Safety assessment for Tris(2,4-di-tert-butylphenyl) phosphite (Irgafos 168) used as an antioxidant and stabilizer in food contact applications.

During and after fabrication of polymeric food contact articles (FCA), polymers undergo oxidation by thermal decomposition processes initiated by oxygen, heat, light, shear, and catalyst residues. To reduce degradation of the polymer, a commonly used secondary antioxidant (AO), Irgafos 168 (I-168), may be included. Use of I-168 in polymeric FCAs presents a potential concern for neurotoxicity due to its phosphate-containing degradation species, I-168ate. As a result, we evaluated dietary exposure and oral toxicity data for I-168 and its degradants when used as an AO in FCAs. Our exposure assessment included extensive review of the U.S. food-contact regulatory history of I-168 resulting in a combined cumulative estimated daily intake (CEDI) of 0.09 mg/kg bw/day for I-168 and I-168ate when used as an AO in FCAs. Our comprehensive literature review of toxicological data and supporting structure activity relationship (SAR) analysis of I-168 reactivity against acetylcholinesterase diminished concern for potential neurotoxic effects of I-168 and its degradants. An acceptable daily intake (ADI) value of 1 mg/kg bw/day for I-168 was derived from a two-year rodent combined chronic toxicity/carcinogenicity study, which is higher than the CEDI and supports the safety of current authorized food contact use levels of I-168.

New insights into the growth response of the macrophyte Vallisneria natans exposed to phosphite.

Renewed interest in phosphite, an analog of phosphate, has increased due to its widespread distribution and increasing abundance in many waterbodies. However, up until recently very little is known about their ecological effects on aquatic organisms. Herein we studied the effects of phosphite via root and foliar exposure on the growth responses of the dominant pioneer macrophyte V. natans. Overall, both exposures of phosphite to V. natans resulted in significant reductions in the leaf length, root length, relative growth rate (RGR) and photosynthetic pigments, suggesting phosphite had an inhibitory effect on the plant growth. Our results further confirmed phosphite could induce the oxidative stresses in the V. natans cells, as indicated by the significantly increased intracellular enzyme activities i.e. superoxide dismutase activity (SOD) and malondialdehyde (MDA). Microscopic evidence also showed phosphite penetrated the cell membrane and destroyed membrane integrity under high phosphite stress. Besides, V. natans leaves exhibited intuitive deterioration symptoms, which seemed to be more sensitive to phosphite toxicity than roots. It is concluded that the increased abundance of phosphite in waterbodies cannot be utilized as a bioavailable P source but impose adverse physiological and metabolic limitations to plant growth, which should be receive more attention in the ecological risk assessment. Our result is necessary to build a comprehensive understanding of phosphite biogeochemical behaviors in aquatic ecosystems.

Considerations when deriving compound-specific limits for extractables and leachables from pharmaceutical products: Four case studies.

Leachables from pharmaceutical container closure systems are a subset of impurities that present in drug products and may pose a risk to patients or compromise product quality. Extractable studies can identify potential leachables, and extractables and leachables (E&Ls) should be evaluated during development of the impurity control strategy. Currently, there is a lack of specific regulatory guidance on how to risk assess E&Ls; this may lead to inconsistency across the industry. This manuscript is a cross-industry Extractables and Leachables Safety Information Exchange (ELSIE) consortium collaboration and follow-up to Broschard et al. (2016), which aims to provide further clarity and detail on the conduct of E&L risk assessments. Where sufficient data are available, a health-based exposure limit termed Permitted Daily Exposure (PDE) may be calculated and to exemplify this, case studies of four common E&Ls are described herein, namely bisphenol-A, butylated hydroxytoluene, Irgafos® 168, and Irganox® 1010. Relevant discussion points are further explored, including the value of extractable data, how to perform route-to-route extrapolations and considerations around degradation products. By presenting PDEs for common E&L substances, the aim is to encourage consistency and harmony in approaches for deriving compound-specific limits.

Evidence for rapid adaptive evolution of tolerance to chemical treatments in Phytophthora species and its practical implications.

Chemical treatments are used widely in agricultural and natural settings to protect plants from diseases; however, they may exert an important selection pressure on plant pathogens, promoting the development of tolerant isolates through adaptive evolution. Phosphite is used to manage diseases caused by Phytophthora species which include a large number of the most economically damaging plant pathogens worldwide. Phosphite controls the growth of Phytophthora species in planta without killing it; as a result, isolates can develop tolerance to phosphite after prolonged exposure. We investigated the inter- and intra-specific variability in phosphite tolerance of eleven Phytophthora species, including P. ramorum, an internationally important, highly regulated pathogen. Phytophthora ramorum is a good model system because it is comprised of multiple genetically homogeneous lineages. Seven species were found to be consistently sensitive to phosphite based on the low Effective Concentration (EC) 50 values of all isolates tested (amount of phosphite required to inhibit mycelial growth by 50% relative to growth in the absence of phosphite). However, P. ramorum, P. lateralis, P. crassamura and P. cambivora showed intraspecific variability in sensitivity to phosphite, with at least one isolate showing significantly higher tolerance than the other isolates. Within the three P. ramorum evolutionarily divergent lineages tested, NA1 was the most susceptible to phosphite, the NA1 and EU1 lineages showed intralineage variability and the NA2 lineage showed a decreased sensitivity to phosphite overall as all isolates were relatively tolerant. This finding is relevant because NA1 is dominant in the wild and can be controlled using phosphite, while the EU1 lineage has recently been identified in the wild and is phosphite-tolerant, making the treatment approach potentially less effective. Phytophthora ramorum, P. lateralis and P. crassamura are either selfing, homothallic species, or are known to reproduce exclusively clonally, indicating tolerance to phosphite can emerge even in the absence of sexual recombination.

A Novel Biocontainment Strategy Makes Bacterial Growth and Survival Dependent on Phosphite.

There is a growing demand to develop biocontainment strategies that prevent unintended proliferation of genetically modified organisms in the open environment. We found that the hypophosphite (H3PO2, HPt) transporter HtxBCDE from Pseudomonas stutzeri WM88 was also capable of transporting phosphite (H3PO3, Pt) but not phosphate (H3PO4, Pi), suggesting the potential for engineering a Pt/HPt-dependent bacterial strain as a biocontainment strategy. We disrupted all Pi and organic Pi transporters in an Escherichia coli strain expressing HtxABCDE and a Pt dehydrogenase, leaving Pt/HPt uptake and oxidation as the only means to obtain Pi. Challenge on non-permissive growth medium revealed that no escape mutants appeared for at least 21 days with a detection limit of 1.94 × 10-13 per colony forming unit. This represents, to the best of our knowledge, the lowest escape frequency among reported strategies. Since Pt/HPt are ecologically rare and not available in amounts sufficient for the growth of the Pt/HPt-dependent bacteria, this strategy offers a reliable and practical method for biocontainment.

Phosphite cannot be used as a phosphorus source but is non-toxic for microalgae.

Phosphorous (P) plays a critical role for all living organisms as a structural component of RNA, DNA and phospholipids. Microalgae are autotrophs organisms that have been reported to only assimilate the fully oxidized phosphate (Pi) as P source. However, there are microorganisms capable of utilizing P reduced compounds (i.e. phosphite (Phi) and hypophosphite) as a sole P source, such as bacteria and cyanobacteria. In this study, we evaluated whether microalgae, such as Chlamydomonas reinhardtii, Botryococcus braunii and Ettlia oleoabundans, are capable of using Phi as a sole P source. Our studies revealed that these three microalgae are unable to use Phi as a sole P source. We also found that when Phi is present at concentrations equal or higher than that of Pi, Phi has an inhibitory effect on C. reinhardtii growth. However, since C. reinhardtii was able to survive for a long period of cultivation in the presence of high concentrations of Phi and to recover cell division capacity after transfer to media containing Pi, we noticed that Phi is not toxic for this microalga. We propose that the inhibitory effect of Phi on C. reinhardtii growth might be caused, at least in part, by a competition between the transport of Pi and Phi.

House dust concentrations of organophosphate flame retardants in relation to hormone levels and semen quality parameters.

BACKGROUND: Organophosphate (OP) compounds, such as tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate (TPP), are commonly used as additive flame retardants and plasticizers in a wide range of materials. Although widespread human exposure to OP flame retardants is likely, there is a lack of human and animal data on potential health effects. OBJECTIVE: We explored relationships of TDCPP and TPP concentrations in house dust with hormone levels and semen quality parameters. METHODS: We analyzed house dust from 50 men recruited through a U.S. infertility clinic for TDCPP and TPP. Relationships with reproductive and thyroid hormone levels, as well as semen quality parameters, were assessed using crude and multivariable linear regression. RESULTS: TDCPP and TPP were detected in 96% and 98% of samples, respectively, with widely varying concentrations up to 1.8 mg/g. In models adjusted for age and body mass index, an interquartile range (IQR) increase in TDCPP was associated with a 3% [95% confidence interval (CI), 5% to 1%) decline in free thyroxine and a 17% (95% CI, 432%) increase in prolactin. There was a suggestive inverse association between TDCPP and free androgen index that became less evident in adjusted models. In the adjusted models, an IQR increase in TPP was associated with a 10% (95% CI, 219%) increase in prolactin and a 19% (95% CI, 30% to 5%) decrease in sperm concentration. CONCLUSION: OP flame retardants may be associated with altered hormone levels and decreased semen quality in men. More research on sources and levels of human exposure to OP flame retardants and associated health outcomes are needed.

Effects of dark-rearing on triphenyl phosphate-induced neuropathy in the visual system of the developing European ferret (Mustela putorius furo).

Results of a previous study in our lab (Tanaka et al., 1994) suggested that the onset of susceptibility to the organophosphorus compound triphenyl phosphite (TPP) in the developing ferret visual system might be closely related to eye opening and the onset of light stimulation. In order to explore this idea further, TPP was administered to ferret kits that had been raised for varying periods of time in total darkness to assess whether a delay in the onset of light stimulation to the visual system might also result in a delay in its susceptibility to TPP. Ferret kits were raised from birth either in total darkness or in open-sided sheds exposed to ambient light, injected subcutaneously with TPP (888 mg/kg body weight) at 5.5, 7.5, 9.5, or 21.5 wk of age, euthanized, and perfused transcardially with a 10% formalin-saline solution 4 d after injection. Brains were sectioned parasagittally at a thickness of 40 microm and subsequently processed with the Fink-Heimer silver impregnation technique to reveal the presence of degenerating axons and terminals, and with cresyl violet stain to delineate nuclear boundaries and cell soma morphology. Comparisons among degeneration patterns present in light-reared and dark-reared kits at the four ages examined revealed that the time of onset, extent, and density of TPP-induced axonal and terminal degeneration seen in the lateral geniculate nucleus and primary visual cortex did not differ significantly between light- and dark-reared groups, with the possible exception of dark-reared kits exposed to TPP at 7.5 wk of age. In addition, neurons in the primary visual cortex showed shrinkage and increased packing densities in kits exposed to TPP in both light and dark environments, as well as in dark-reared non-injected kits. The results of this study indicate that dark-rearing does not delay the onset or lessen the severity of TPP-induced axonal and terminal degeneration in the developing visual system of the ferret. Data suggest that light activation and stimulation of the retino-geniculo-striatal visual pathway is not a necessary prerequisite for the onset of visual system susceptibility to the axonopathic effects of triphenyl phosphite.

Cholinesterase activity in quails of neuropathy caused by organophosphates.

It is known that some organophosphates produce not only well-known acute toxicity but also characteristic delayed neurotoxicity. Tri-ortho-tolyl phophate (TOTP), which was formerly named Tri-ortho-cresyl phosphaete (TOCP), was first noticed in an incident of poisoning as the compound which produced organophosphate induced delayed neurotoxicity (OPIDN). It is said that triphenyl phosphite (TPP) is also one of the organophosphates which possesses OPIDN. However, it is thought that TPP-induced delayed neurotoxicity (TPP-DN) is not identical with classical OPIDN. An intermediate syndrome was later proposed as the third neurotoxicity caused by organophosphates. We think that TPP is a model chemical of the third neurotoxicity. We compared TOTP with TPP using Japanese quails. We measured cholinesterase (ChE) activity and clearly demonstrated the difference between the two chemicals, that is to say, the activity recovered after 72 hrs from the administration of TPP, whereas the inhibition continued for more than 11 days after the administration of TOTP.

Acute effects of a bicyclophosphate neuroconvulsant on monoamine neurotransmitter and metabolite levels in the rat brain.

Naive male Sprague-Dawley rats were injected intraperitoneally (i.p.) with the bicyclophosphate convulsant trimethylolpropane phosphate (TMPP) at dose levels from 0.2 to 0.6 mg/kg. Rats were observed for convulsive activity, and were sacrificed 15 min posttreatment. Levels of the monoamine neurotransmitters norepinephrine (NE), epinephrine (EPI), dopamine (DA), and serotonin (5-HT) and the major metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) were assayed in forebrain, midbrain, hindbrain, cerebellum and brainstem regions. Neurotransmitter and metabolite levels were compared between control rats and rats that did and did not experience seizures. TMPP administration induced significant decreases in levels of measured neurotransmitters that varied as a function of brain region, dose, and expression of the seizure activity. These results show that tonic or tonic-clonic seizures induced by TMPP administration (0.6 mg/kg) are reliably associated with regional decreases in serotonin, dopamine, and norepinephrine. Convulsive activity resulting from lower dose administrations (0.2-0.4 mg/kg) of TMPP result only in decreased regional levels of serotonin.

Triphenyl phosphite and diisopropylphosphorofluoridate produce separate and distinct axonal degeneration patterns in the central nervous system of the rat.

This study compared the neurotoxic effects of triphenyl phosphite (TPP) in the rat with those seen after exposure to diisopropylphosphorofluoridate (DFP), a compound known to produce organophosphorus-induced delayed neurotoxicity (OPIDN). Animals received either three subcutaneous injections of TPP (1184 mg/kg body wt each dose) administered at 3-day intervals or a single subcutaneous injection of DFP (4 mg/kg body wt). TPP-induced clinical signs were initially observed 2 to 18 days after the last injection and included ataxia, flaccid paresis, stereotyped alternating side-to-side movements, and circling behavior. Axonal and terminal degeneration were present in the cerebellum, vestibular nuclear complex, cochlear nuclei, and superior and inferior colliculi. The subthalamic nucleus, substantia nigra, septal region, hypothalamus, thalamus, hippocampus, and cerebral cortex also contained degenerating axons and terminals. Degeneration was particularly evident in the sensorimotor cerebral cortex, mediodorsal, ventromedial, and medial geniculate thalamic nuclei and in the magnocellular preoptic and medial mammillary nuclei of the hypothalamus. Very light degeneration was present in the gracile fasciculus and nucleus. In contrast, rats injected with DFP showed moderate degeneration in the gracile fasciculus and nucleus but did not display degeneration in any other brain region. Injections of DFP did not produce delayed onset clinical signs. The results indicate that in the rat, different central nervous system cell groups are affected by these two organophosphorus compounds and that TPP affects nuclei and tracts at all levels of the neuraxis, including those associated with higher-order processing and cognitive functions. In addition, the distinct degeneration patterns produced by these two compounds support the view that TPP-induced neurotoxicity should not be considered as a type of OPIDN, but rather as a separate category of organophosphorus-induced neurotoxicity.

[A review of studies of the delayed neurotoxicity induced by organophosphorus esters].

Organophosphorus esters have been used in the plastics industry as antioxidants and plasticizers, in agriculture as insecticides, and in the military as nerve agents. Some of these compounds have organophosphorus ester-induced delayed neurotoxicity (OPIDN) different from the acute toxicity caused by the acetylcholine esterase inhibiting activity. this review describes recent progress in studies on OPIDN and, discusses the future direction of studies. OPIDN is characterized by a more than 7 day incubation period, lower limb paralysis accompanied by axonal degeneration, and age- and species-specificity. Younger animals and rodents are not very sensitive to OPIDN. As well as fast recovery of inhibited neurotoxic esterase or neuropathy target esterase (NTE) in the sciatic nerve, detoxicating mechanisms including carboxylesterases are contributing to age- and species-specificity for OPIDN. Although, anterograde axonal transport does not seem to be affected by OPIDN, slow down of retrograde axonal transport was observed. Inhibition of NTE, and aging of inhibited NTE has been thought to be responsible for OPIDN, but there are some arguments against the role of NTE in OPIDN. Phosphorylation of cytoskeletal proteins by kinases such as calcium dependent-calmodulin kinase II and/or high affinity neurotoxic compound binding site(s) are possible candidates for the initiation of OPIDN. Triphenyl phophite (TPP), a compound commonly used in the plastics industry, has delayed neurotoxicity that is somewhat different from OPIDN. The onset of TPP-induced neuropathy is earlier than that of OPIDN, and rodents are sensitive to TPP. In addition to the axonal damage, cell damage is observed in TPP-induced neuropathy. Mitochondrial energy metabolism-related enzymes could be the target of this neuropathy. Future studies should be focused on the relation of OPIDN to the phosphorylation of cytoskeletal proteins and high affinity binding site(s), and on the development of rodent models. These studies would answer the questions related to OPIDN, and further contribute toward elucidating the pathogenesis of degenerative neuronal diseases.

Triphenyl phosphite-induced impairment of spatial alternation learning.

Triphenyl phosphite (TPP) is a weak acetylcholinesterase inhibitor and a type II organophosphorus compound-induced delayed neurotoxic agent. The current study examined the cognitive effects of a single 250 mg/kg ip dose of TPP administered to either 3-mo- or 1-yr-old male Sprague-Dawley rats. Starting 4 d after TPP administration, the rats began training on a T-maze spatial alternation task for food reinforcement. Over five sessions of acquisition training, the TPP-treated rats showed significantly lower alternation scores than controls. There was no difference in spatial alternation performance in the first session, when both groups were performing at near-chance levels. In sessions 2-5, the controls improved dramatically to an average of 85.3 +/- 3.2% correct, while the TPP-treated rats did not significantly change, with 69.7 +/- 3.1 percent correct. During sessions 2 and 3 there was a significant TPP treatment-related deficit. This TPP-induced choice accuracy deficit was persistent in that it was seen well after the acute exposure. With continued training the TPP-exposed rats were able to learn the task as well as controls. There were no significant TPP effects on response latency. These data show that acute TPP administration has persistent effects of impairing T-maze learning that do not appear to result from effects on motor function.

Organophosphorus-induced delayed neurotoxicity: a comparative study of the effects of tri-ortho-tolyl phosphate and triphenyl phosphite on the central nervous system of the Japanese quail.

The neurotoxic effects of single oral doses of tri-ortho-tolyl phosphate (TOTP) (500 mg/kg body weight) or single subcutaneous injections of triphenyl phosphite (TPP) (62.5-500 mg/kg body weight) were investigated in the Japanese quail (Coturnix coturnix japonica). Oral doses of TOTP resulted in no detectable clinical signs while injections of TPP resulted in mild ataxia to severe paralysis depending upon the dosage level. At 24 hr postdosing, whole-brain activity of neuropathy target esterase (NTE) was inhibited by 90% in birds exposed to TOTP and by 11-87% in birds injected with TPP. Oral doses of TOTP resulted in only sparse Fink-Heimer silver-impregnated degeneration in the white matter of the cerebellum with no degeneration noted in any other region of the brain. Injections of TPP resulted in widespread degeneration in large numbers of brainstem nuclei and tracts and in all cerebellar foliae and deep nuclei. These results indicate that the Japanese quail responds differentially to exposure to TOTP and TPP. Oral doses of TOTP do not result in clinical signs or in significant amounts of degeneration in the brain even though NTE activity is inhibited by 90%. In contrast, injections of TPP at higher dosage levels yield severe clinical signs, widespread axonal and terminal degeneration in the CNS, and significant inhibition of NTE activity. This sharp dichotomy in relative sensitivity to TOTP and TPP in the Japanese quail suggests that each compound may have its own unique effect on CNS structure and function. In addition, the relationship between levels of NTE inhibition and the onset of clinical signs or neuropathology remains unclear.

Triphenylphosphite neuropathy in hens.

Single doses of triphenyl phosphite (TPP), a triester of trivalent phosphorus, cause ataxia and paralysis in hens. Characteristics of neurotoxicity were described as somewhat different from organophosphate induced delayed polyneuropathy (OPIDP), which is caused by triesters of pentavalent phosphorus. The onset of TPP neuropathy was reported to occur earlier than that of OPIDP (5-10 versus 7-14 days after dosing, respectively), and chromatolysis, neuronal necrosis and lesions in certain areas of the brain were found in TPP neuropathy only. Pretreatment with phenylmethanesulfonyl fluoride (PMSF) protects from OPIDP, but it either partially protected from effects of low doses or exacerbated those of higher doses of TPP. In order to account for these differences with OPIDP, it was suggested that TPP neuropathy results from the combination of two independent mechanisms of toxicity: typical OPIDP due to inhibition of neuropathy target esterase (NTE) plus a second neurotoxicity related with other target(s). We explored TPP neuropathy in the hen with attention to the phenomena of promotion and protection which are both caused by PMSF when given in combination with typical neuropathic OPs. When PMSF is given before neuropathic OPs it protects from OPIDP; when given afterwards it exaggerates OPIDP. The former effect is due to interactions with NTE, the latter to interactions with an unknown site. The time course of NTE reappearance after TPP (60 or 90 mg/kg i.v.) inhibition showed a longer half-life when compared to that after PMSF (30 mg/kg s.c.) (10-15 versus 4-6 days, respectively). The clinical signs of TPP neuropathy (60 or 90 mg/kg i.v.) were similar to those observed in OPIDP, appeared 7-12 days after treatment, correlated with more than 70% NTE inhibition/aging and were preceded by a reduction of retrograde axonal transport in sciatic nerve of hens. TPP (60 mg/kg i.v.) neuropathy was promoted by PMSF (120 mg/kg s.c.) given up to 12 days afterwards and was partially protected by PMSF (10-120 mg/kg s.c.) when given 24 h before TPP (60 or 90 mg/kg i.v.). The previously reported early onset of TPP neuropathy might be related to the higher dose used in those experiments and to the resulting more severe neuropathy. The lack of full protection might be explained by the slow kinetics of TPP, which would cause substantial NTE inhibition when PMSF effects on NTE had subsided. Since PMSF also affects the promotion site when given before initiation of neuropathy, the resulting neuropathy would then be due to both protection from and promotion of TPP effects by PMSF. No promotion by PMSF (120 mg/kg s.c.) was observed in TPP neuropathy (90 mg/kg i.v.) partially protected by PMSF (10-30 mg/kg s.c.). This might also be explained by the concurrent effects on NTE and on the promotion site obtained with PMSF pretreatment. We conclude that TPP neuropathy in the hen is likely to be the same as typical OPIDP. The unusual effects of combined treatment to hens with TPP and PMSF are explained by the prolonged pharmacokinetics of TPP and by the dual effect of PMSF i.e. protection from and promotion of OPIDP.

Age-related effects of triphenyl phosphite-induced delayed neuropathy on central visual pathways in the European ferret (Mustela putorius furo).

The objective of this study was to investigate the relationship between the maturation of visual system neurons and the onset of their susceptibility to triphenyl phosphite (TPP)-induced delayed neurotoxicity in the European ferret. We administered single subcutaneous doses of TPP (1184 mg/kg body wt) to 1- to 10-week-old ferret kits to assess the effects on connections and neurons of the developing lateral geniculate thalamic nucleus (LGN) and primary visual cortex. Brains were processed with a modified Fink-Heimer silver-impregnation method. Axonal and terminal degeneration were first noted in the LGN of kits injected at 5 weeks of age. The severity of the degeneration increased in kits injected at later ages and reached adult densities and configurations in ferrets injected at 10 weeks of age. Degenerating neuronal cell bodies were also present in the LGN of kits injected at 7 weeks of age and older. In the visual cortex, axonal and terminal degeneration were consistently present in kits injected at 8 weeks of age and attained adult-like densities in kits injected at 10 weeks of age. Previous studies have reported that the ferret visual system appears to reach anatomical maturity (as defined by mature LGN lamination patterns, the location and density of axon terminals originating from neurons in the retina and LGN, and the migration and synaptic connections of cortical neurons) by 4-5 weeks of age. A temporal comparison of these normal developmental data with the degeneration data obtained in the present study suggests that immature neurons in the visual system of the ferret are not susceptible to TPP-induced delayed neurotoxicity but only become so after they have achieved some degree of maturity. Whether the LGN neurons undergoing degeneration are directly affected by TPP or are showing a transneuronal response to loss of afferent input remains unresolved.