Safety assessment of Mpp75Aa1.1, a new ETX_MTX2 protein from Brevibacillus laterosporus that controls western corn rootworm.

The recently discovered insecticidal protein Mpp75Aa1.1 from Brevibacillus laterosporus is a member of the ETX_MTX family of beta-pore forming proteins (ß-PFPs) expressed in genetically modified (GM) maize to control western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). In this manuscript, bioinformatic analysis establishes that although Mpp75Aa1.1 shares varying degrees of similarity to members of the ETX_MTX2 protein family, it is unlikely to have any allergenic, toxic, or otherwise adverse biological effects. The safety of Mpp75Aa1.1 is further supported by a weight of evidence approach including evaluation of the history of safe use (HOSU) of ETX_MTX2 proteins and Breviballus laterosporus. Comparisons between purified Mpp75Aa1.1 protein and a poly-histidine-tagged (His-tagged) variant of the Mpp75Aa1.1 protein demonstrate that both forms of the protein are heat labile at temperatures at or above 55°C, degraded by gastrointestinal proteases within 0.5 min, and have no adverse effects in acute mouse oral toxicity studies at a dose level of 1920 or 2120 mg/kg body weight. These results support the use of His-tagged proteins as suitable surrogates for assessing the safety of their non-tagged parent proteins. Taken together, we report that Mpp75Aa1.1 is the first ETX-MTX2 insecticidal protein from B. laterosporus and displays a similar safety profile as typical Cry proteins from Bacillus thuringiensis.

Tiered application of the neutral red release and EpiOcular™ assays for evaluating the eye irritation potential of agrochemical formulations.

Agrochemical formulations have been underrepresented in validation efforts for implementing alternative eye irritation approaches but represent a significant opportunity to reduce animal testing. This study assesses the utility of the neutral red release assay (NRR) and EpiOcular™ assay (EO) for predicting the eye irritation potential of 64 agrochemical formulations relative to Draize data. In the NRR, formulations with an NRR50 value ≤ 50 mg/mL were categorized as UN GHS Cat 1 and those >250 mg/mL were classified as UN GHS Non Classified (NC). The accuracy, sensitivity, and specificity were 78, 85 and 76% and 73, 85 and 61% for identifying UN GHS 1 and NC formulations, respectively. Specificity was poor for formulations with NRR50 > 50 to ≤250 mg/mL. The EO (ET-40 method) was explored to differentiate formulations that were UN GHS 1/2 and UN GHS NC. The EO resulted in accuracy, sensitivity, and specificity of 65%, 58% and 75% for identifying UN GHS NC formulations. To improve the overall performance, the assays were implemented using a tiered-approach where the NRR was run as a first-tier followed by the EO. The tiered-approach resulted in improved accuracy (75%) and balanced sensitivity (73%) and specificity (77%) for distinguishing between irritating and non-irritating agrochemical formulations.

Application of the KeratinoSens™ assay for assessing the skin sensitization potential of agrochemical active ingredients and formulations.

Assessment of skin sensitization potential is an important component of the safety evaluation process for agrochemical products. Recently, non-animal approaches including the KeratinoSens™ assay have been developed for predicting skin sensitization potential. Assessing the utility of the KeratinoSens™ assay for use with multi-component mixtures such as agrochemical formulations has not been previously evaluated and is a significant need. This study was undertaken to evaluate the KeratinoSens™ assay prediction potential for agrochemical formulations. The assay was conducted for 8 agrochemical active ingredients (AIs) including 3 sensitizers (acetochlor, meptyldinocap, triclopyr), 5 non-sensitizers (aminopyralid, clopyralid, florasulam, methoxyfenozide, oxyfluorfen) and 10 formulations for which in vivo sensitization data were available. The KeratinoSens™ correctly predicted the sensitization potential of all the AIs. For agrochemical formulations it was necessary to modify the standard assay procedure whereby the formulation was assumed to have a common molecular weight. The resultant approach correctly predicted the sensitization potential for 3 of 4 sensitizing formulations and all 6 non-sensitizing formulations when compared to in vivo data. Only the meptyldinocap-containing formulation was misclassified, as a result of high cytotoxicity. These results demonstrate the promising utility of the KeratinoSens™ assay for evaluating the skin sensitization potential of agrochemical AIs and formulations.

Concurrent evaluation of general, immune, and genetic toxicity endpoints as part of an integrated testing strategy.

Integrated testing strategies involve the assessment of multiple endpoints within a single toxicity study and represent an important approach for reducing animal use and streamlining testing. The present study evaluated the ability to combine general, immune, and genetic toxicity endpoints into a single study. Specifically, this study evaluated the impact of sheep red blood cell (SRBC) immunization, as part of the T-cell dependent antibody response (TDAR) assay, on organ weights, micronuclei (MN) formation (bone marrow and peripheral blood), and the Comet assay response in the liver of female F344/DuCrl rats treated with cyclophosphamide (CP) a known immunosuppressive chemical and genotoxicant. For the TDAR assay, treatment with CP resulted in a dose-dependent decrease in the antibody response with a suppression of greater than 95% at the high dose. Injection with SRBC had no impact on evaluated organ weights, histopathology, hematology, and clinical chemistry parameters. Analysis of MN formation in bone marrow and peripheral blood revealed a dose-dependent increase in response to CP treatment. Injection with SRBC had no impact on the level of MN in control animals and did not alter the dose response of CP. There was a slight increase in liver DNA damage in response to CP as measured by the Comet assay; however, injection with SRBCs did not alter this endpoint. Overall these data provide strong support for the concurrent assessment of general, immune, and genetic toxicology endpoints within a single study as part of an integrated testing strategy approach.