Tracey Goodband, Senior Manager of Aquatic Ecotoxicology, presented the webinar
“Navigating the Challenges of the Medaka Extended One Generation Reproduction Test (MEOGRT).”
Read below for responses to the questions asked during the audience Q&A.
Q: The aerated hatching chambers were partly intended to synchronize hatching. How variable is the time to hatch when using crystalizing dishes?
A: In general, the eggs start to hatch between days 7 and 9 and completion of hatching takes 3 to 5 days. The variability between control replicates is low, i.e. start and end of hatch are generally within ± 1 day between replicates.
Q: In a theoretical dietary study would you expect toxicity to affect appetite and thus exposure?
A: Yes, this is possible especially if testing up to the MTC. Loss of appetite is often observed as a result of toxicity and, yes, if this occurred then exposure would be reduced.
Q: Are your background findings of XX males or XY females in line with the rates reported in the guideline?
A: Yes. Of the six completed studies, the incidence of XX males in the control group ranged from 0 to 4.5% which was within the <5% required. To date we have not seen any XY females in the controls for the MEOGRT studies.
Q: In zebrafish there can also be external factors influencing sexual development, e.g. temperature. How is this with Medaka?
A: With most fish species, environmental factors can influence development including temperature, space (ie loading rate), feeding etc. This can also be true for medaka.
Q: Is there a reason sex can’t be genetically identified in zebrafish/fat head minnows?
A: Currently no specific sex gene has been identified for zebra fish or fathead minnow. The dmy gene is specific to the medaka and only expressed on the Y chromosome allowing for genetic sexing of the fish.
Q: Are histology examinations performed in-house?
A: No. Macroscopic observation of the gonads is performed during termination, but histology/histopathology is sub-contracted. We work with several specialist pathology laboratories.
Q: What method(s) are typically used to prepare solvent-free exposures with poorly soluble materials?
A: For insoluble substances that are hydrolytically stable, Smithers often uses column saturators to dose the test systems. As described in the OECD Guidance No 23 Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, this method is especially recommended for low solubility substances. Smithers has a long history of success with this type of solvent-free method in endocrine studies. However, substances that are dosed using column saturators are typically highly adsorptive, therefore, there are many potential binding sites in the exposure system, such as the glassware, silicone, waste, and biofilm buildup, or the animals themselves from bioaccumulation. Increased cleaning of the system and/or increased flow rates can help mitigate this potential. A solid/liquid saturator system, also described in OECD 23, could also be used to prepare a saturated solution although the volume required could be problematic for this method dependent on the test concentrations used.
Q: Is the scoring of anal fin rays still part of the MEORGT guideline?
A: Yes, this is conducted on all F1 generation fish (non-breeding and breeding fish).
Q: Are dietary exposures possible for substances with low water solubility?
A: In theory this could be possible. However, an alternative food source would need to be investigated as generally medaka are fed live artemia and flake food. Neither of these would be suitable for spiking with the test substance. They will however take trout pellets as we have used these to feed small fry although these need to be ground to feed newly hatched/young fry and have not been used to feed medaka throughout their life. In addition, unlike a bioaccumulation study which uses juvenile fish and includes weight measurements as part of the sampling to allow for recalculation of the feed supplied based on growth, the MEOGRT includes fish at all stages of the life cycle. This would make calculation of the amount of feed required problematic without constant weighing of the live fish during the course of the study.
Q: Do you have experience regarding animal welfare concerns linked to the fin-Clipping procedure?
A: This was discussed by our animal welfare committee (AWERB) before we started performing the procedure. Although the guideline suggests that this is conducted under anaesthetic, following discussion with our VET, it was agreed that anaesthetising small fish can be very problematic and also that there is a certain amount of evidence that anaesthetics can adversely affect the mucus layer of the fish. We also decided that taking a ‘v’ clip from the tail fin was better for the fish as it avoids the main outer fin rays of the tail. The procedure was practiced extensively on dead fish before conducting on live fish. With the experience gained, this procedure now takes seconds and the fish recover quickly.
Q: If you wanted to better link/unlink the VTG levels to breeding, given the issue of only measuring VTG in non-breeders, would you suggest having additional animals in the existing dose groups, maybe having a satellite group, where they have bred but you then remove some for histopath?
A: Additional animals or satellite groups would not be necessary. We could simply preserve the non-breeding fish for potential histopath and also sample the breeding fish for VTG. This way both VTG analysis and histopathology can be conducted on all F1 generation fish (breeding and non-breeding) allowing for comparison of VTG and histopathology for both non-breeding and breeding fish.
Q: The guideline is designed to test single compounds at the time, what adaptations need to be considered to test environmental, complex matrix samples?
A: The dosing of the system and analytical issues would be the same as for any other aquatic test although the size and duration of the test adds further complexity. Ideally the test should be conducted using flow-through conditions which can be problematic for complex mixtures especially if the compound is poorly water soluble. Whilst a semi-static design would not be impossible, it adds an additional variable of potential increased stress on the animals due to constant handling. In addition, the challenge would be interpreting the results ie are any effects due to one or more of the components, possible synergistic/antagonistic effects especially if the components show different solubilities etc.
Q: Any detriments/ challenges to changing the dawn cycle?
A: No. We still use the same light/dark cycle (i.e. 16 hours light/8 hours dark) but have just moved where the dawn/dusk occurs. All fish labs including the stock fish lab are on the same cycle so there is no change to the diurnal rhythm.
Q: Are you seeing an increase in demand for the MEOGRT test and are there any issues with capacity for these studies?
A: Yes. Mainly from the pharmaceutical industry but also from the industrial chemical and plant protection sectors. The studies do take up a reasonable amount of laboratory space for ca 5 months therefore in both the UK and US we are currently expanding our laboratory space to increase capacity.
Q: Do you have any feedback from regulators who are reviewing these studies, and do they have sufficient knowledge and experience with these study types?
A: We are now starting to see feedback from the regulators. From some of the comments received, it would suggest that they do not appreciate the complexity of the test or the fact that it is primarily designed to evaluate potential endocrine activity and that it is important to consider results from all endpoints and not assess them on an individual basis.
Q: What are the reasons that the LADGA is considered a level 4 test whereas the MEOGRT is a level 5 test?
A: The LAGDA only assesses a part of the life cycle. The MEOGRT assesses not only the full life cycle but also includes additional generations.
Q: You mentioned you don't use substrates...they aren't recommended in the TG?
A: No, the OECD 240 guideline does not recommend adding substrates for medaka. Other guidelines, e.g. OECD 229 recommend using spawning substrates for fathead minnow and zebra fish but not medaka. The female medaka carries the eggs on the body near the vent prior to depositing onto a substrate if one was available. Adding any substrates can complicate matters especially if the test substance has adsorptive properties.
Q: Have you run this study type with any other fish species, e.g Fathead minnows or zebra fish
A: Zebra fish no. However, both the UK and US sites both have experience using fathead minnow (FHM) as an alternative species for this study type. As there is currently no guideline for the conduct of a FEOGRT, this becomes a bespoke test design which would need agreement from the authorities before conducting. Smithers has conducted a FEOGRT following the design of the MEOGRT but the replication was problematic for a couple of reasons 1) the space required (FHM need larger vessels compared to the medaka) and 2) the number of fish required (FHM are usually bred 2:4 males to females compared to 1:1 for medaka although we did reduce the FHM to 1:2). A more realistic design, which again has been used at Smithers, would be to reduce the replication to 4 replicates as per other fish guidelines and the proposed ZEOGRT design.
Q: You said that there is limited experience with the MEOGRT. What are some example cases where this test would be required?
A: For endocrine active pharmaceuticals, or those which indicate possible endocrine activity from mammalian studies, these studies are now becoming routine. For other chemicals (industrial and plant protection), they can be triggered if effects are observed in a FSTRA or FSDT to further investigate whether or not the chemical has endocrine disruption properties in fish.
Q: What do you consider appropriate level of malformations in the study control organisms?
A: Ideally, malformations would not be observed in any of the control organisms. However, realistically, due to the number of animals used for the study a small number may show malformations/abnormalities due to natural variation. Anything higher than 10% of the animals showing deviations from the norm would be a little worrying and bring into question the suitability or robustness of the batch of animals.
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