Having spear-headed the development of this test, we are world-renowned experts in its performance! We are moreover the first service provider globally to offer this test under GLP.

The RTgill-W1 cell line assay comprises a 24-well plate format acute toxicity test based on the permanent cell line from rainbow trout (Oncorhynchus mykiss) gill. After 24 h of exposure to a test chemical or product at six concentrations, cell viability is assessed based on three fluorescent indicator dyes. Data are expressed as percent cell viability of unexposed controls versus the test chemical concentration. The resulting concentration-response curves serve to determine the effective concentrations causing 50% loss in cell viability (EC50 values). The lowest observed EC50 value is taken as proxy to predict the fish acute toxicity (LC50 value).

Aside from being an animal-free alternative to the high severity traditional fish test (e.g. according to OECD TG203), the gill cell line assay requires about three orders of magnitude less testing material, making it the perfect choice for screening, e.g. in new chemical synthesis and product development.

We were part of the development of this test guideline and hence have extensive experience in carrying out this test. We are certified to offer this test under GLP.

The Fish Embryo Acute Toxicity test is a 24-well plate-based test method in which newly fertilized zebrafish (Danio rerio) eggs are exposed to a test chemical or product at five concentrations for a period of 96 h. Exposed and control embryos are studied every 24 h for apical observations indicative of mortality. Data are expressed as percent mortality of exposed vs. unexposed embryos. The resulting concentration-response curves serve to determine the effective concentrations causing 50% lethality (LC50 values), which are taken as proxy to predict the fish acute toxicity (LC50 value).

Beyond the OECD TG236, we routinely measure sub-lethal endpoints as well, which include malformations, edemas and uncontrolled movement, providing additional information on developmental toxicity. In jurisdictions such as Europe, in which animal protection laws exclude early life stages until independent feeding, the test can moreover be extended to 120 h exposures, which allows for better resolution of impacts on behavior. Compared to the traditional acute fish test (e.g. according to OECD TG203), the FET test requires about two orders of magnitude less testing material.

We have an ever expanding list of primers to analyse the expression of a wide variety of genes, such as related to biotransformation, metal detoxification, cell proliferation, oxidative stress response and immune regulation. We frequently add new primers based on clients’ needs.

Gene expression analysis can be performed in fish cell lines as well as zebrafish embryos, using a concentration-response, as well as time-resolved, experimental design. These tests are usually preceded by an acute fish toxicity test with either the cell line of choice or the embryos to ensure that gene expression is indeed studied at non-acutely toxic concentrations.

Concentration-dependent gene expression analysis allows the derivation of Points-Of-Departure (POD), which are increasingly used to derive estimated low effect levels for chronic toxicity assessments.

Hypothesizing that reduced fish growth on chemical exposure is due to a lower number of cells, research in the Schirmer group at Eawag has been able to demonstrate that a fish cell proliferation assay, based on the RTgill-W1 cell line as representative of richly perfused tissue, can be used to predict the reduced growth of fish as traditionally determined using the Fish Early Life Stage Test (FELST; OECD TG210).

Cells are exposed to the chemical over the course of five days and cell number determined, from which cell weight can be calculated as input parameter for the Bertalanffy growth model. The proof-of-concept study has yielded excellent results, showing concordance to in vivo results obtained after months of experimentation with fish DOI: 10.1126/sciadv.1500302.

As functional units of organs and tissues, cells comprise the different mechanisms that collectively lead to ADME (Adsorption-Distribution-Metabolism-Elimination) of chemicals. Hence, we can assess bioaccumulation of chemicals in cells along with chemical depletion and the potential formation of biotransformation products. As well, the influence of the test chemical on the activity of biotransformation enzymes (such as EROD activity as proxy for CYP1A) can be measured.

Please refer to the following examples as illustration of possibilities:

• Ranking of chemicals for their potency to induce EROD activity as proxy for CYP1A: DOI: 10.1006/eesa.1999.1808
• Bioaccumulation, transformation and derivation of a bioconcentration factor: DOI: 10.1021/acs.est.7b04548
• Fish cell barrier model to assess transfer of organic chemicals in vitro: DOI: 10.1021/acs.est.9b04281
• Estimation of bioaccumulation of anionic compounds in fish based on the liver cell line, RTL-W1: DOI:10.1016/j.envint.2023.107798

We collaborate with well established contract research organisations who specialize in chemical analysis, including under GLP. If chemical analysis is desired, e.g. to verify exposure concentrations or biotransformation products, we offer bundled packages, which include co-ordination for a streamlined workup and reporting.

Samples can also be shipped to a laboratory of the client’s choice or for in-house analysis.