In Vivo Toxicity Testing

Toxicity Screening Using Zebrafish & C. elegans

Toxicity studies using mammalian models are expensive and time-consuming1, 2, and meta-analyses indicate that rodent models predict specific toxic effects in humans only about 50% of the time3, 4, 5. Using small animal model systems such as nematodes or zebrafish can increase predictivity to 70%, dramatically reducing costs through early screening and elimination6.

While we do not suggest replacing mammalian models entirely with C. elegans or zebrafish assays for early safety and hazard evaluation, we believe an integrative approach can improve prediction of human outcomes while reducing the cost, time and use of mammals in toxicity assessments.

Why Use Zebrafish?

Zebrafish are increasingly recognized as an indispensable tool in toxicity testing. For companies seeking to test acute toxicity and teratogenicity, assays in larval zebrafish provide predictive answers within a few short weeks.


Studies have confirmed that mammalian and zebrafish toxicity profiles are strikingly similar. In addition, zebrafish organs are similar to human in cellular composition, function, signaling, and response to injury as well as the cellular processes that mediate organ-specific diseases. Many genes are highly conserved between humans and zebrafish, making them a useful system to study the basic mechanisms of organ-specific diseases, such as heart, liver or neurological disorders.

Time Saving:

Zebrafish develop rapidly; all of their major organ systems become fully formed during the early larval stages, making it a recognized standard model to assess organ toxicity. Even at an early stage, zebrafish show concordance at a metabolic pathway level, confirming its utility as a model to screen the toxic potential of compounds on major organs *.

  • Zebrafish Acute Toxicity Study Demonstrates Concordance with Rat Fetal Embryotoxicity.

Zebrafish Toxicity Testing Offerings

InVivo Biosystems provides services and expertise across the zebrafish workflow including genetic modification, molecular characterization, and behavioral testing. Our toxicity testing is performed using automation and customized software for data collection and analysis. Our main testing services include:

Acute Toxicity & Teratogenicity Screening

What: Acute developmental toxicity studies in zebrafish can predict mammalian exposure levels associated with embryotoxicity and teratogenicity. 

Goal/Result: By testing a range of concentrations, we define concentrations such as the LD50, NOEL, and MNLD. 

How: Our team tests the teratogenicity of compounds on a battery of morphological development endpoints, as well as two behavioral outcomes, for zebrafish embryos between 24 and 120 hours post fertilization (hpf). This protocol is more comprehensive than the set of 4 endpoints prescribed by the OECD FET 236.

Data Collection: All toxicity screening data is collected in an automated, high-throughput workflow that combines experimental endpoints collected into a developmental hazard profile report, while retaining individual animal-level details and providing raw data files for user analysis.

  • Assessed at 24 hpf: mortality, developmental progression, spontaneous movement, notochord development.
  • Assessed at 120hpf: total mortality, body axis (body axis non-linearity, eye edema, yolk sac edema and pericardial edema and brain malformation), otic vesicle, pectoral fin, caudal fin, pigmentation, blood circulation, swim bladder formation, not present or not inflated.
  • Embryo Photomotor Response (EPR) behavior. EPR is used as an early, rapid and sensitive predictor of generalized adverse outcomes later in life, not restricted to behavioral endpoints. The EPR assay is used as an early sensor of chemical developmental hazard at 22-24 hpf. 
  • Larval Photomotor Response (LPR) behavior. LPR can reveal neuromuscular effects of compounds that may not be detected with morphological malformations. At 120 hpf (5 days post fertilization) zebrafish are free swimming larvae and the photomotor response assayed total movement (swim distance) in response to multiple light/dark transitions. 

Molecular Testing

What: We will perform gene expression studies to characterize mechanisms of action. 

Goal/Result: Provide mechanistic insight into toxicity responses in developing zebrafish at a whole transcriptome level to deliver a high depth, high resolution analysis of the underlying biology of your compound of interest.

How: Perform single embryo RNA-seq analysis on a range of conditions, beginning at exposure and RNA isolation to sequencing and bioinformatic analysis, in a high throughput, automated workflow.

Other Zebrafish Testing Services

  • Embryo Acute Toxicity Test

    Determine the acute or lethal toxicity of chemicals on embryonic stages of fish (Danio rerio).

  • Zebrafish behavioral phenotyping

    Assess photomotor response, measure anxiety behaviors, or understand learning and memory mechanism.

  • Early-life Stage Toxicity Test

    Determine the lethal and sub-lethal effects of chemicals on the early life stages of the fish tested.

  • Custom Testing Project

    Still cannot find what you are looking for? We can work with you to create a custom in vivo testing solution to get you the answers you need.

Why Use C. elegans?

  • C. elegans is a small nematode that can be maintained at low cost and handled using standard in vitro techniques.
  • Unlike toxicity testing using cell cultures, C. elegans toxicity assays provide data from a whole animal with intact and metabolically active digestive, reproductive, endocrine, sensory and neuromuscular systems.
  • Toxicity ranking screens in C. elegans have repeatedly been shown to be as predictive of rat LD50 ranking as mouse LD50 ranking. Additionally, many instances of conservation of mode of toxic action have been noted between C. elegans and mammals6.

The most toxic compounds in worms will also be the most toxic in mammals. 

Drug development frequently involves starting with an initial lead compound and systematically testing a range of structurally related compounds or analogs.  The utility of worms for drug toxicity testing is evident in the conservation of drug toxicity ranking.  Within a list of compounds with different toxicities, the most toxic compounds in worms will also be the host toxic in mammals.  This is especially useful for screening classes of related compounds as an assay in C. elegans can quickly and inexpensively determine which among dozens of related compounds are least likely to have toxic effects7.

Pathways. C. elegans possess nearly all of the key cellular pathways that operate in humans.

Despite the lack of heart, liver, lungs, etc. C. elegans still possess nearly all of the key cellular pathways that operate in humans.  For many pathways that control vertebrate-specific organ systems, C. elegans possess a different or simplified output. We refer to the different outputs as ‘phenologs’—phenotypes that manifest differently in a model system despite sharing a common cellular mechanism with mammals or humans. 

For example, C. elegans do not possess a heart, yet they eat by rhythmic pumping of their pharynx which is controlled through cellular mechanisms analogous to those regulating cardiac muscle in higher organisms. 

  • Worms on drugs: How well can C. elegans predict drug toxicity in mammals?

C. elegans Toxicity Testing Offerings

Predictive toxicology requires a balance between catching all the compounds that pose a hazard and excluding those that do not pose a risk. When testing drug toxicity and dosage in C. elegans, we use a combination of two assays:

Larval growth assay

What: A highly sensitive assay that picks up minor perturbations in the developmental pathway, and we can be assured that negative-testing compounds or treatments will not adversely affect development in downstream assays or other systems.

Goal/Result: Worms are highly susceptible to developmental toxins. Using high resolution images, we can precisely measure the size of hundreds to thousands of worms over the course of development.

How: To determine whether conditions or mutations affect the development of the C. elegans, approximately 50 age-synchronized stage 1 larvae (L1s) are grown on standard plates with a bacterial food source. 72 hours after plating the worms are assessed for size and life stage. 

Data Collection: Data of worm size (measured as length and area) are collected over different life stages including larval stage (L2, L3, L4) and/or young adult.

Egg viability assay

What: A highly selective assay to identify compounds that result in egg viability have a higher probability of being true positives and are likely toxic in mammals. We measure the percentage of viable progeny produced by worms treated with the drug.

Goal/Result: Mutations and compounds can affect animal viability. By quantifying embryo viability, this assay can reveal developmental effects of mutations and compound treatments.

How: Adult C. elegans are allowed to lay eggs on a plate for 2 hours. A set number of embryos are moved to a new plate. After 24 hours of incubation at 20℃, the number of unhatched embryos is counted.

Data Collection: The percentage of embryo lethality is calculated from the unhatched embryos divided by the total number of embryos plated. Three replicates are tested.

By combining a highly sensitive larval growth assay with a highly selective egg viability assay (Figure 1), we can refine the pool of possible compounds or dosages to those least (or most) likely to be toxic (Figure 2).

Figure 1.  Two complementary approaches to testing developmental toxicity in C. elegans. (A) Larval growth assay violin plot shows distribution of worm length measured by high resolution imaging and automated worm detection. Darker blue indicates higher drug concentration. Day 1 = L2/L3, day 2 = L4, day 3 = adult. (B) Egg viability assay showing the average proportion outcomes for 50 embryos 3 days after laying. EC = effective concentration.

Figure 2. Combining egg viability and larval growth to determine the toxicity of a compound. Egg viability and larval growth assay have contrasting sensitivity and selectivity properties that help refine the boundary between a toxic or non-toxic treatment. The blue histogram represents the responses of the population adversely affected by the treatment; the yellow histogram represents the responses of the population for which treatment has no adverse effect. The red-shaded area indicates a refined cutoff range extrapolated from the two assays.

Our Process

Understand Your Goal

We will set up a 30-minute call with you to understand your needs. During this call, we will define the scope of your project, answer your practical questions and help you assess whether our service is a good fit for you.

Create A Personalized Catalog

After we decide on the scope of the project, we will create a proposal that includes cost and time estimate for each experiment proposed. You will be able to personalize your project plan.

Finalize Your Project Design

We will send you a final statement of work and payment schedule. Once we receive your first PO, we will start the experiments and give you a defined timeline for your project.

Report Out
Maintain Clarity And Transparency

We will keep you updated every two weeks on the status of your project. At the end of your project, we provide you with a report that includes comprehensive findings and key takeaways.



  1. Nass R, Hamza I. 2007. The nematode C. elegans as an animal model to explore toxicology in vivo: solid and axenic growth culture conditions and compound exposure parameters. Curr. Protoc. Toxicol. Chapter 1: Unit1 9 doi:10.1002/0471140856.tx0109s31.
  2. Tralau T, Riebeling C, Pirow R, Oelgeschlager M, Seiler A, Liebsch M, Luch A. 2012. Wind of change challenges toxicological regulators. Environ. Health Perspect. 120: 1489–1494.
  3. Hartung T. 2009. Toxicology for the twenty-first century. Nature 460: 208–212.
  4. Knight AW, Little S, Houck K, Dix D, Judson R, Richard A, McCarroll N, Akerman G, Yang C, Birrell L, Walmsley RM. 2009. Evaluation of high-throughput genotoxicity assays used in profiling the US EPA ToxCast chemicals. Regul. Toxicol. Pharmacol. 55: 188–199.
  5. Olson H, Betton G, Robinson D, Thomas K, Monro A, Kolaja G, Lilly P, Sanders J, Sipes G, Bracken W, Dorato M, Van Deun K, Smith P, Berger B, Heller A. 2000. Concordance of the toxicity of pharmaceuticals in humans and in animals. Regul. Toxicol. Pharmacol. 32: 56–67.
  6. Hunt, P. R. The C. elegans model in toxicity testing. J. Appl. Toxicol. 37, 50–59 (2016).
  7. Harlow, P. H. et al. The nematode Caenorhabditis elegans as a tool to predict chemical activity on mammalian development and identify mechanisms influencing toxicological outcome. Sci Rep 1–13 (2016). doi:10.1038/srep22965
  8. Ferguson, Boyer, M. S. & Sprando, R. L. A method for ranking compounds based on their relative toxicity using neural networking, C. elegans, axenic liquid culture, and the COPAS parameters TOF and EXT. OAB 139–6 (2010). doi:10.2147/OAB.S13466
  9. Li, Y. et al. Correlation of chemical acute toxicity between the nematode and the rodent. Toxicol. Res. 2, 403–10 (2013).
  10. Boyd, W. A. et al. Developmental Effects of the ToxCast™ Phase I and Phase II Chemicals in Caenorhabditis elegans and Corresponding Responses in Zebrafish, Rats, and Rabbits. Environmental Health Perspectives 124, 586–593 (2016).

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