Toxicological safety evaluation of an aqueous lemon balm (Melissa officinalis) extract.

Melissa officinalis (lemon balm) has a long history of safe use as an aromatic herb, flavoring, tea, food supplement, and traditional medicine. An aqueous extract of the leaves of M. officinalis is intended for use as a food ingredient, however the existing safety database does not contain any high quality toxicological studies to support safe consumer exposure. Therefore, a standard tier 1 genotoxicity battery (bacterial reverse mutation and in vitro mammalian cell micronucleus tests) and a 90-day repeated dose oral toxicity study in rats were conducted in accordance with GLP and OECD guidelines. The genotoxicity studies confirmed that aqueous lemon balm extract is not genotoxic at up to the highest concentrations tested (5000 µg/plate or 5000 µg/mL). A non-GLP 14-day dose range finding study was conducted prior to the 90-day study to confirm dietary administration of aqueous lemon balm extract at concentrations of 0, 0.5, 1.6, or 5.0%. The 90-day study was conducted using the established dietary concentrations and no test substance-related adverse effects on clinical, hematological, biochemical, macroscopic, or histopathologic parameters were reported. Thus, the no-observed-adverse-effect-level was determined to be at least 3046.1 and 3720.9 mg/kg body weight/day (the highest doses tested) for male and female rats, respectively.

Biotransformation Changes Bioaccumulation and Toxicity of Diclofenac in Aquatic Organisms.

Biotransformation plays a crucial role in regulating the bioaccumulation potential and toxicity of organic compounds in organisms but is, in general, poorly understood for emerging contaminants. Here, we have used diclofenac as a model compound to study the impact of biotransformation on the bioaccumulation potential and toxicity in two keystone aquatic invertebrates: Gammarus pulex and Hyalella azteca. In both species, diclofenac was transformed into several oxidation products and conjugates, including two novel products, that is, diclofenac taurine conjugate (DCF-M403) and unexpected diclofenac methyl ester (DCF-M310.03). The ratios of biotransformation products to parent compound were 12-17 for DCF-M403 and 0.01-0.7 for DCF-M310.03 after 24 h exposure. Bioconcentration factors (BCFs) of diclofenac were 0.5 and 3.2 L kgww-1 in H. azteca and G. pulex, respectively, whereas BCFs of DCF-M310.03 was 164.5 and 104.7 L kgww-1, respectively, representing a 25- to 110-fold increase. Acute toxicity of DCF-M310.03 was also higher than the parent compound in both species, which correlated well with the increased bioconcentration potential. The LC50 of diclofenac in H. azteca was 216 mg L-1, while that of metabolite DCF-M310.03 was reduced to only 0.53 mg L-1, representing a 430-fold increase in acute toxicity compared to diclofenac. DCF-M403 is less toxic than its parent compound toward H. azteca, which may be linked to its slightly lower hydrophobicity. Furthermore, the transformation of diclofenac to its methyl ester derivative was explored in crude invertebrate extracts spiked with an S-adenosylmethionine cofactor, revealing possible catalysis by an S-adenosylmethionine-dependent carboxylic acid methyltransferase. Methylation of diclofenac was further detected in fish hepatocytes and human urine, indicating a broader relevance. Therefore, potentially methylated metabolites of polar contaminants should be considered for a comprehensive risk assessment in the future.

Bioaccumulation, Biotransformation, and Synergistic Effects of Binary Fungicide Mixtures in Hyalella azteca and Gammarus pulex: How Different/Similar are the Two Species?

Aquatic organisms are consistently exposed to a mixture of micropollutants that can bioaccumulate, undergo biotransformation, and may exert mixture effects. However, little is known on the underlying mechanisms and species-specificity. Herein we investigated bioaccumulation, biotransformation and synergistic effects of azole (i.e., prochloraz) and strobilurin (i.e., azoxystrobin) fungicides in the two aquatic invertebrate species, Hyalella azteca and Gammarus pulex. Bioaccumulation of azoxystrobin was similar, whereas bioaccumulation of prochloraz was slightly different in the two species but was still significantly below the REACH criteria for bioaccumulative substances. Similar biotransformation patterns were observed in both species, and only a few unique biotransformation reactions were detected in H. azteca such as malonyl-glucose and taurine conjugation. Toxicokinetic modeling additionally indicated that biotransformation is a more important elimination pathway in H. azteca. In mixtures, no-observed-adverse-effect levels of prochloraz decreased the LC50s of azoxystrobin in both species which correlated well with increased internal azoxystrobin concentrations. This synergistic effect is partly due to the inhibition of cytochrome P450 monooxygenases by prochloraz which subsequently triggered the reduced biotransformation of azoxystrobin (lower by five folds in H. azteca). The largely similar responses in both species suggest that the easier-to-cultivate H. azteca is a promising representative of invertebrates for toxicity testing.