Organophosphorus compounds (OPs) are widespread in both the natural and industrial worlds. Being major components of DNA and cell membranes, their diverse chemical properties are fundamental for the biology of life. But they also find uses in a range of anthropogenic applications, for example, as flame retardants, plasticisers, antioxidants and, perhaps most famously, as pesticides.
OP pesticides and acetylcholine esterase inhibition
OPs are among the most widely used pesticides around the world. They all function by blocking acetylcholine esterase, a crucial enzyme responsible for removing acetylcholine after this chemical messenger has done its job and transmitted a nerve signal. When this enzyme is not functional, acetylcholine will overstimulate the nerves, potentially ultimately leading to paralysis and death of the organism. This mechanism is very well characterised and is the reason why so many OPs have a high degree of acute toxicity.
This high degree of acute neurotoxicity has been acknowledged in the realm of human health risk assessment. For example, OPs were removed from the dataset of Kroes et al. (2004) when deriving their oral Threshold of Toxicological Concern (TTC) values based on Cramer classifications. Later, the European Food Safety Authority (EFSA) proposed a new (and very low) TTC especially for OPs. In a Technical Specification from the International Organization for Standardization (ISO), providing guidance on the use of the TTC in the risk assessment of chemical constituents from medical devices, OPs are regarded as a cohort of concern, and thus are exempt from the standard TTC approach.
But in addition to these acute cholinergic effects, some OPs can cause other harms. One such effect is known as Organophosphorus Induced Delayed Neurotoxicity (OPIDN).
History of OPIDN
Back in the prohibition era in the US, in an attempt to find a substitute for alcoholic beverages, many Americans consumed tri-ortho-cresyl phosphate (TOCP), a gasoline additive considered to have a low degree of acute toxicity. But as the weeks passed, tens of thousands of TOCP consumers found that they were suffering from persistent muscle weakness and abnormal gait. Although some consumers recovered within a few years, a number of unfortunate individuals eventually lost the ability to walk. It became clear that TOCP was capable of inducing severe delayed neurotoxicological effects. Moreover, this delayed neuropathy appeared to be acting independently of acetylcholine esterase inhibition.
Since then, there have been thousands more cases of OPIDN associated with exposure to TOCP as well as other OPs.
Characterising OPIDN
OPIDN is characterised by a delayed manifestation of ataxia (poor muscle control and lack of coordination) and axonal degeneration. Humans appear to be very sensitive to the effects of OPIDN compared to other species including rats and mice; chickens and cats on the other hand seem to be sensitive to OPIDN.
A variety of OPs are capable of causing OPIDN, including OPs with phosphorus in both common oxidation states of +3 and +5. In some cases the chemical configuration appears critical; while TOCP (the ortho-isomer) is a potent inducer of delayed neuropathy, the meta- and para-isomers of tricresyl phosphate are not. On the other hand, triphenyl phosphite (without any ortho-methyl ring substituents) does induce OPIDN.
Established test methods
Organisation for Economic Co-operation and Development (OECD) officials have published two Test Guidelines (TGs) specifically for the identification of OPIDN. In these assays, chickens are the recommended test species. In TG 418, a single non-lethal oral dose is administered and the hens are observed for 21 days to detect signs of ataxia. Histopathology examinations are then performed to identify any nerve damage. In addition, an assay is performed to detect inhibition of Neuropathy Target Esterase (NTE), as inhibition of this enzyme in brain and spinal cord reportedly correlates well with the clinical and morphological effects seen 10-20 days later. If evidence of OPIDN is identified, TG 419 (an analogous study involving repeated administrations to groups of chickens for 28 days) can be used in order to define a No-Observed-Adverse-Effect Level (NOAEL). TOCP is the recommend positive control substance for both test methods.
When are such tests required?
According to the European Biocidal Products Regulation (BPR), these OECD test methods should be conducted for any OP being registered as an active substance. As noted above, OPs have been very popular in pesticide and biocide products but, given their history of causing a range of adverse health effects including delayed neurotoxicity, there is clearly a need for the identification of OPIDN-inducing compounds. Moreover, the more traditional repeated dose oral toxicity studies in rats and mice (also a requirement for biocides and pesticides) are not able to reliably detect OPIDN effects. These test methods should also be considered when registering an OP under REACH.
But apart from in the context of BPR and REACH, OPIDN seems to have fallen under the radar in the hazard and risk assessment of OP compounds.
This is particularly concerning given that OPs are common additives in (and potential leachables from) polymeric materials that are then used in a variety of applications, including in food-contact materials, medical devices and pharmaceutical packaging.
As a result of their widespread use, certain OPs are commonly identified as extractables and/or leachables.
Irgafos 168
One common OP extractable is tris(2,4-di-tert-butylphenyl) phosphite, otherwise known as Irgafos 168. In a recently published safety assessment of Irgafos 168 authored by US FDA scientists, the potential for this OP (and its oxidation product) to cause delayed neurotoxicity was acknowledged. However, as the authors remarked that OPIDN was a result of OPs’ interaction with acetylcholine esterase, there is clearly some confusion surrounding the topic, even amongst regulatory toxicologists. Nevertheless, based on the results of an oral study in hens, the FDA scientists were reassured that OPIDN was not a concern for Irgafos 168 as a food-contact material additive (Markley et al., 2023).
Irgafos 168 has also been identified as a common extractable in pharmaceutical products. In a publication authored by scientists from the Extractables and Leachables Safety Information Exchange (ELSIE) consortium, a Permitted Daily Exposure (PDE) of 2.9 mg/day was derived for the parenteral route of exposure based on a long-term oral toxicity study in rats (Parris et al., 2020). Although the authors were reassured that neurotoxicity was not a concern (again, based on the same oral study in hens), given that Irgafos 168 is very poorly absorbed via the gastrointestinal tract, do Irgafos 168 and its oxidation product have the potential to cause OPIDN when administered via injection? As Irgafos 168 is so prevalent in pharmaceutical and medical devices E&L studies, this possibility seems worth investigating.
Hazard and risk assessment of OPs
Many other OPs are not so well studied, so their OPIDN potential remains unknown. Structure-Activity-Relationship (SAR) analysis could possibly provide a useful method for OPIDN identification in the future, but this is an area that requires further research.
In any case, the potential for OPs to cause OPIDN needs to be addressed in human health risk assessment. But how?
Data generated with TOCP, the recommended positive control material for the OECD OPIDN test methods, might provide a useful benchmark for OP risk assessors. In a key hen study, delayed neuropathy was induced in chickens exposed to TOCP at 2.5 mg/kg bw/day for 90 days, and ataxia was observed at higher doses. A NOAEL of 1.25 mg/kg bw/day was established. German scientists from the Deutsche Forschungsgemeinschaft (DFG) considered this NOAEL as a key Point of Departure (PoD) when deriving their candidate Maximum Workplace Concentration (MAK) values for TOCP.
In a US Environmental Protection Agency (EPA) evaluation of diisopropyl methylphosphonate (DIMP), it was noted that an unsteady gait (indicative of OPIDN) was observed in hens exposed to DIMP at various dose levels. Although blood effects seen in a 90-day study in mink were used as the basis of a subchronic oral Reference Dose (RfD) of 1 mg/kg bw/day, the EPA decided to incorporate a factor of 3 into the RfD derivation to account for the neurotoxicity potential of DIMP.
The future of OP assessment
OPIDN is an important toxicological feature worth considering for all OPs. The characteristic delayed neuropathy may be missed in standard repeated dose toxicity studies in rodents, hence the preference for hens as the test species of choice in dedicated OECD TGs. In addition, OPIDN should be treated as a separate phenomenon to OP-induced cholinergic toxicity, and the two mechanisms of action are not mutually exclusive. More research is required to investigate OPIDN and develop hazard identification methods (e.g. SAR), and OPs probably deserve more attention in health risk assessment, where OPIDN is often overlooked or overshadowed by their more infamous acute cholinergic neurotoxicity.
If you need support in the human health risk assessment of phosphorus-containing organic compounds then please don’t hesitate to get in touch with us.
References
Kroes R, Renwick AG, Cheeseman M, Kleiner J, Mangelsdorf I, Piersma A, Schilter B, Schlatter J, van Schothorst F, Vos JG and Würtzen G (2004). Structure-based thresholds of toxicological concern (TTC): guidance for application to substances present at low levels in the diet. Food and Chemical Toxicology 42(1), 65-83.
Markley LC, González Bonet AM, Ogungbesan A, Bandele OJ, Bailey AB and Patton GW (2023). Safety assessment for Tris(2,4-di-tert-butylphenyl) phosphite (Irgafos 168) used as an antioxidant and stabilizer in food contact applications. Food and Chemical Toxicology 178:113877.
Parris P, Martin EA, Stanard B, Glowienke S, Dolan DG, Li K, Binazon O, Giddings A, Whelan G, Masuda-Herrera M, Bercu J, Broschard T, Bruen U, Callis CM, Stults CLM, Erexson GL, Cruz MT and Nagao LM (2020). Considerations when deriving compound-specific limits for extractables and leachables from pharmaceutical products: Four case studies. Regulatory Toxicology and Pharmacology 118:104802.