Human Exposure Routes. 

Agrochemicals are widely used to increase both food yield and quality by protecting crops from pests such as fungi, insects or weeds.  Also known as pesticides, plant protection products, fungicides, herbicides, and insecticides – they go by many names.  There are three primary potential routes of human exposure to agrochemicals: a) to the workers applying the product, b) bystanders that are nearby when the product is being used, and c) those eating food on which the product was used.  All of these scenarios need to be taken into account when assessing the safety of agrochemicals.

Testing Requirements. 

Agrochemical active ingredients are some of the most intensively toxicologically tested chemicals in the world and, as such, are assessed for genotoxicity among many other endpoints.  However, it’s not only the active ingredient itself that needs to be evaluated for safety.  Impurities in the active ingredient technical material, mammalian metabolites (either potential human metabolites or formed in animals eating the crop), metabolites occurring in the field or during food processing, or metabolites entering groundwater have to be assessed for safety and regulatory compliance.  Before considering that all chemicals used to synthesise the active substance may be covered under regulations such as REACH.

Typically, plant protection products are mixtures of the active ingredient and other chemicals in a formulation, including solvents, wetters, plant safeners, and other active ingredients.  Formulation safety is usually assessed based on the parts and potential additive effects if some substances have the same mode of action or toxicological target.  However, there are some exceptions, such as glyphosate formulations used in Europe, which had additional mixture genotoxicity testing considerations added when they were most recently reviewed.

Changes in Regulatory Requirements. 

Genotoxicity testing requirements for active ingredients are broadly similar across the different regions of the world. However, no globally harmonised organisation (such as the International Council for Harmonisation (ICH) for pharmaceuticals) standardises the approach.  However, taking into account all of the country and regional requirements, a minimum base set of Ames test, in vitro micronucleus assay, mammalian gene mutation assay (typically a mouse lymphoma assay or HPRT), and an in vivo micronucleus assay should be enough for global acceptance unless positive results in these or other studies indicate the need to generate follow-up data.  An example of this could be conducting a Comet assay in a tissue that showed an increase in tumours to determine whether a DNA-damaging mode action was likely responsible.

The lack of an ICH equivalent means that the change in genetic toxicology testing of agrochemicals is glacially slow.  As many authorities will only accept non-OECD, non-GLP studies for follow-up investigative purposes, only OECD guideline studies are typically conducted and used for regulatory submissions to ensure acceptability in all key regions.  As such, the in vitro micronucleus and in vivo Comet assays, despite being used in other industry sectors for many years before the OECD guideline was adopted, have only recently been used in the agrochemical sector.  Likewise, newer in vivo assays such as Pig-a have the same difficulties.

However, issuing and updating OECD guidelines in recent years has resulted in some changes, as evidenced by scientific investigations.  The one mainstay has been the venerable Ames test, which hasn’t been updated since the 1997 guideline was issued.  However, there have been changes in mammalian in vitro studies.  The introduction of the in vitro micronucleus guideline in 2010 eventually filtered through into most regulations and regulatory guidance, and it has now largely replaced the in vitro chromosome aberration assay.  In addition to being easier to score, the in vitro micronucleus assay has the advantage of detecting aneugens and clastogens, which results in a complete assessment.

The revisions of the HPRT assay test guideline have given it a new lease of life after the number of cells scored has been substantially increased, thereby reducing the concern of a potential lack of sensitivity compared to the mouse lymphoma assay (MLA).  HPRT studies were favoured by some companies over the mouse lymphoma assay long before the change; however, as it was still an OECD guideline study, they had a history of conducting it in preference to the mouse lymphoma assay – feeling that it resulted in less misleading positive results.  The mouse lymphoma assay detects a broader range of genotoxicants than the HPRT assay, as it can detect clastogens as well, typically detected as small colonies rather than large ones.  Given that clastogens should be detected by the in vitro micronucleus assay, this is essentially a redundant feature and can complicate the follow-up of an in vitro positive (perform a Comet or in vivo micronucleus, or both?).

The main changes for agrochemicals have come in the in vivo studies.  Until the OECD guideline for the transgenic rodent assay was adopted in 2013, the only realistic globally acceptable follow-up in vivo studies for an Ames test or mammalian gene mutation assay was the unscheduled DNA synthesis (UDS) assay, as the transgenic and Comet assays (and pig-a) didn’t have OECD guidelines in place at the time.  Adopting the transgenic assay was particularly relevant as the sensitivity of the UDS assay for genotoxic carcinogens had been questioned in the preceding years based on a comparison with the Comet assay.

Currently, for in vivo follow-up of a positive Ames, HPRT or mouse lymphoma assay, a transgenic or Comet assay is available, with the transgenic being considered more of a gold standard assay. Still, the Comet (DNA damage rather than point mutation assay) is faster, cheaper and more readily available.  Comparisons of Comet and transgenic studies have typically shown similar sensitivity (i.e. the ability to detect a positive response); however, these datasets are heavily biased towards positive compounds (already known mutagens) and look at few negative studies.  However, anecdotal evidence from the industry suggests that several unexpected positive Comet results have been seen, which don’t tie in nicely with existing repeat dose and carcinogenicity data, so they are considered to be misleading positives.  Pig-a is another in vivo assay that may be useful in the future but is in the early stages of developing an OECD guideline.

Regional differences exist in agrochemical genotoxicity testing. For instance, if an in vitro study produces a positive response, India requires two follow-up in vivo studies rather than a single study that would be expected to detect the same endpoint as the in vitro study (e.g., clastogenicity). Unfortunately, this results in greater use of animals, which would be required to satisfy the data requirements of most other nations.

Whilst in most regions, genotoxicity testing on formulated products is not required (on the basis the components have to be tested separately), Brazil require an Ames test and micronucleus test on formulations.  Recently, in vitro micronucleus studies have been considered acceptable and have replaced the minimum requirement for conducting an in vivo micronucleus assay.  An in vivo study may be required for follow-up and in vitro positive response.  In vivo positives are incredibly rare because the industry does not intentionally include in vivo positive substances in formulated products.

Advances in Testing Methods. 

Changes in study interpretation and an increasingly conservative evaluation of studies in recent years have resulted in many genotoxicity studies that were, in the past, or likely would previously be considered harmful, being called positive or equivocal by some European regulatory authorities.  In some cases, this is a result of using non-standard evaluation criteria or for in vitro mammalian studies considering anything that does rigidly meet the OECD guideline criteria as being not ‘clearly negative’ as being insufficient.  In many cases, there appears to be a reluctance to rely on expert scientific judgement and an overreliance on statistical methods meant to enhance rather than replace scientific interpretation.

Where data have been considered insufficient, the industry often has little option but to consider performing extra (and sometimes, in their judgment, unnecessary) animal testing to satisfy authorities.  Considering the number of genotoxicity studies conducted to satisfy agrochemical registration requirements, this can lead to substantial increases in animal testing.  Making sure your studies are performed professionally and with a high degree of rigour is essential.

Challenges and Future Directions. 

Despite the slow rate of change, genotoxicity requirements for agrochemicals have gradually evolved.  While it seems unlikely, there will be seismic changes any time soon, and that highly conservative assessment by some regulators will continue, there may be scope in future for new guidelines for the likes of Pig-a and ToxTracker to add to the interpretation of genotoxicity data.  A well-thought-through data generation strategy and evaluation of data by expert and experienced hands is essential for a successful registration.

Gentronix Services. 

Gentronix offers GLP and OECD guideline testing services and has extensive experience in the agrochemical sector. For more information, visit Gentronix.