Toxicity Studies - Science topic

In general, regulatory bodies such as the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA) in Europe usually require comprehensive pre-clinical testing to ensure the safety of new products before they can advance to clinical trials. Toxicity studies are also performed. To determine the potential harmful effects of the encapsulated substance on various organs and systems of the body.

Hello Priyansha Singh

It was Marroquin et al. (2007) who proposed a model of HepG2 cells to mitochondrial toxicants by replacing glucose with galactose in cell culture media. You may want to refer to his paper attached below for more information.

Not every cell line is amenable to culture in galactose. Therefore, for mitochondrial toxicity studies, HepG2 cells are used.

Let me explain further.

When you carry out in vitro anticancer studies on immortalized cancer cells in media containing high glucose, the mechanistic studies investigating the role of mitochondria in cancer cells cannot be studied due to the Crabtree effect. The Crabtree effect decreases oxidative phosphorylation in response to increasing glucose concentration and allows cancer and proliferating cells to adjust their energy metabolism depending on substrate availability. So, the mitochondria are not sufficiently active for energy homeostasis in cancer cells in high glucose-conditioned media.

By replacing glucose with galactose in cell culture media, HepG2 cells are forced to produce ATP by shifting from the cytosol to the mitochondria, consequently making HepG2 cells vulnerable to mitochondrial toxicity. When considering that mitochondria play an essential role in biomass synthesis, including fatty acids, amino acids, and nucleotides, which are required for the growth and proliferation of cancer cells, it becomes essential to investigate the interactions of anticancer drugs with possible regions or structures in mitochondria by using HepG2 cells as they are vulnerable to mitochondrial toxicity.

So, when HepG2 cells are forced to rely on mitochondrial oxidative phosphorylation rather than glycolysis by substituting galactose for glucose in the growth media, oxygen consumption doubles in galactose grown HepG2 cells and their susceptibility to canonical mitochondrial toxicants correspondingly increases. Similarly, toxicity of several drugs with known mitochondrial liabilities is more readily apparent in aerobically poised HepG2 cells compared to glucose-grown cells.

So, for mitochondrial toxicity studies using HepG2 cells, you have to replace glucose with galactose in the cell culture media.

Best.

Hi Sweatha, I get the impression, reading your question that the chemicals are poorly soluble and are already coated on sand. If this is the case then I quite understand that by just putting the sand in a fish tank you may get uneven distribution of the chemicals and fail to reach water saturation of the chemicals with a large amount remaining undissolved on the sand. If you start using mechanical methods to increase the diffusion of the dissolved chemical fraction into water then you risk creating a physical disturbance in the aquaria that could have an adverse effect on you fish by clogging or damaging their gills. Fot this reason, I would recommend a two step process where you place the sand into a generator column prior to exposure to the fish. Ideally, you will allow the water (not deionised, but the mineral media you will use to expose the fish) to turn in the generator until saturation is reached. This process could take several days depending on the water solubility of the chemicals on the sand and the time for them to reach equilibrium with the water. The best way to know when your solution is ready to test is by analysing the chemical(s) which are on your sand in the water fraction and only stopping the process when you have reached equilibrium. There are a certain number of factors to consider when using this process: The columne is a closed circuit with water pumped around through the sand so volatility of your test substance is not a factor. On the other hand you may get some adsorption onto the glassware itself so make sure you put enough sand into the column that this factor is minimised. Next, beware the potential for autoxidation of the substance if you are turning the column for several days. Consider doing this preparation step in the dark or covering the column with aluminium foil. Consider also the potential for biodegradation so make sure that you use sterile apparatus. Once you have saturated tyour solution then you can allow the sand to sink to the bottom for an hour or so and transfer your solution directly to aquaria or (if only one test substance is present on the sand) you can serial dilute if the solution is highly toxic. If you are dealing with a mixture then you will have to use a slighlty modified technique by adding known quantities of sand to your column (e.g., 10 g/L, 5 g/L etc)) in separate generators. Hope that helps.

Correct. When plotted or calculated, the LC50 has the least variability of data compared to points near the outside like the LC90 or LC10. The LC50 has been used extensively by governments in regulatory work, but doesn't have much meaning to fish farmers. This is why my research includes the NOEC (No Observable Effect Concentration). See Straus et al 2018 - Toxicity of PAA to fish - variation among species and impact of water chemistry.

Good toxicity studies are more difficult to do than most people realize and many of them are done incorrectly or do not investigate the correct parameters. Good luck!!

Hi Prabhat! I'm assuming you have seen no toxicity with your extracts up to at least 2000 mg/kg. I would suggest you do the following if your plant/extract has been used in any traditional system of medicine for any indication (this is very likely) 1)Identify the human dose 2) Calculate the animal equivalent dose using the appropriate conversion factor for rats/mice. 3) Since you need three doses consider that as the median dose and the other two doses as half and double respectively. This may increase your chances of seeing efficacy in your animal studies. All the Best!

(Nair AB, Jacob S. A simple practice guide for dose

conversion between animals and human. J Basic Clin Pharma 2016;7:27-31.)

The dose concentration selected for the chronic tests should be based on the results of the acute test. The concentration chosen should have a noticeable effect on the test organisms but not cause lethal effects. The selection of the appropriate concentration will depend on the toxicology of the substance being tested and the species used in the test.

Suppose the acute test results indicate that the substance has high toxicity. If the substance has low toxicity, the dose concentration selected for the chronic test can be higher. In that case, the chosen dose concentration for the chronic test should be at or below the no observed effect concentration (NOEC) identified in the acute test.

It is important to consider the purpose of the chronic test and the endpoints being evaluated when selecting the dose concentration. For example, if the test is focused on reproductive effects, a concentration that results in a noticeable impact on reproduction but does not cause lethal effects should be selected.

In general, choosing a range of concentrations that cover the range from the NOEC identified in the acute test to a concentration that causes noticeable effects is a general practice. This will allow for a more thorough evaluation of the chronic toxicity of the substance.

It is also important to consider the test species used in the acute and chronic tests. Suppose the species used in the acute test differ from those in the chronic test. In that case, the acute test results may not be directly applicable to the chronic test, and the dose concentration may need to be adjusted accordingly.

Not that I know of.

Even the ADMET study in computational work is a prediction, though it may be very close to what we see in vivo or in vitro analysis.

Hi,

As in your next question, both substances are described in the TGA document:

Please check the document by keyword "ALC" and Ctrl+F, the number of hits is 91.

Best regards

Tomasz

Mean body weight, Feed conversion ratio, and hematobiochemical analysis are some of the fundamental analyses that should be carried out following the administration of the test dose of plant extract in order to analyze its effect.

That way you have only a value for each group... and cannot calculate the average and SD. If you repeat it two times more (at least) then it will be better.

You can go for sas software using probit model.

what about the age why not using old rats rather than adult

Several OECD RDT guidelines / Technical guidance documents mention the opportunity of a limit test in the appropriate animal model. Earlier versions spend more words on dose spacing than the current versions (because nowadays we mostly have PD/PK data from dose range finders before performing the ultimate studies ). Because it seems that you have none, you should follow the guideline recommendations for: 1.) slight toxicity (e.g. body weight decrease not more than 20 %) in the high dose, 2.) a potential NOEL in the low dose and 3.) a mid dose evenly spaced in between. The dose spacing (from experience) therefore mostly equals a factor of 4-5. No guideline will actually provide you with pre-selected dose levels, as they are completely dependent on the substance and their properties, the selected animal model and the duration of treatment.

All the best

Geertje

Hello,

You should try the CALculate IBR Interface (Calibri, https://liec-univ-lorraine.shinyapps.io/calibri/) proposed by the Laboratory for Continental Environments (LIEC) of Lorraine University, it is a user-friendly website.

There are no guidelines for i.v./i.p. route. The OECD guidelines for oral route were developed for testing of chemicals, which were also used in pharmaceutical testing.

You need to decide the doses on the basis of exposures in inital PK studies, and further trials by yourself. ICH M3(R2) - "Non-clinical safety studies for the conduct of human clinical trials for pharmaceuticals" is relevant guideline for pharmaceuticals safety testing.

Hello Dennis, Check out https://doi.org/10.1021/acs.est.9b07657

Dear Olushola Awoyemi,

In general sigmoidal .... is the model. That means you have chosen a model, and it means that the selection has been evaluated by parameters assessing the fit of the model (eg AIC, BIC etc). So you have appropriate justification of the model based on relevant criteria. If you decide that best model is for example sigmoid Emax model with a baseline effect parameter (WNL model 106) then 0 is not a defined value. In many studies nonzero baseline effect is present (heart rate for example). If it your case and in your model baseline nonzero effect exist it cant be stated that 0 is the control. 0 could be element of the model or not its depend on your model justification and selection process. It should be your argument for discussion with Editor.

Best regards

Tomasz

As per the literature mangiferin slightly soluble in ethanol sparingly soluble in methanol and water

Practically insoluble acetone.

Yeah I was wondering the same thing, that the correlation is not good. Thanks for your input Thomas!

According to the USEPA 2000, oral reference doses (mg/kg/day) for several metals are follows. Cr (III)=1.5 , Fe = 0.7, Cu = 0.04, Zn = 0.3, As = 0.014, Cd = 0.001, Pb = 0.004, Ni = 0.91

Hello

The same answer of source article.

Because the tow are not in the same trophic level and the man present an important way of exposure.

Your problem is, in theory there is no threshold. Fortunately, for you governments usually set practical thresholds. Personally, I would not go above pico levels, but that's just my impression, nothing legal.p

It is possible, but highly unlikely base on current regulatory guidelines. For a given dose value, the human can have a better bioavailability and exposure than the animal model used preclinically. However, the preclinical models are dosed at several multiples until toxicity or maximum acheivable dose is reached. Human Ph1 trials start at a fraction of the NOEL reported preclinically. At these starting doses, exposures are compared to the animal data so guidance is available for the escalating dose studies. So it is unlikely that you would ever be in a position that human clinical trials were achieving exposures that were in excess of any reported for preclinical studies. If preclinical studies are not designed correctly, then it is a possible scenario that clinical doses could provide unexpected exposures but in that case the company has wasted much time and money and should rethink their position as a drug company.

The Up-and-Down procedures for oral toxicity begins with 3 rats and dose 2000mg/kg. Any case - use the OECD guidelines

Chemical materials leave a great negative and harmful impact on human beings life and causes the diseases too.

Hi, 6 months study is a good data to derive OEL, rest depends on the choice of your uncertainty/safety factors. You can check TWA data available in public domain for your compound (this is actually OEL, and is available for most of the compounds..mostly in safety data sheets or MSDS. If it is not available for any particular compound, then you can check TWA value for the analogues or compounds of similar class).

There is lot of literature available (I have attached some), which mentions the control banding (1-4 or 1-5) on the basis of OEL. The safety measures can be decided on the basis of banding where the OEL falls, mean that whether the compound can be handled in open or under isolation or use of selected PPEs etc.

Dear Karanam Abiram

Generally, PAHs has low degree of acute toxicity to humans. It's predominant toxicity endpoint is cancer. Increased incidences of lung, skin, and bladder cancers are associated with occupational exposure to PAHs.

Note that exposure to PAHs are always in mixtures rather than in singlet form. Thus it is difficult to say categorically if there is another compound more toxic than PAHs in your soil specimen. Again, I'll suggest you review the individual PAH content of your sample to know which is mostly present. Determine the heavy metals content and health risk assessment study. These will give a leadway link to the observed health/toxicity concerns. Hope this was helpful.

Thank you.

Hello Hong,

You have given four different studies here for Cefdinir. Ideally, the lowest NOAEL/LOAEL available value should be taken, if several values are available.

-Here, taking LOEL of 10 mg/kg/d for calculation of PDE, values of safety factors will be F3= 1 (whole period of organogenesis is covered), F4= 1 (they have mentioned matenal toxicity but nothing about fetal toxicity, but they mentioned it as non-teratogenic also )

-If you go for the LOEL of 1000 mg/kg, then F3=10, F4=1

-If you go for LOEL of 32 mg/kg, then F3=1 (whole period of organogenesis is covered), F4=5

Dear Prosper,

Look at the following link:

Best regards

Thomas

Thanks for the clarification Varun.

I agree with Gupta. Cat fishes are hardy, able to survive under conditions that many other fishes cannot; this makes them a favourable choice among fish farmers. In the tropics there can be very wide variations in dissolved oxygen for farmed fishes, as a result cat fish is the main fish cultivated.

Then, how long were the fishes exposed to the toxicant?

If you analyse some parts of the fishes for toxicant residue and some organs for residues and metabolites of the toxicant, it will reveal more.

My thoughts

All da best

I use my cells between 4 and 8 passage (not more), in high passage you can get a risk that cells come with some abnormal "behaviour" and much gene senescence or damaged gene, these genes could interfere in your assays.

thread here https://www.researchgate.net/post/Problems_with_Amyloid_Beta_Oligomer_Preparation

Not sure how to design your study, but when using Ag, be careful of the Cl- and other ions that can form Ag precipitates.

Dear Laxit

The answer coild be short: yes, absolutely.

The reason is that toxicokinetics may give an answer to the justified question as to whether systemic uptake will take place and if so, in what quantities to estimate systemic toxicity.

I agree with Dr Clemens Guenther’s comments.

Wishing you success, best regards,

Frederik

Rotary evaporator may be workout.

Hi Ron

Thank you very much. I will consider your resourceful response.

Mam

I want to do in vitro on cell line. Getting ethical number is itself an issue.Moreover my minimum period is also over Therefore I prone to carry out my toxicity study in normal liver and kidney cells.

We have recently provided an online application for the evaluation of skin sensitization which you could try:

I agree with Janice' comments. Extensive non-clinical studies does not seem to be necessary; however minimum investigations including local tolerance of the patch are required to ensure safety of clinicla trial subjects. good luck!

The size of the nanoparticles may have a greater impact on their activity due to more

accumulation inside the cell membrane and the cytoplasm. Comparative study on the antibacterial effect of both micron and nano scale particles of ZnO revealed that ZnO NPs have a greater antibacterial effect.

Please see below the link of a review article for more details

www.omicsonline.org/peer-reviewed/zinc-oxide-na..

I think that sodium phosphate may be suitable for your study because of the stadium ion levels with in the cell remain constant .

There are freely available softwares to predict mutagenicity (e.g Toxtree, OECD toolbox). Interpretation of data may not be straightforward and requires specific expertise.

There is another waf dealing with safety of impurities, which is the application of the treshold of Toxicological concern (TTC). There are different TTCs according to structure and whether there is an alert for genotoxicity (see Kroes et al., 2004). TTC is for life time exposure, but there are ways to adhust them when shorter duration of exposure can be documented (some relevant info in Schilter et al., 2014, reg tox Pharm, 68:275-296). Obviously to apply the TTC requires to get an estimmate of exposure.  

Manures  are pasteurized rather than sterilized. Pasteurization allows certain beneficial bacteria to survive in the substrate, which prevents harmful bacteria or mold from growing. To pasteurize a substrate it must be kept at a temperature of 160-180F for 1-3 hours. Going any higher than 18⁰F will kill the beneficial bacteria and greatly increase your chances of contamination.There are many different methods by which a substrate can be pasteurized such as:

Filling a pillow case with substrate and soaking in hot water. Then removing the pillow case from the water and allowing it to drain and cool. Pillow Case Pasteurization Tek

Filling a plastic "oven bag" with substrate an

With steam pasteurization, weed seeds are sterilized. As shown the auger circulates the hot (140 +,- degree) material. With this technique the soil is thoroughly composted. This is how one  can claim  weed free.

Check polarity.

There are two major differences between acute and subchronic: Objective and Duration

Acute study is intended to test the effects after single administration (or multiple administration within 24 hr). As the effect of any chemical depends on the dose and the duration, in acute studies, you will have to test higher doses to see effects. The data helps to understand what can happen in human incases of accidental or deliberate overdose. This also helps to select dose levels for the repeated dose studies. In subacute studies, the objective is to determine what effects the compound may produce when given daily for up to 4 weeks in rodents (incase of subchronic, up to 13 weeks and chronic, for >6 months). In these studies lower dose levels are used to ensure that the animals tolerate the dose for the duration of the study. The main objective is to see what effects compound may produce on different organs in the body (target organ effects).

The dose response of a CNS active chemical depends on the target, and it is to be noted that some drugs show a reverse effects. For example, CNS stimulation at lower dose levels and CNS depression at higher dose levels (example: Ethyl alcohol)

It indicates that the extract used in the experiment is toxic to fluks at the concentration 100 and 200 mg/ml.

Hello Nyoman, I think you mean 'maternal' instead of 'material'. To answer your question, whether it is toxicity or teratogenicity, if the effects are seen in the presence of maternal toxicity then it is possible that the foetal effects were secondary to the effects on the mother. In the case when no toxic effects were observed in the mother, then it demonstrates a toxic mechanism that is specific to the development process in the foetus. Furthermore, there is generally more concern about teratogenic effects than for general toxicity, mainly because of the hangover from the thalidomide experience many decades ago.

It's difficult to think of something that meets all your criteria. How about liposomal photodynamic therapy agents?

I find this question interesting. It may deserve to be re-looked. 

This question involves / is associated with  the following aspects:  (a) Degradation of the plastic used (as mentioned by Graymer earlier in his answer) (b) Leaching of additives, that could be present in the concerned plastic (c) Impurities present in stored water (these might catalyse the processes of Degradation / Leaching)

It would be nice if some experts in this field / fields could participate in this discussions. 

See, herein Rath et all. 2011.

Thank you very much Supriya,

The food consumption is toxicological end point for many studies. as per guideline this parameter is mandatory for (oral) gavage studies. therefore, my question is that what is the important of this practically inaccurate measure parameter

Most of medicine contain tweet 80 as excepient. and allow to use pregnant women. so upto certain concentration is is consider to be safe.

Thank you so much William for your detailed explanation!!

Loss of conjuctival goblet cells is a very well known phenomenon seen with the use of tear drops containing BAK as preservative while it is least seen with oxychloro complex based drugs.

Dear Wirasak

To be honest with you, your mail is not clear to me. It is quite confusing, Will you try the animal fat oil whether it is toxic on rat ?

I think, acute toxicity can be different from the sytem to system. If you give the "animal fat oil" to the rats by gavage, and look for defacation amount, count, blood stain in stool or histologicaly detection of microvillus damage etc. within 2-3 hours , you can get idea about the oils acute gastrointestinal toxicity. But it does not mean cardiovascular system has been immidiately affected. It depends on your hypothesis, what are you looking for? 

Dear Jyoti Gavali

Pls. find the attached files

Lewis S. Nelson ; Richard D. Shih ; Michael J. Balick ; Andrew Weil. 2007. Handbook of Poisonous and Injurious Plants . Springer Science & Business Media, L.R. Goldfrank (Foreword) 340 pages. ISBN-10: 0387312684 ISBN-13: 978-0387312682

Hoping this will be helpful

Regards

Prof. Houda Kawas

Dawki podesle na priv.:) 

and http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2013.3143/pdf

DMSO is highy toxic ultimately it will show toxic effect on cells. In order to to solubulize your test compound first dissolve the test compound in little quantity( known volume) of 100% DMSO and finally make the volume to the required conc. in your case 0.25mg/ml with with distill water in such away that final you should be able to get 1ml of 0.1% DMSO containing 0.25mg  of your test item

what do you mean by "extract" and "challenged by lps" ? I don't understand what you are asking.

which chemical you have used?

Check USEPA Iris.

Hi Harsha

As you have loaded the drug to a recombinant protein, it is vital to assess the pharmacotoxicity of the drug prior to toxicity study of the formulated protein to identify the safe doses of the drug. In this case you can follow the standard OECD guidelines for in vivo toxicity studies starting with genotoxicity and clastogenicity studies. Following preliminary toxicity studies of the drug itself, the in vivo biodistribution and biotransformation of the protein and the drug-protein complex can be analyzed separately to identify the dose of your recombinant protein. The attached links might help.

These two strains of rats are different, but rats are often used in toxicological studies. You need to make your own choice based on previous work (some other researchers or your own). Personally I would choose Wistar.

It is just what data you have fed and what was expected from it. Soumya and Ganesh have explained well.There should not be confusion now.

Thank you so much for all the answers!

Here are two references with human drug information that might be useful.  Both are free downloads.  Note that the second reference is from 2003 so won't have information on new drugs.  

Benet, Leslie Z., Fabio Broccatelli, and Tudor I. Oprea. "BDDCS applied to over 900 drugs." The AAPS journal 13.4 (2011): 519-547.

 

Schulz, M., and A. Schmoldt. "Therapeutic and toxic blood concentrations of more than 800 drugs and other xenobiotics." Die Pharmazie-An International Journal of Pharmaceutical Sciences 58.7 (2003): 447-474.

I am not sure what other species you need to deal with in Iran, but if you are just needing to distinguish D. pulex from D. magna, they are quite different from one another. In particular, D. magna has a distinctive deeply sinuate postabdomen (see images at the links, below). Otherwise, you will probably want to get hold of Benzie JAH (2005) Cladocera: the genus Daphnia (including Daphniopsis). Leiden, The Netherlands, Backhuys

Publishers, 376 pp

See some images here for D. magna: http://cfb.unh.edu/cfbkey/html/Organisms/CCladocera/FDaphnidae/GDaphnia/Daphnia_magna/daphniamagna.html

vs D.pulex:

 http://cfb.unh.edu/cfbkey/html/Organisms/CCladocera/FDaphnidae/GDaphnia/Daphnia_pulex/daphniapulex.html

For Gadolinium, see nephrogenic systemic fibrosis in patients with kidney disease.

It is to understand 1) if the compound is "active", 2) if active at which concentration, and 3) what is the mechanism of action on cancer cell at the IC50 concentration.

For example cytotoxic pro-apoptotic drugs do not more meet unmet oncological need because cancers associated with dismal prognoses (such as metastatic cancers) are resistant to pro-apoptotic insults.

Thus, the in vitro data bring you a rather pretty good view about the effects of a given drug on cancer cells.

The problem is that a cancer is much more complex than the sole cancer component (because of the tumor microenvironment) and actual metastatic process cannot be studied in vitro.

Thus, in vitro studies can eliminate compounds that are not interesting and/or promising.

In vivo data are mandatory to study the effects of a drug in "real cancer biological conditions", including the metastatic process and the tumor microenvironment, and among others cancer heterogeneity, cancer stem cells, metastasis dormancy, etc..., etc....

The attached articles illustrate some of these facts.

Best regards

Robert

The design of any study depends on the question you are asking. This principle also applies to choosing the route of administration. If the purpose of the study is mechanistic, and the objective is simply to get the test compound into the circulation, then the route of administration is of secondary importance. However, if the objective is risk assessment, it is important to use the route of administration that would mirror actual exposures. For example, if the purpose of the study is to assess the toxicity of a skin lotion that is a personal care product, then the study should employ dermal administration. However, in reality, exposures might occur by multiple routes. Nevertheless, in order to simplify interpretation of results, it may be best to limit exposures to one route of administration at a time. It should also be kept in mind that when a study design employs one route, such as dermal administration, the actual exposures might be mixed if, for example, the animals were able to lick the area of skin to which the substance had been applied then the exposure would be a mix of dermal and oral.

Hi Deepika,

Many cancer derived cell lines, such as HL-60 (derived from from a 36-year-old Caucasian female with acute promyelocytic leukemia) are often used because these types of cells are robust, they grow well and often have sped up metabolism that enables these types of tests. However, it is worth keeping in mind that cancer derived cells may not reflect what is actually happening in vivo. I would say they are good in order to establish/estimate mechanisms of activation on the compound itself, but one needs to be very careful in drawing conclusions and comparisons to in vivo processes or even those in normal/non-cancerous cells. Ergo; they are good to see in what way xenobiotics may be activated, perhaps what enzymes may participate in the action, in addition, to establish reactive intermediates of transformed xenobiotics and various types of damages (DNA damage, oxidative stress etc), but keep in mind that these processes may not correspond 100% to normal cells. Examples we often see in our labs are that whilst cancer derived cell lines are efficient metabolisers of certain PAHs, stem cells or normal cells require external activation (such as S9-mix) or that the level of DNA damage is lower in normal cells as they have more efficient DNA repair.

Take care,