Efficacy and safety profile of a subtilisin protease produced by fermentation in Bacillus licheniformis to be used as a feed additive.

The efficacy and safety of a novel Subtilisin protease from a Bacillus sp. produced in Bacillus licheniformis was investigated in broiler chickens, and a range of toxicological tests, respectively. The B. licheniformis production strain culture supernatant was not found cytotoxic in a Vero cell assay. Subtilisin was non-mutagenic and non-clastogenic in in-vitro tests, and did not exhibit irritating potential to the eye or skin in ex-vivo/in-vitro models. Oral administration of Subtilisin to rats did not cause any adverse effects in a 13-week sub-chronic toxicity study. In addition, a 35-day dose response broiler performance trial conducted with Subtilisin (30,000 and 60,000 NFP/kg diet), showed a significant linear improvement in both body weight gain and feed conversion ratio up to 35 days of protease supplementation. In conclusion, there are no safety concerns using this novel Subtilisin as a feed additive, and the protease is efficient in improving broiler growth performance, making it a good candidate for use as a feed additive.

Safety assessment of Weissella confusa - A direct-fed microbial candidate.

Weissella confusa is part of the lactic acid bacteria genera and a member of an autochthonous microbiota of humans and livestock. W. confusa has been proposed to have potential as a direct-fed microbial product, however, there is a lack of studies assessing its safety. A toxicological safety assessment of W. confusa was performed using a battery of in vitro, ex vivo and in vivo studies, testing for genotoxicity, skin and eye irritation and general toxicity. The bacterial reverse mutation (Ames) study did not reveal any genotoxicity in the presence and absence of metabolic activation (S9) at concentrations up to 5000 µg/mL. Moreover, an in vivo mammalian erythrocyte micronucleus study did not reveal any biologically relevant or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes in mice, when tested at concentrations up to 2000 mg/kg body weight. W. confusa did not exert any skin irritation potential when tested in reconstructed skin membranes. When tested for eye irritation using an ex vivo model of isolated chicken eyes, mild irritation was observed. The 90-day sub-chronic oral toxicity (gavage) study was performed using Sprague Dawley rats at concentrations up to 92 × 108 cfu/kg body weight/day (nominal). The results showed that W. confusa were well tolerated, and no signs of toxicity were seen. The No Observed Adverse Effect Level (NOAEL) for both female and male animals was the highest concentration administered, 92 × 108 cfu/kg body weight/day (nominal). In conclusion, the toxicological studies performed confirmed W. confusa to be safe, making it a good candidate as a direct-fed microbial product.

Regulatory ecotoxicity testing of nanomaterials - proposed modifications of OECD test guidelines based on laboratory experience with silver and titanium dioxide nanoparticles.

Regulatory ecotoxicity testing of chemicals is of societal importance and a large effort is undertaken at the OECD to ensure that OECD test guidelines (TGs) for nanomaterials (NMs) are available. Significant progress to support the adaptation of selected TGs to NMs was achieved in the context of the project MARINA ( http://www.marina-fp7.eu/ ) funded within the 7th European Framework Program. Eight OECD TGs were adapted based on the testing of at least one ion-releasing NM (Ag) and two inert NMs (TiO2). With the materials applied, two main variants of NMs (ion releasing vs. inert NMs) were addressed. As the modifications of the test guidelines refer to general test topics (e.g. test duration or measuring principle), we assume that the described approaches and modifications will be suitable for the testing of further NMs with other chemical compositions. Firm proposals for modification of protocols with scientific justification(s) are presented for the following tests: growth inhibition using the green algae Raphidocelis subcapitata (formerly: Pseudokirchneriella subcapitata; TG 201), acute toxicity with the crustacean Daphnia magna (TG 202), development toxicity with the fish Danio rerio (TG 210), reproduction of the sediment-living worm Lumbriculus variegatus (TG 225), activity of soil microflora (TGs 216, 217), and reproduction of the invertebrates (Enchytraeus crypticus, Eisenia fetida, TGs 220, 222). Additionally, test descriptions for two further test systems (root elongation of plants in hydroponic culture; test on fish cells) are presented. Ecotoxicological data obtained with the modified test guidelines for TiO2 NMs and Ag NM and detailed method descriptions are available.

Influence of pH and media composition on suspension stability of silver, zinc oxide, and titanium dioxide nanoparticles and immobilization of Daphnia magna under guideline testing conditions.

In aquatic toxicity testing of engineered nanoparticles (ENPs) the process of agglomeration is very important as it may alter bioavailability and toxicity. In the present study, we aimed to identify test conditions that are favorable for maintaining stable ENP suspensions. We evaluated the influence of key environmental parameters: pH (2-12) and ionic strength using M7, Soft EPA (S EPA) medium, and Very Soft EPA (VS EPA) medium; and observed the influence of these parameters on zeta potential, zeta average, and acute immobilization of Daphnia magna for three different ENPs. Despite being sterically stabilized, test suspensions of silver (Ag) ENPs formed large agglomerates in both VS EPA and M7 media; and toxicity was found to be higher in VS EPA medium due to increased dissolution. Low-agglomerate suspensions for zinc oxide (ZnO) could be obtained at pH 7 in VS EPA medium, but the increase in dissolution caused higher toxicity than in M7 medium. Titanium dioxide (TiO2) ENPs had a point of zero charge in the range of pH 7-8. At pH 7 in VS EPA, agglomerates with smaller hydrodynamic diameters (~200nm) were present compared to the high ionic strength M7 medium where hydrodynamic diameters reached micrometer range. The stable suspensions of TiO2 ENPs caused immobilization of D. magna, 48-h EC50 value of 13.7mgL(-1) (95% CI, 2.4mg-79.1mgL(-1)); whereas no toxicity was seen in the unstable, highly agglomerated M7 medium suspensions, 48-h EC50 >100mgL(-1). The current study provides a preliminary approach for methodology in testing and assessing stability and toxicity of ENPs in aquatic toxicity tests of regulatory relevance.

Methodological considerations for using umu assay to assess photo-genotoxicity of engineered nanoparticles.

In this study we investigated the feasibility of high-throughput (96-well plate) umu assay to test the genotoxic effect of TiO2 engineered nanoparticles (ENPs) under UV light (full spectrum) and visible light (455 nm). Exposure of TiO2 ENPs to up to 60 min of UV light induced a photocatalytic production of ROS. However, UV light itself caused cytotoxic damage to Salmonella typhimurium at exposures >15 min and a genotoxic effect at exposures >0.5 min; and use of UV filters did not lower this effect. No genotoxicity of TiO2 ENPs was observed under visible light conditions at concentrations up to 100 µg mL(-1); or under dark conditions at concentrations up to 667 µg mL(-1), though cytotoxicity was seen at the higher concentrations. Additionally, the growth factor calculation was influenced by a shading effect due to ENPs, and was corrected by considering the pre-incubation OD readings of Plate B. Recommendations provided in this paper, as well as investigation of the effect of the light sources should be considered when using the umu assay to quantify the photo-genotoxicity of engineered nanomaterials.

Techniques and Protocols for Dispersing Nanoparticle Powders in Aqueous Media-Is there a Rationale for Harmonization?

Selecting appropriate ways of bringing engineered nanoparticles (ENP) into aqueous dispersion is a main obstacle for testing, and thus for understanding and evaluating, their potential adverse effects to the environment and human health. Using different methods to prepare (stock) dispersions of the same ENP may be a source of variation in the toxicity measured. Harmonization and standardization of dispersion methods applied in mammalian and ecotoxicity testing are needed to ensure a comparable data quality and to minimize test artifacts produced by modifications of ENP during the dispersion preparation process. Such harmonization and standardization will also enhance comparability among tests, labs, and studies on different types of ENP. The scope of this review was to critically discuss the essential parameters in dispersion protocols for ENP. The parameters are identified from individual scientific studies and from consensus reached in larger scale research projects and international organizations. A step-wise approach is proposed to develop tailored dispersion protocols for ecotoxicological and mammalian toxicological testing of ENP. The recommendations of this analysis may serve as a guide to researchers, companies, and regulators when selecting, developing, and evaluating the appropriateness of dispersion methods applied in mammalian and ecotoxicity testing. However, additional experimentation is needed to further document the protocol parameters and investigate to what extent different stock dispersion methods affect ecotoxicological and mammalian toxicological responses of ENP.

The influence of natural organic matter and aging on suspension stability in guideline toxicity testing of silver, zinc oxide, and titanium dioxide nanoparticles with Daphnia magna.

The present study investigated changes in suspension stability and ecotoxicity of engineered nanoparticles (ENPs) by addition of Suwannee River natural organic matter and aging of stock and test suspensions prior to testing. Acute toxicity tests of silver (Ag), zinc oxide (ZnO), and titanium dioxide (TiO2 ) ENPs with Daphnia magna were carried out following Organisation for Economic Co-operation and Development test guidelines. Daphnia magna was found to be very sensitive to Ag ENPs (48-h 50% effective concentration 33 µg L(-1) ), and aging of the test suspensions in M7 medium (up to 48 h) did not decrease toxicity significantly. Conversely, the presence of Suwannee River natural organic matter (NOM; 20 mg L(-1) ) completely alleviated Ag ENP toxicity in all testing scenarios and did not aid in stabilizing suspensions. In contrast, addition of Suwannee River NOM stabilized ZnO ENP suspensions and did not decrease toxicity. Aging for 48 h generated monotonous concentration-response curves in the presence and absence of Suwannee River NOM. At concentrations up to 100 mg L(-1) TiO2 ENPs did not cause immobilization of D. magna under any of the tested conditions. Presence of Suwannee River NOM caused agglomeration in stock suspensions. The authors' results suggest that aging and presence of Suwannee River NOM are important parameters in standard toxicity testing of ENPs, which in some cases may aid in gaining better control over the exposure conditions but in other cases might contribute to agglomeration or elimination of ENP toxicity. Therefore, modifications to the current guidelines for testing ENPs should be evaluated on a case-by-case basis. Environ Toxicol Chem 2015;34:497-506. © 2014 SETAC.