles before you start to think about a toxicological test?

Accepted Answer

1) Purpose of testing 2) Prior to testing: Nanoparticle characterization 3) Prior to testing: Choice of the test model, in vitro / in vivo testing 4) Nanoparticle dispersion/ Mode of application 5) Dosimetry and Dose Range 6) Other issues (e.g. interferences)

Question 2

a) What kind of purposes of test are existing for NP? b) What kind of guideline are existing and what is the main difference between them?

Accepted Answer

a) •Screening and Prioritization •Basic Science •Regulatory Purposes b) •OECD: adequate for testing and evaluation of chemicals => regulatory purpose •ICH: developed for the testing of pharmaceuticals => Screening, Prioritization and basic science purposes

Question 3

a) What are aspects of the extrinsic (system dependent) properties you might have to think about before testing? b) How can you differentiate the different views on stability? c) What are aspects of the intrinsic properties you might have to think about before testing? d) Which of the intrinsic properties determines the toxicology of TiO2 particles in the strongest manner?

Accepted Answer

a) •Chemical Composition (Impurities?) •Stability b) •Chemical (dissolution) •Physical (agglomeration) •Thermodynamic (Phase transition) c) •(altering) Size & Size distribution•Coating •Crystallinity•(altering) Surface Reactivity •Shape•(altering) Surface Area d) •Crystallinity (3 modifications: anatase, rutil and brookit => most toxic effects in anatase modification)•particle size: microform pigment and in nanoform transparent (more toxic)

Question 4

a) What are the main questions for choosing a test model? b) What are general recommendations before choosing a test model?

Accepted Answer

a) •Which (How many) parameters to assess? •What is the minimal requirement? (There may be endpoint specific different requirements) •Which techniques to use? (for most there are several possibilities) b) •1) Characterization should be as complete as possible. •2) If possible, different techniques should be applied. •3) Characterization of NP „as synthesized“ is only a starting point but clearly not sufficient! •4) Characterization in the relevant biological test matrix is needed as well •5) Stability of the NP inside the test system must be addressed

Question 5

a) What should you might think about before dispersing NP into a medium? b) Which methods do you know?

Accepted Answer

a) •method of dispersion can influence the toxicity•the stability of the system should be checked over time•NP surfaces or Proteins can be changed due to the high energy intake b) •ultrasonication based methods•stirring•vortexing

Question 6

a) Can you count the four 50%-Dose-Effect-Ratios? b) What are NOAEL and LOAEL? c) How is the margin of safety determined?

Accepted Answer

a) •ED50 => dose at which 50% of population therapeutically responds •TD50 => dose at which 50% of population experiences toxicity •LD50 => dose at which 50% of population dies •IC50 => dose at which 50% of the effect is inhibited b) •NOAEL: no observed adverse effect level => This is the maximum dose, which does not cause an adverse effect•LOAEL: lowest observed adverse effect level => This is the lowest dose which does show an adverse effectc) •difference between the dose-response curve for the therapeutic effect and toxic effect determines the “margin of safety”

Question 7

a) What is in vitro, in vivo and ex vivo? b) Why is a performance of test methods in ex vivo mode more advantageous than in in vivo? c) What can be measured in vitro? d) What is the main problem of these validated tests, if you want to test NP?

Accepted Answer

a) •In vitro: in cultured cells •In vivo: in living organism •Ex vivo: in organs or pieces of organs (but testing is done in a cell culture dish) b) •more options in testing are possible•adaption to in vivo is relatively easy•CaCo 2 conditions are reached c) Endpoints are always adverse •Cytotoxicity, Cell Viability, Cell Proliferation (e.g. LDH, MTT, colony formation) •Genotoxicity/ Mutagenicity (e.g. COMET or micronucleus assay, gene mutation assays) •Stress response (e.g. ROS formation) •inflammation (e.g. cytokine release) d) •there are no existing validated test for NP

Question 8

a) List at least two cell types of each kind! b) Why are cell lines also called “immortalized”? c) What kind of cell types are more sensitive: primary or cell lines? d) Is it possible to develop a in vitro test method with carcinogenesis as an endpoint?

Accepted Answer

a) Primary Cells => keratinocytes, monocytes Cell lines => HaCaT (immortal keratinocytes), THP-1 (monocytes), CaCo-2 (epithelial cancer) b) •the cell lines are growing endlessly c) •primary cells (Endpoint of testing is the cytotoxicity) d) •no, this can be only measured in vivo

Question 9

a) Can you briefly explain the difference between cytotoxicity, inhibited cell proliferation and cell viability? b) Cytotoxicity test are usually not so accurate as they should be. How can you distinguish between cytotoxicity and inhibited cell proliferation? c) Cell viability can be not observed only by counting of cells, because the cell division is not impaired. How can you distinguish between healthy cells and viable cells?

Accepted Answer

a) •Cytotoxicity: cell division is stopped => cells are dying•Cell Viability: cell division is normal, but the cells are in different stadia•Cell Proliferation: cell division is stopped => cells are staying at their same number b) •by using an in vitro dye method (e.g. LDH => Only necrotic effects will be observed) => only dead cells are dyed, so the counting should show the difference c) •by using the ATP test => due to their different stadia, the cells should have different ATP level

Question 10

a) What are common in vivo endpoints? b) What is the main problem, if you think of the endpoints in vitro studies?

Accepted Answer

a) •General Toxicology •Dermal Toxicity (Irritation/ Photoirritation => usually this is done in vitro nowadays) •Inhalation Toxicity •Neurotoxicity •Immunotoxicity => e.g. Sensitization (accepted in vitro tests exists) •Developmental & Reproductive Toxicity •Effects on the Endocrine Systeme•Genotoxicity (accepted in vitro tests exist) •Carcinogenicity •Toxicokinetics b) •for many endpoints no in vitro alternative exists

Question 11

a) If we are thinking of the study procedure: What kind of differences do you know? b) What kind of difference do we make between animal species?

Accepted Answer

a) •Inhalation=> inhalation studies (whole body chambers or head/ nose only) •Oral (Oral gavage or voluntarily Uptake via food/drinking water •Dermal => occluded, semi-occluded, non occluded b) •rodent studies: mice, rats, guinea pigs •non-rodent studies: mini-pigs, dogs

Question 12

How long is the study duration of: a) acute studies? b) sub-acute studies? c) sub-chronic studies? d) chronic studies? e) What kind of difference is important for the application of material?

Accepted Answer

a) • short term effects (typically up to 14d) => typically single dose studies to determine maximum tolerated doses, for dose finding for longer studies or to initially determine possible target organs b) •28d c) •90d d) •should represent „the life span of an animal“ (depends on animal type typically 12 month rats, mice) e) •single doses or repeated doses

Question 13

What is a proper design for a test report?

Accepted Answer

1) Information about the test substance (composition, purity, phys.-chem properties etc) 2) Vehicle (which vehicle was used, including a justification) 3) Test animals (species, strain, number, age, sex, start weight etc.) (information on housing conditions) 4) Study protocol (which guideline) this includes single/ repeat dose, application route, application frequency/ duration postexposure observation time what was analyzed and how 5) Results and Interpretation

Question 14

What kind of read out-parameters are usually used in in vivo studies?

Accepted Answer

Mortality Body weight and weight of organs Clinical parameters: Hematology, Urine analysis Histopathology

Question 15

a) What should you think about before choosing a in vitro-test system? b) Why are 95% of the results in in intro tests false positive?

Accepted Answer

a) •Which application route is relevant for your NP of interest? •What is a relevant cell model/organ that is represented by your test system? •What is the origin of the cells (which species)? •Cell line (which) or use primary cells? (Some cell lines may be of tumor origin) •Single cultures or co-cultures? •Differentiated cells? 3D cell cultures? b) in reality they're defence and repair mechanisms in the organism, so they're not adapted in vitro

Question 16

What is depending on choosing a relevant cell model?

Accepted Answer

•relevant for the target organ •consider species differences •consider that many cell cells are of tumor origin •consider relevant application route (if possible) •consider relevant biofluid/ corona

Question 17

a) Can you briefly explain the principle of the Sulforhodamine B-test? b) Can you briefly explain the principle of the Crystal Violet-test? c) Can you briefly explain the principle of the Neutral red-test?

Accepted Answer

a) •the cells are dyed with the marker → measurement with fluorescence spectroscopy → concentration of absorbed dye is linear to the number of cells per “Well”•indication for the Cell Proliferation (Total protein synthesis rate) b) •same as Sulforhodamine test c) •neutral red can be only incorporated from living cells into the lysosomes → concentration of absorbed dye is linear to the number of cells per “Well” •lysosomal activity can be analyzed as well the membrane permeability

Question 18

a) Can you briefly explain the principle of the MTT test? b) What kind of inference can you draw from a MTT test? c) What kind of drawbacks are existing and can you bypass them?

Accepted Answer

a) •Measures enzymatic activity in mitochondria => MTT reduction: quantity of formazan (reduction product of MTT) is proportional to number of living (viable) cells b) •Viability assay => the darker the dye of the cells, the higher is the viability c) •solubility: a dilution of the dyed molecular is needed => XTT, MTS and WTS are eluding this step by forming a water-soluble product (otherwise a measurement is not possible)•robustness: NP can have an influence in the reduction of MTT => this must be tested before (if there is an impact => other assay)

Question 19

a) Can you briefly explain the principle of the LDH release-test? b) What kind of inference can you draw from a LDH test? c) Is it possible to perform a LDH test in vivo?

Accepted Answer

a) •When cell membranes are damaged → intracellular enzymes such as LDH can be released → transforms pyruvate into lactate → Diaphorase is using the new originated NADH-H to reduce the dye to a formazan (highly absorbent) b) •Membrane Permeability => LDH activity is a surrogate for the damaged cells after NP exposure (Cell viability can be assessed) c) •this is also possible

Question 20

a) Can you briefly explain the principle of the ATP-test? b) What kind of inference can you draw from a ATP test? c) Is it possible to perform a ATP test in vivo?

Accepted Answer

a) •ATP is released over cell dying → Luciferase uses the ATP to form a luminescent → the emission of light is measured (linear to the ATP level) b) •the impact of cell toxicity can be assessed c) •this is also possible

Question 21

Which of these sentences is wrong? 1) Not all carcinogens are genotoxic compounds. There are also non-genotoxic carcinogens. 2) Not all mutagenic agents are genotoxic-but all genotoxic agents are mutagen. 3) Not all genotoxic agents are mutagens- but all mutagens are genotoxic agents. 4) All damages of DNA are irreversible.

Accepted Answer

Which of these sentences is wrong? 1) Not all carcinogens are genotoxic compounds. There are also non-genotoxic carcinogens. 2) Not all mutagenic agents are genotoxic-but all genotoxic agents are mutagen. 3) Not all genotoxic agents are mutagens- but all mutagens are genotoxic agents. 4) All damages of DNA are irreversible.

Question 22

a) What kind of genotoxicity is usually measured employing the standard test methods? b) Why is the other kind of genotoxicity not so important to be tested? c) What is the difference between an indicator and a mutagenicity test method?

Accepted Answer

a) •secondary genotoxicity => implies a pathway of genetic damage resulting from the oxidative DNA attack by reactive oxygen/nitrogen species (generated during particle-elicited inflammation) b) •Primary genotoxicity is defined as genetic damage elicited by particles in the absence of pulmonary inflammation (barely happens) c) •indicator test => primary DNA damages (theoretically reversible) → detection through Comet-Assay•mutagenicity test => secondary DNA damages (irreversible) → detection through Re-or forward mutation (Ames-Test) or by observing aberration of chromosomes (MN-Test)

Question 23

a) Can you briefly explain the principle of the comet test? b) What kind of inference can you draw from a comet test? c) What is the main pitfall in interpretation of the results?

Accepted Answer

a) •can be used for cells containing a nucleus → cells are lysed in a salt fluid (destruction of the nuclear envelope) → proteins, RNA, membranes and cytoplasmic and nucleoplasmic constituents migrating through a electric field of agarose → chromosomal DNA in it’s entirety is to big to migrate in a electric field → only possible if the DNA is broken, then it can migrate in a electric field (DNA pattern resembles a comet)→ by employing fluorescence microscopy the dyed damaged cells can be analyzed b) •Clastogens (poisons which are causing a chromosome aberration) can be determined by counting the chromosomes which are disrupted or broken c) •usually 2-5% of the cells are dead before performing the comet assay => must be observed by use of a cell model

Question 24

a) Can you briefly explain the principle of the ames test? b) What kind of inference can you draw from a ames test?

Accepted Answer

a) •bacteria are spread on an agar plate with small amount of histidine (growing for initial time) → a point mutation is a characteristic of auxotrophic bacteria → they can’t produce an Histidine, so they require it for growth → by exposure with a potent mutagen a specific amount of bacteria return to a "prototrophic" state, so that the bacteria can grow by producing their own Histidine (occurs through point-mutation or frame-shift) b) •mutagenicity (frame-shift or point mutations) of a substance is proportional to the number of colonies observed

Question 25

a) Can you briefly explain the principle of the micronucleus test? b) What kind of inference can you draw from a micronucleus test? c) Can you perform the MN in vitro or in vivo?

Accepted Answer

a) •mouse bone marrow erythroblast develops into a polychromatic erythrocyte → the main nucleus is extruded from the erythrocytes → through an impact of mutagens, chromosomes in whole or fragments of them are remaining in eth erythrocytes (called micronuclei) → Visualization of micronuclei is facilitated in these cells because they lack a main nucleus => increase in the frequency of micronucleated erythrocytes in treated animals is an indication of induced chromosome damage b) •Aneugenic Effects (cause aneuploidy, i.e. altering the number of chromosomes) and clastogenic events (breakage of chromosomes) can be observed => genotoxicity and mutagenicity c) •can be performed in living cells (in vivo) or in vitro in cell lines (e.g. CHO, V79, TK6) or in primary cells (z. B. human lymphocytes)

Nanotoxikologie (Fach) / Teil 3 (Lektion)