Agrochemical Formulations Research Articles

Engineering and use of proteinoid polymers and nanocapsules containing agrochemicals

To address global challenges such as population growth and climate change, introduction of new technologies and innovations in agriculture are paramount. Polymer-based formulations of agrochemicals have received much attention in recent years, and there is strong motivation to develop agrochemicals that are not harmful to the environment. Proteinoid polymers are produced by thermal step-growth polymerization of natural and unnatural amino acids. Under suitable gentle conditions, the proteinoid polymers may self-assemble to form nano-sized hollow proteinoid nanoparticles (NPs) of a relatively narrow size distribution. Agrochemical molecules may be encapsulated within these hollow proteinoid NPs, integrated in the crude proteinoid shell, or bound covalently/physically to the NP surface. In the present manuscript we prepared and characterized four model proteinoid polymers and NPs: P(KEf), P(KF), P(EWH-PLLA) and P(KWH-PLLA), where Ef denotes the unnatural herbicidal amino acid glufosinate. The NPs were fluorescently labeled and loaded with agrochemicals such as the plant hormone auxin. In addition, the NP surface was hydrophobized by covalent conjugation of dodecyl aldehyde via its surface primary amine groups. Following treatment of the plants with the different fluorescent-labeled NPs, fluorescent microscopic techniques enabled to localize the NPs and observe the accumulation in the plant’s vascular system. Next, using genetically modified plants, which express fluorescent protein and are responsive to the level of auxin, we demonstrated the possibility to deliver encapsulated agrochemicals into cells. We also illustrated that the proteinoid NPs are non-toxic to human umbilical vein endothelial cells, and apart from P(KEf) also to lettuce plants.

Innovative strategy based on mechanisms to substitute animal testing for ocular toxicity assessment of agrochemical formulations market in Brazil

Considering the successful employment of alternative methods for eye toxicity assessment of products for regulatory purposes, and the recent advances in Brazilian legislative scenario, which adopted the UN GHS classification system for agrochemical formulations toxicity assessment, there is an emerging demand for strategies that allow the evaluation of such products. Based on this, the present study aimed to address the applicability of a mechanistic-based defined approach for eye toxicity assessment of agrochemical formulations. It was investigated the opacity/permeability, depth and location of corneal injury in bovine cornea, and vascular events in chorioallantoic membrane induced for different Brazilian agrochemicals using a Sequential Testing Strategy (STS). Cytotoxicity induced by the agrochemical formulations was evaluated by Short Time exposure (STE) (OECD TG 491) assay (step 1), corneal injury was investigated by standard Bovine Corneal Opacity and Permeability (BCOP) (OECD TG 437) followed by histopathological evaluation (step 2), and Hen Chorionic-allantoic Membrane test (HET-CAM) was used to evaluate vascular injury (step 3). The results demonstrated that the proposed defined approach enabled a classification corresponding UN GHS classification of agrochemical formulations while minimizing the use of live animals. Therefore, this approach may be useful for categorization of agrochemicals in Brazil according to the new regulatory scenario.

Evaluation of extraction procedures applied to apprehended formulations of agrochemicals

Illicit formulations of agrochemicals comprise hazardous substances that have to be fully identified for forensic purposes. Nonetheless, analysis may be hampered by their complex matrix and few available methodologies. In this sense, the aim of this work was to evaluate the extraction of three apprehended agrochemicals using dichloromethane or acetonitrile associated with liquid–liquid (LLE), ultrasonic bath (UBAE), ultrasonic probe (UPAE) and microwave-assisted extraction (MAE) by means of chromatographic approaches. Results showed that, in certain conditions, dichloromethane could not extract the active ingredient, which was possible using acetonitrile. Moreover, LLE and UBAE extracted, respectively, 0.55 ± 0.03 and 2.86 ± 0.19 mg mL−1 of quinclorac and were generally inefficient compared to UPAE and MAE that reached, respectively, 3.54 ± 0.18 and 3.31 ± 0.16 mg mL−1 of quinclorac. Therefore, solvent and extractive protocol were crucial factors that influenced the extraction efficiency of agrochemicals, which have to be carefully evaluated in order to obtain adequate results.

An integrated transcriptomic- and proteomic-based approach to evaluate the human skin sensitization potential of glyphosate and its commercial agrochemical formulations

We investigated the skin sensitization hazard of glyphosate, the surfactant polyethylated tallow amine (POEA) and two commercial glyphosate-containing formulations using different omics-technologies based on a human dendritic cell (DC)-like cell line. First, the GARD™skin assay, investigating changes in the expression of 200 transcripts upon cell exposure to xenobiotics, was used for skin sensitization prediction. POEA and the formulations were classified as skin sensitizers while glyphosate alone was classified as a non-sensitizer. Interestingly, the mixture of POEA together with glyphosate displayed a similar sensitizing prediction as POEA alone, indicating that glyphosate likely does not increase the sensitizing capacity when associated with POEA. Moreover, mass spectrometry analysis identified differentially regulated protein groups and predicted molecular pathways based on a proteomic approach in response to cell exposures with glyphosate, POEA and the glyphosate-containing formulations. Based on the protein expression data, predicted pathways were linked to immunologically relevant events and regulated proteins further to cholesterol biosynthesis and homeostasis as well as to autophagy, identifying novel aspects of DC responses after exposure to xenobiotics. In summary, we here present an integrative analysis involving advanced technologies to elucidate the molecular mechanisms behind DC activation in the skin sensitization process triggered by the investigated agrochemical materials. SignificanceThe use of glyphosate has increased worldwide, and much effort has been made to improve risk assessments and to further elucidate the mechanisms behind any potential human health hazard of this chemical and its agrochemical formulations. In this context, omics-based techniques can provide a multiparametric approach, including several biomarkers, to expand the mechanistic knowledge of xenobiotics-induced toxicity. Based on this, we performed the integration of GARD™skin and proteomic data to elucidate the skin sensitization hazard of POEA, glyphosate and its two commercial mixtures, and to investigate cellular responses more in detail on protein level. The proteomic data indicate the regulation of immune response-related pathways and proteins associated with cholesterol biosynthesis and homeostasis as well as to autophagy, identifying novel aspects of DC responses after exposure to xenobiotics. Therefore, our data show the applicability of a multiparametric integrated approach for the mechanism-based hazard evaluation of xenobiotics, eventually complementing decision making in the holistic risk assessment of chemicals regarding their allergenic potential in humans.

Effect of Carrier Oil and Co-Solvent on The Formation of Clove Oil Nanoemulsion by Phase Inversion Technique

Development of nanoemulsion is gaining considerable attention for use in delivering hydrophobic constituents such as clove oil in foods and agriculture system. The small size of the oil droplets in the nanoemulsion system offers many advantages such as high stability, optical clarity, and improved water solubility and bioactivity. This research was aimed at investigating the effect of incorporation of carrier oil and co-solvent on the formation of clove nanoemulsion. Clove oil-loaded nanoemulsions were prepared by a low energy – phase inversion technique involving a carrier oil (medium chain triglyceride/MCT), at different ratios to the clove oil (1:2, 1:1, 2:1), a co-solvent (glycerol) at ratios of 0 and 1:1 to the mixture of clove oil and MCT and a non-ionic surfactant (Tween 80)at a ratio of 1:1, with two concentration levels of mixture of clove oil and MCT (5% and 10%). The formation and characteristics of nanoemulsion were evaluated including particle size, polydispersity index, zeta potential, and freeze-thaw stability, as well as their possible mechanisms of destabilization. Particle sizes ranged from 45.98 to 220 nm with narrow ranges of polydispersity index (0.072 – 0.286) and zeta potentials (-12.8 and - 22.6 mV). Incorporation of carrier oil at low proportions gave smaller size of oil droplets, and the presence co-solvent enhanced nanoemulsion stabilization. Creaming accompanied by oiling- off was found upon destabilization of nanoemulsion with different rate and appearance as influenced by nanoemulsion composition. This study provides important information about stabilization of nanoemulsion by incorporating carrier oil and co-solvent suitable for foods and agrochemical formulation.

Effects of herbicide mixtures on freshwater microalgae with the potential effect of a safener

Freshwater microalgae are primary producers and cosmopolitan species subjected to the effects of herbicides. In this work, thein vitroalgal growth inhibitory effects of 11 agrochemicals (9 herbicides, 1 metabolite, and 1 safener) were quantified. Chemical compounds were applied singly and in specific mixtures. Three species were used in axenic condition: the green algaDesmodesmus subspicatus(Chodat), the diatomsNitzschia palea(Kützing) W. Smith andNavicula pelliculosa(Kützing) Hilse. When exposed to single compounds,N. paleaandN. pelliculosawere only sensitive to atrazine/desethylatrazine and the safener benoxacor (BE), respectively.D. subspicatuswas equally sensitive to four herbicides including atrazine and its metabolite and significantly more sensitive to iodosulfuron-methyl-sodium (IODO). The mixture of these five compounds induced a significantly higher growth inhibition of about 1.5-fold than IODO alone, which could be attributed to the four other herbicides. The mixture of all compounds was twofold less toxic than IODO onD. subspicatus. A halogen atom is present in IODO as in the herbicides to which the safener BE − known to induce glutathione-S-transferases − is associated in agrochemical preparations. We then showed that IODO was less toxic when combined with non-toxic concentrations of BE. These results indicated that the toxicity of the most active herbicide studied was decreased by a non-herbicide compound present in agrochemical formulations of other herbicides. These results suggest the importance to take into account the chemistry and the mechanisms of action for each compound in a risk assessment approach of a complex mixture.

Negative Effects of Copper Oxide Nanoparticles on Carbon and Nitrogen Cycle Microbial Activities in Contrasting Agricultural Soils and in Presence of Plants.

Metal-oxide nanoparticles (NPs) such as copper oxide (CuO) NPs offer promising perspectives for the development of novel agro-chemical formulations of pesticides and fertilizers. However, their potential impact on agro-ecosystem functioning still remains to be investigated. Here, we assessed the impact of CuO-NPs (0.1, 1, and 100 mg/kg dry soil) on soil microbial activities involved in the carbon and nitrogen cycles in five contrasting agricultural soils in a microcosm experiment over 90 days. Additionally, in a pot experiment, we evaluated the influence of plant presence on the toxicity of CuO-NPs on soil microbial activities. CuO-NPs caused significant reductions of the three microbial activities measured (denitrification, nitrification, and soil respiration) at 100 mg/kg dry soil, but the low concentrations (0.1 and 1 mg/kg) had limited effects. We observed that denitrification was the most sensitive microbial activity to CuO-NPs in most soil types, while soil respiration and nitrification were mainly impacted in coarse soils with low organic matter content. Additionally, large decreases in heterotrophic microbial activities were observed in soils planted with wheat, even at 1 mg/kg for soil substrate-induced respiration, indicating that plant presence did not mitigate or compensate CuO-NP toxicity for microorganisms. These two experiments show that CuO-NPs can have detrimental effects on microbial activities in soils with contrasting physicochemical properties and previously exposed to various agricultural practices. Moreover, we observed that the negative effects of CuO-NPs increased over time, indicating that short-term studies (hours, days) may underestimate the risks posed by these contaminants in soils.

Functional Supramolecular of Inclusion Complex of Herbicide Fluroxypyr with HPβCD.

In this study, hydroxypropyl-β-cyclodextrin (HPβCD) was used to form an inclusion complex with fluroxypyr to enhance water solubility and thermal stability. The inclusion complex was prepared by a saturated solution method and characterized by FT-IR, SEM, TGA, and XRD. All results indicated that fluroxypyr successfully entered the HPβCD cavity. In addition, the study of phase solubility identifies that the water solubility of fluroxypyr was greatly improved after the formation of the inclusion complex, and TGA analysis suggested that the formation of the inclusion complex improved the thermal stability. Bioactivity assay tests showed that the inclusion complex still had strong herbicidal activity. Our research showed that HPβCD was a promising carrier for improving the properties of fluroxypyr and, thus, expanding its use in agrochemical formulations.

Novel self-assembling conjugates as vectors for agrochemical delivery

BackgroundModern agricultural practises rely on surfactant-based spray applications to eliminate weeds in crops. The wide spread and indiscriminate use of surfactants may result in a number of deleterious effects that are not limited to impacts on the crop and surrounding farm eco-system but include effects on human health. To provide a safer alternative to the use of surfactant-based formulations, we have synthesised a novel, self-assembling herbicide conjugate for the delivery of a broad leaf herbicide, picloram.ResultsThe synthesized self-assembling amphiphile–picloram (SAP) conjugate has three extending arms: a lipophilic lauryl chain, a hydrophilic polyethylene glycol chain and the amphiphobic agrochemical active picloram. We propose that the SAP conjugate maintains its colloidal stability by quickly transitioning between micellar and inverse micellar phases in hydrophilic and lipophilic environments respectively. The SAP conjugate provides the advantage of a phase structure that enables enhanced interaction with the hydrophobic epicuticular wax surface of the leaf. We have investigated the herbicidal efficiency of the SAP conjugate compared against that of commercial picloram formulations using the model plant Arabidopsis thaliana and found that when tested at agriculturally relevant doses between 0.58 and 11.70 mM a dose-dependent herbicidal effect with comparable kill rates was evident.ConclusionThough self-assembling drug carriers are not new to the pharmaceutical industry their use for the delivery of agrochemicals shows great promise but is largely unexplored. We have shown that SAP may be used as an alternative to current surfactant-based agrochemical formulations and has the potential to shift present practises towards a more sustainable approach.

Agricultural adjuvants may impair leaf transpiration and photosynthetic activity

Adjuvants such as surfactants are commonly incorporated into agrochemical formulations to enhance the biological efficiency of foliar sprays by improving the wetting behavior of the spray and/or the penetration of the active ingredients into the leaf tissues. Penetration accelerating adjuvants are known to increase the cuticular permeability and may alter the cuticular barrier to water loss. However, none or very little emphasis has been given to the impacts of adjuvants on crop water balance or drought tolerance, a very important factor affecting crop performance under water scarcity. Two model crops with strongly varying leaf traits, kohlrabi (Brassica oleracea) and apple (Malus domestica) seedlings were grown in controlled environments. Three adjuvants with varying solubility in the cuticle, i.e. octanol-water partition coefficients (logKow) were selected: rapeseed methyl ester (RME) and the surfactants alkyl polyglycoside (APG) and polyoxyethylated tallow amine (POEA). The higher the logKow of the adjuvant, the stronger was the increase of minimum epidermal conductance (gmin, an essential parameter describing plant drought tolerance). However, such effects depended on the physio-chemical properties of the leaf surface. In comparison to kohlrabi, the adjuvant effects on gmin of apple leaves were relatively weak. The increase of gmin was associated with a decrease in contact angle and with an alteration of the wax microstructure. Furthermore, POEA affected photochemical efficiency of kohlrabi leaves. Some adjuvants could have a temporal influence on transpirational water loss and gmin. At repeated applications, they might alter the effective water use and possibly reduce drought tolerance of some horticultural crops.

GHS additivity formula: can it predict the acute systemic toxicity of agrochemical formulations that contain acutely toxic ingredients?

In vivo acute systemic testing is a regulatory requirement for agrochemical formulations. GHS specifies an alternative computational approach (GHS additivity formula) for calculating the acute toxicity of mixtures. We collected acute systemic toxicity data from formulations that contained one of several acutely-toxic active ingredients. The resulting acute data set includes 210 formulations tested for oral toxicity, 128 formulations tested for inhalation toxicity and 31 formulations tested for dermal toxicity. The GHS additivity formula was applied to each of these formulations and compared with the experimental in vivo result. In the acute oral assay, the GHS additivity formula misclassified 110 formulations using the GHS classification criteria (48% accuracy) and 119 formulations using the USEPA classification criteria (43% accuracy). With acute inhalation, the GHS additivity formula misclassified 50 formulations using the GHS classification criteria (61% accuracy) and 34 formulations using the USEPA classification criteria (73% accuracy). For acute dermal toxicity, the GHS additivity formula misclassified 16 formulations using the GHS classification criteria (48% accuracy) and 20 formulations using the USEPA classification criteria (36% accuracy). This data indicates the acute systemic toxicity of many formulations is not the sum of the ingredients’ toxicity (additivity); but rather, ingredients in a formulation can interact to result in lower or higher toxicity than predicted by the GHS additivity formula.

Iron/NPK agrochemical formulation from superabsorbent nanocomposite based on maize bran and montmorillonite with functions of water uptake and slow-release fertilizer

Slow-release character of hydrogel nanocomposite makes it efficient in amending Fe nutritional disorder, fertilizer loss, and crops growth and yield.

Rерrotoxiсity profiling of Bisрyribас Sodium, Pinoxaden and Spinosad Pesticides on Drosoрhilа mеlаnogаstеr

Present research exploration is attributed to reproductive toxicity evaluation of three synthetic agro-chemical pesticidal formulations viz: Bisрyribас Sodium, Pinoxaden and Spinosad, by implementing, test model, Drosophila melanogaster (2n=8). The target of present exploration is to evaluate reprotoxicity induced by selected pesticides. To accomplish the objectives of present research analysis, standardization of semilethal concentration LC 20 , had been considered by exposing second instar larvae to serial dilution of selected pesticides, for continuation of 24 hrs. Thereafter, considering co-relation between exposed concentration and mortality rate, exact values of LC 20 , had been calculated by probit analysis,which correspond to values 4.4 pl/ml for Sodium Bispyribac and 7.1pl/ml for Pinaxoden pl/ml for Bisрyribас Sodium, and 2 pl/ml for Spinosad. Subsequently, second instar larvae of fruit fly were exposed to sub lethal concentration, LC 20 of selected pesticides, for 24 hrs, by means of nutrition, adding to culture medium. Afterward, larvae molted into imago in normal culture medium, which were allowed to cross mate in three separate experimental sets, in triplicate with respective negative controls. In first set, both pesticide exposed male and female cross-mated. In second experimental set, treated male and normal female flies were allowed to crossmate, whereas in third experimental set, pesticide exposed females were allowed to mate with normal males. After comparison of fecundity rate in all experimental sets with natural population, it had been concluded that selected pesticides induced statistical significant decrease in fecundity, when scrutinized by Z-test (p<0.05). Additionally, it had been analysed that the male fruit flies were more susceptible than female fruit flies to selected pesticides.

Surfactant-induced enhancement of droplet adhesion in superhydrophobic soybean (Glycine max L.) leaves

This study performed with soybean (Glycine max L.), one of the most important crops for human and animal nutrition, demonstrates that changes in the leaf surface structure can increase the adhesion of applied droplets, even on superhydrophobic leaves, to reduce undesirable soil contamination by roll-off of agrochemical formulations from the plant surfaces. The wettability and morphology of soybean (Glycine max L.) leaf surfaces before and after treatment with six different surfactants (Agnique® SBO10 and five variations of nonionic surfactants) have been investigated. The leaf surface structures show a hierarchical organization, built up by convex epidermal cells (microstructure) and superimposed epicuticular platelet-shaped wax crystals (micro- to nanostructure). Chemical analysis of the epicuticular wax showed that 1-triacontanol (C30H61OH) is the main wax component of the soybean leaf surfaces. A water contact angle (CA) of 162.4° (σ = 3.6°) and tilting angle (TA) of 20.9° (σ = 10.0°) were found. Adherence of pure water droplets on the superhydrophobic leaves is supported by the hydrophilic hairs on the leaves. Agnique® SBO10 and the nonionic surfactant XP ED 75 increased the droplet adhesion and caused an increase of the TA from 20.9° to 85° and 90°, respectively. Scanning electron microscopy showed that surfactants with a hydrophilic–lipophilic balance value below 10 caused a size reduction of the epicuticular wax structures and a change from Cassie–Baxter wetting to an intermediate wetting regime with an increase of droplet adhesion.

Limited applicability of in vitro skin corrosion and irritation tests for agrochemical formulations

Limited applicability of in vitro skin corrosion and irritation tests for agrochemical formulations

Regulatory accepted but out of domain: In vitro skin irritation tests for agrochemical formulations

Several in vitro methods have gained regulatory acceptance for the prediction of skin irritation and corrosion. However, the test guidelines for the majority of in vitro methods do not state whether they are applicable to agrochemical formulations. Hence, we would like to share the results from our routine skin corrosion and irritation testing of agrochemical formulations in which both in vitro (according to OECD TG 431 and OECD TG 439) and in vivo (according to OECD TG 404) tests were conducted as regulatory requirements. The in vitro skin irritation test did not correlate well with the CLP classification by in vivo results (44% sensitivity, 60% specificity, and 54% accuracy, based on 65 data pairs). This indicates a lack of applicability of the current protocol of the in vitro skin irritation test for agrochemical formulations. The data set did not contain formulations which were skin corrosive in vivo and hence its applicability could not be assessed. The correlation of in vitro skin corrosion testing to formulations which were not corrosive in vivo was, however, high (95% specificity based on 81 data pairs).

A retrospective analysis of in vivo eye irritation, skin irritation and skin sensitisation studies with agrochemical formulations: Setting the scene for development of alternative strategies

Analysis of the prevalence of health effects in large scale databases is key in defining testing strategies within the context of Integrated Approaches on Testing and Assessment (IATA), and is relevant to drive policy changes in existing regulatory toxicology frameworks towards non-animal approaches. A retrospective analysis of existing results from in vivo skin irritation, eye irritation, and skin sensitisation studies on a database of 223 agrochemical formulations is herein published. For skin or eye effects, high prevalence of mild to non-irritant formulations (i.e. per GHS, CLP or EPA classification) would generally suggest a bottom-up approach. Severity of erythema or corneal opacity, for skinor eye effects respectively, were the key drivers for classification, consistent with existing literature. The reciprocal predictivity of skin versus eye irritation and the good negative predictivity of the GHS additivity calculation approach (>85%) provided valuable non-testing evidence for irritation endpoints. For dermal sensitisation, concordance on data from three different methods confirmed the high false negative rate for the Buehler method in this product class. These results have been reviewed together with existing literature on the use of in vitro alternatives for agrochemical formulations, to propose improvements to current regulatory strategies and to identify further research needs.

Degradation of bis-p-nitrophenyl phosphate using zero-valent iron nanoparticles

Phosphate esters are employed in some agrochemical formulations and have long life time in the Environment. They are neurotoxic to mammals and it is very difficult to hydrolyze them. It is easy to find papers in the literature dealing with transition metal complexes used in the hydrolysis processes of organophosphorous compounds. However, there are few reports related with degradation of phosphate esters with inorganic nanoparticles. In this work bis-4-nitrophenyl phosphate (BNPP) was used as an agrochemical agent model. The BNPP interaction with zero-valent iron nanoparticles (ZVI NPs), in aqueous media, was searched. The concentration of BNPP was 1000 times higher than the ZVI NPs concentration. The average size of the used iron nanoparticles was 10.2 ± 3.2 nm. The BNPP degradation process was monitored by means of UV-visible method. Initially, the BNPP hydrolysis happens through the P-O bonds breaking-off under the action of the ZVI NPs. Subsequently, the nitro groups were reduced to amine groups. The overall process takes place in 10 minutes. The reaction products were identified employing standard substances in adequate concentrations. The iron by-products were isolated and characterized by X-RD. These iron derivatives were identified as magnetite (Fe3O4) and/or maghemite (γ-Fe2O3) and lepidocrocite (γ-FeOOH). A suggested BNPP degradation mechanism will be discussed.

In vitro assessment of skin sensitization, photosensitization and phototoxicity potential of commercial glyphosate-containing formulations

This study evaluated the applicability of a modified Direct Peptide Reactivity Assay (DPRA) (OECD N° 442C, 2015) through the 10-fold reduction of reaction volume (micro-DPRA, mDPRA) for skin sensitization evaluation of six commercial glyphosate-containing formulations. In addition, another modification of DPRA was proposed by adding a UVA (5J/cm2) irradiation step, namely photo-mDPRA, to better characterize (photo)sensitizer materials. The phototoxicity profile of pesticides was also evaluated using the 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) (OECD N° 432, 2004). The mDPRA could represent an environmentally acceptable test approach, since it reduces costs and organic waste. Peptide depletion was greater in photo-mDPRA and changed the reactivity class of each test material, in comparison to mDPRA. Thus, the association of mDPRA with photo-mDPRA was better for correctly characterizing human (photo)sensitizer substances and pesticides. In general, cysteine depletion was greater than that of lysine for all materials tested in both mDPRA and photo-mDPRA. Furthermore, while 3T3-NRU-PT is unable to predict (photo)sensitizers, it was capable of correctly identifying the phototoxic potential of the tested agrochemical formulations. In conclusion, mDPRA plus photo-mDPRA and 3T3-NRU-PT seem to be preliminary non-animal test batteries for skin (photo)sensitization/phototoxicity assessment of chemicals, agrochemical formulations and their ingredients.

Lacking applicability of in vitro eye irritation methods to identify seriously eye irritating agrochemical formulations: Results of bovine cornea opacity and permeability assay, isolated chicken eye test and the EpiOcular™ ET-50 method to classify according to UN GHS

In vitro methods have gained regulatory acceptance for the prediction of serious eye damage (UN GHS Cat 1). However, the majority of in vitro methods do not state whether they are applicable to agrochemical formulations. This manuscript presents a study of up to 27 agrochemical formulations tested in three in vitro assays (three versions of the bovine corneal opacity and permeability test (BCOP, OECD TG 437) assay, the isolated chicken eye test (ICE, OECD TG 438) and the EpiOcular™ ET-50 assay). The results were compared with already-available in vivo data. In the BCOP only one of the four, one of five in the ICE and six of eleven tested formulations in the EpiOcular™ ET-50 Neat Protocol resulted in the correct UN GHS Cat 1 prediction. Overpredictions occurred in all assays. These data indicate a lack of applicability of the three in vitro methods to reliably predict UN GHS Cat 1 of agrochemical formulations. In order to ensure animal-free identification of seriously eye damaging agrochemical formulations testing protocols and/or prediction models need to be modified or classification rules should be tailored to in vitro testing rather than using in vivo Draize data as a standard.