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, 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 which the product was used on.  All of these scenarios need to be taken into account when assessing the safety of agrochemicals.

Testing of agrochemicals

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, its not only the active ingredient itself that needs to be assessed 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.  And that is before considering that all of the 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 – which may include solvents, wetters, plant safeners, and quite often other active ingredients.  Formulation safety is usually assessed based on the component parts, and potential additive effects if some of the 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 it was most recently reviewed.

Genotoxicity testing requirements for active ingredients are broadly similar across the different regions of the world, although there is no globally harmonised organisation (such as the International Council for Harmonisation (ICH) for pharmaceuticals) that 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 which showed an increase in tumours to determine whether a DNA damaging mode action was likely to be responsible.

Changes in regulatory requirements

The lack of an ICH equivalent means that 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, typically only OECD guideline studies are conducted and used for regulatory submissions, to ensure acceptability in all key regions.  As such the likes of 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 in more recent time been used in the agrochemical sector.  Likewise, the use of newer in vivo assays such as Pig-a have the same difficulties.

However, issuing and updating of OECD guidelines in recent years have resulted in some changes, as has evidence from 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 the 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.  As well as being easier to score, the in vitro micronucleus assay has the advantage of being able to detect aneugens as well as clastogens, so results in a more 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 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 and 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, which are typically detected as small colonies, rather than large.  Given that clastogens should be detected by the in vitro micronucleus assay this is largely a redundant feature, and can complicate follow-up of an in vitro positive (perform a Comet or in vivo micronucleus, or both?).

Changes in in vivo genotoxicity studies

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.  The adoption of the transgenic assay was particularly relevant as sensitivity of the UDS assay for genotoxic carcinogens had been questioned in the preceding years, based on comparison with the Comet assay.

Currently, for in vivo follow-up of a positive Ames, HPRT or mouse lymphoma assay, use of a transgenic or Comet assay are available, with the transgenic being considered more of a gold standard assay, but 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. ability to detect a positive response); however, these datasets are heavily biased towards positive compounds (already known mutagens) and look at few negative studies.  Anecdotal evidence from industry, however, suggests that a number of unexpected positive Comet results have been seen, which don’t tie in well 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 of use in future but is in the early stages of getting an OECD guideline developed.

Diverging regional requirements

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

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

Challenges in Europe

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 negative, being called positive or equivocal by some Europeans regulatory authorities.  In some cases, this is as 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 which are meant to enhance rather than replace scientific interpretation.

Where data have considered insufficient this often leaves industry with little option but to consider performing extra (and sometimes in their judgement unnecessary) animal testing in order 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 conducted professionally and to a high degree of rigour is essential.

The future for agrochemical genotoxicity testing

Despite the slow rate of change, genotoxicity requirements for agrochemicals has 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 adding 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 are a specialist genotoxicity testing services laboratory and have provided GLP and OECD guideline testing services for sectors including the agrochemical industry for several years, and screening services for over a decade.  In addition, several of the team have experience of working in the agrochemical industry in the past, including in global toxicology department roles.

For more information on this subject, please read Gentronix overview of the regulatory requirements for genotoxicity assessment of plant protection product active ingredients, impurities, and metabolites.