Are there any toxicology endpoints for which you have high confidence in NAMs being able to currently replace animal testing for assessing safety? (Please explain and feel free to add citations)

Do not know enough to comment

I believe investigating developmental effects are complicated as they require longitudinal methodologies and, in some cases, might be sensitive from an ethical viewpoint. Nonetheless, this is not a good enough reason to turn towards animal models as these models are unreliable (and on top of biological differences, the same methodological problems remain in animal models).

endpoints vary with the clinical effects, I don't know if endpoints are available for NAMs.

Moving in to use NAMs is not expected to be a one-step process and we may have to depend on animals to draw comparisons with NAMs. In the first place, it is important to understand, that by using modern molecular biology technologies, the number of animals needed for every test can be greatly reduced, and one of the first parameters to look into will be the inflammatory pathways and any alterations of the gut microbiota. These two can function as highly sensitive predictors of long´-term adverse effects.

I am not able to give a qualified answer.

Biomarkers in cell-based assays can be easily monitored for the above ticked reactions that would indicate for possible adverse effects from tested substances.

none

some allergic reactions

Not very sure to comment on the same but yes there are several studies done and further meta analysis required to set such point particularyl in physiological levels for example many of the disease diagnosis are on the basis of clincal range

These are short term effects that do not require the overall organism.

All effects probably related to a tissue/cell damage or a clear functional parameter are easier to use as a replacement. A major weakness in NAMs is the lack of good toxicokinetic modeling in the database. Furthermore, any complex interactions may be missed by simple NAMs. Here, an agenda for the use of NAMs with a decision tree would help.

For certain experiments, NAM is applicable only. Not much helpful for physiological level.

The ones check off are already using NAMs. so what are you on about?

As above

It is possible that NAM might be able to generate data related to topical applications such as Eye and skin.

may be reproductive effects?

See EU legislation

These toxicological endpoints have proven to be reliable in NAM's and do not require further pain and suffering to animals.

DART is hard as we need to identify the mechanisms and create AOPs and then tests.

effects on the intracellular activity can be visible from cell cultures, however effects of this activity often impact neighbouring cells via many signalling pathways.All what include interactions between cells and organs would require whole organisms and NAMs seem to be curently limited.
See further information in the review below.

Doke SK, Dhawale SC. Alternatives to animal testing: A review. Saudi Pharm J. 2015;23(3):223-229. doi:10.1016/j.jsps.2013.11.002

Respiratory tox

No love for pulmonary?

None, these are things that can not be predicted without living systems

it's one of the most advance domain by using organoids and spheroids from initial tissue

I believe any toxicology endpoints help. But, those for establishing irritants are more reliable. The number of variables is smaller and the analysis environment more controllable.

computational (in silico) toxicological (ADMET) methods may be adopted.

La risposta risente della variabilità di cui al punto precedente.Non.tralascerei lo stress ossidativo:sbilanciamento fra capacità antioxidant endogena dell'organismo e concentrazione dei radicali liberi ainteragenti

The chosen effects must be tested on a whole organisms, at specific stages of development.

For biological systems exposed to xenobiotics directly from the environment such as the skin and the eye non-animal methods can be sufficient to reasonably ascertain human safety of xenobiotic compounds. For the other effects mentioned above non-animal methods are progressing, but as yet are not sufficient to safely exclude serious damage to human.

Validated in vitro test methods, stem cell research and test methodologies, as well as transcriptomic signatures are enabling data production for categorical conclusions on the types of organ and system toxicities that could occur in vivo. "Categorical" means "Likely", "Possible", or "Unlikely" and may not identify a NOAEL or POD.

Allergic reactions are far to complex and still poorly understood. Hepatic effects require organ testing. 3D cell culture as well as bioprinting and other cell tech solutions are important developments into the right direction, but to my knowledge they cannot yet sufficiently replace native organs. Reproductive effects can only be investigated properly with multi-generational testing. As non-animal procedures assays with invertebrates are the only possibility, but inter-species differences add a lot of uncertainty, also in terms of exposure paths. Neurological effects are again very complex and inter-species differences are certainly an issue. Same goes for cancer, plus that cancer is often a multistressor effect, which would be pretty hard to impossible to simulate. Until we have molecular assays which resemble the entire cascade of events leading to tumor formation, this remains a challenge. AOPs are certainly key for this. However, also animal testing can only shed light on a small part of the process. For irritation we have the HET-CAM and some other, more advanced cell tests giving reliable results. Developmental effects have good vertebrate, non-animal models, primarily the zebrafish. Endocrine effects are a case for the precautionary principle: if a substance disturbs hormone signalling it is likely to have some effect. Hence, cell-based elucidation of endocrine disruption should always raise a flag. On the other hand, we still need to much better understand the entire hormone system to sufficiently assess substances based on their endocrine activity.

Skin and eye are not as dependent on pharmacokinetics as the other endpoints.

NAMs likely to be of utility for acute findings.

currently, no

From what I have seen NAM methods at this time can be a means to prescreen chemicals for potential problems. That is if the NAMs detect a significant response (i.e. a red flag) to a chemical, the company can halt further development and reduce animal testing. However, owing to the complexity associated with PK/PD and other factors some forms of animal testing need to be conducted. Although not currently required as far as I know, the results of NAMs should be submitted to the regulatory agencies with a petition to reduce /replace certain types of animal testing. If the NAMs show an effect and waiver for animal testing is made, how will the result in dose level (or in vitro concentrations) be extrapolated in the risk models?

Since the human may be uniquely sensitive to a chemical whereas both NAM and animal studies may miss a significant toxic response, it may become more necessary to demand occupational and environmental chemical epidemiology studies to continuously assure that the chemicals are not harmful. If NAMs eventually predominate such epi testing will become increasingly important. In this regard, the standards for chemical epi conducted be more uniform across the regulatory agencies and their need to be better systematic standards for their review by fully focused experts. The combination of more chemical epi studies and the means for the systematic review may prove to be more expensive and problematic since they involve humans and this includes problems with privacy. Please see: Doherty, JD Toxicological Sciences 2020 177(1):156-157 and Doherty (2021) EC Pharmacology and Toxicology 9(8):92-96 and Doherty, SOT 2021 and Doherty Toxicology Forum January 25, 2022).

There are just too many factors involved to eliminate animal testing before 2035. Based on my experience about 13 years in the regulatory world is very fast considering the magnitude of exposure to humans.

However, in spite of my current concerns as above, I favor research efforts to reduce animal testing. Currently some cancer studies are conducted with what I feel are exceedingly high dose levels (i.e. > 1000 ppm) where the defense mechanisms are overwhelmed and the dose is far far greater than what people will be exposed to. More work should be done in revising the guidelines for when animal cancer studies are needed and what doses are meaningful. This is one way to reduce animal testing.

I have expert in oxidative stress, DNA damage and cytotoxicity studies.

Not sure

Currently, not all systems can be easily modelled with NAMs yet, and I think particularly developmental and reproductive effects are a challenge bc we have to mimick life and not just organs.

None that I am aware of to date. Further research is required.

Chronic systemic toxicity endpoints still require much development and evaluation. For cancer endpoints, the use of short term in vitro and non mammalian systems available, can be used to screen and prioritize so animal testing is only done in specific cases with strong justification.

The data for comparison, inter lab studies, and repeat studies doesn’t exist for chemical testing.

Those I have checked, I do so with some confidence, perhaps also the reproductive and developmental targets as well. much less so with the neuro endpoints as the the adverse outcome pathways have little utility in functional determinations which are complex beyond organic markers. Already the animal test systems undervalue many neurophysiological assessments.

I have utilized different labs to perform the same NAM test. They give different results. The lack of replicability across different labs is appalling.

I do not have high confidence that there are current NAM approaches used now that can be used to be applied across the board that replace animal testing to inform regulatory decisions although there are some in use now that I do have high confidence for specific applications. A summary of the status of NAM use by EPA can be found at https://www.epa.gov/assessing-and-managing-chemicals-under-tsca/alternative-test-methods-and-strategies-reduce

I believe there are some in vitro cell assays for skin irritation via corrosion that might be useful, though they are not able to accurately test for immunological-based irritation. There are estrogen and testosterone receptor binding assays that can identify potential endocrine disruptors, but they don't account for pharmacokinetics or the resulting endpoint in a live animal which could, and likely will be more complex than just activating a sex hormone receptor. Many chemicals have multiple mechanisms of action.

To the best of my knowledge, only irritation (and possibly allergic reactions) no longer need animal testing. Hepatic effects are probably the next area where we could confidently rely on NAMs.

Irritation being a local (i.e., not a systemic) reaction probably would be the only endpoint I consider sufficiently validated to be assessed in NAM's. All other endpoints are compklex systemic reactions which depend on manifold factors (ADME, local organ availability etc) which - at the moment - cannot be fully assessed by alternative methods. In the case of allergic reactions, skin sensitization may be the only endpoint that reasonably can be assessed by NAM's; respiratory allergy, however, again is a complex case and there is no widely accepted alternative test system available.

Non-animal experiments have proven useful in highlighting mechanisms of carcinogenesis and endocrine disruption, but a living organism is more complex. Interactions at the receptor level and circadian rhythms in the endocrine and metabolic pathways cannot be easily replicated by non-animal lab experiments.

The foregoing effects are too vague to be helpful. For example, how can the effect of cancer be ascertained without using animals? It is conceivable when the mechanism of a carcinogenic effect is commonly accepted, one may test the effects at the cellular, and/or molecular level.

NAMs are available for certain acute hazard endpoints. The reason I checked all the boxes is to make the point that existing knowledge on a chemical class may enable a chemical-to-chemical extrapolation. Read-across is a nonanimal method which has been used in regulatory frameworks. And as with any type of extrapolation there will be some level of uncertainty. Confidence can be increased if high quality data are available on the structurally similar chemicals for the endpoint of interest, and there is good coverage of the chemical space. Additionally, the similarity hypothesis/rationale could be strengthened with PK and mechanistic in vitro data. Although more work is needed on the systemic endpoints, an approach might be used that integrates in silico/in vitro information with some animal testing (reduced).
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While a lot of progress has been made in developing NAMS, upon their application a lack of concordance with in vivo data continues to become apparent - especially in the cases of chemical mixtures (e.g. formulated products vs single chemicals). While I believe their is value in NAMS, and the supplementary data they provide, at this point I do not believe that any single NAM can reliably replace in vivo testing. In order to rely solely on NAMS (or a NAM battery) better integration and understanding of their associated AOPs is critical.

receptor mediated bioactivity profiling to yield PODs that can then be compared to exposure estimates (or even biomonitoring results) for MOE determinations.

The immune sustem is a highly individual and genetically self-varying system. Any in-vitro system lacks intrinsic biological adaptation. Some repro effects might be testable, but not all. Developmental tox is highly complex and time-of-exposure dependent. I can't see how that can be effectively copied in vitro. Neuro effects are complex, and are about the interaction and balance of many many variables, not something that is reasonably copied in vitro. Some carcinogenicity is predicatable, but some is just a surprise.

I have not selected any box. For some substaces where there is good in vitro and in vivo of mechanism of action then the combination of in vitro and minimal in vivo data can be adequate. The point is surely that every chemical assessment should provide a logic tree that explains the basis of the assessment.

Its not that I don't believe we can do NAMs for all of these, but I am not totally familiar with the state of the art with all of these. For certain, skin irritation and hepatic effects I know about and have tremendous confidence.

I think we can have a reasonable information about irritation and allergic reactions using NAM given corneal and dermal cells can be used (like caco-2 cells) in the case of irritation and determining the binding of haptins to proteins that are known to cause an immune response leading to allergic reactions. Other systems are difficult to replicate in vitro. However, x-year cancer studies are likely not needed as we get enough information from in vitro and short-term animal studies indicating whether a carcinogenic output is possible or not in longterm cancer studies.

These effects are more localized to the target organs and less impacted by the whole body interconnected interactions.

Simpler toxicological endpoints with defined MOA can most readily be addressed by NAMs. Complex toxicities will require much more work, partly due to complexity (needing systems approach?), multiple MOAs, and chronic effects.

None of these at the moment.

For these endpoints the quality and reproducibility of the assays allows for interpretation with some confidence

Except for molecular entities or drug evaluation where reductionist approaches may be applied or tested by NAMs, complex disease models require animals,

Limited mechanistic information available. Test systems and designs may require refinement

Too soon to call

Skin irritation and sensitization have suitable NAMs which have OECD guidelines and ECVAM acceptance, so should be formally recognized by regulatory bodies. The same can be said for genetic tox assays to inform cancer risk.

It would be interesting to see the rank order of these based on experts' inputs. For myself I do not believe NAMs can or will replace any of these endpoints in the near future, let alone 13 years from now.

Excellent progress in these areas already.

Not sure NAMs could adequately replicate reproductive effects

Veterinary medicine

Certain effects can not be experience in people hence NAMs should play the role

None that I know of.

Any effects that are multi-determinate probably are a bit further away, developmental tox is s good example. Reproductive tox also

Certain Genetox endpoints, maybe dermal sensitization and acute lethality.

No.

Using methods with single cells or tissues.

I don't have that confidence that NAM would replace animals

none

Kleinstreuer et al., 2018 - Non-animal methods to predict skin sensitization (II): an assessment of defined approaches *. Crit Rev Toxicol. 2018 May;48(5):359-374. doi: 10.1080/10408444.2018.1429386. Epub 2018 Feb 23. PMID: 29474122; PMCID: PMC7393691.

Although NAMs represent great and greatly needed help in toxicity estimation unfortunately we still cannot completely renounce using animals in toxicity testing as the complexity of the organism cannot be modeled completely.