22The rise in antimicrobial resistance has prompted the development of alternatives, 23 such as plant-derived compounds, to combat bacterial infections. Bald's eyesalve, a 24 remedy used in the Early Medieval period, has previously been shown to have efficacy 25 against Staphylococcus aureus grown in an in vitro model of soft tissue infection. This 26 remedy also had bactericidal activity against methicillin-resistant S. aureus (MRSA) in 27 a chronic mouse wound. However, the safety profile of Bald's eyesalve has not yet 28 been demonstrated, and this is vital before testing in humans. Here, we determined 29 the safety potential of Bald's eyesalve using in vitro, ex vivo, and in vivo models 30 representative of skin or eye infections. We also confirmed that Bald's eyesalve is 31 active against an important eye pathogen, Neisseria gonorrhoeae. Low levels of 32 cytotoxicity were observed in eyesalve-treated cell lines representative of skin and 33 immune cells. Results from a bovine corneal opacity and permeability test 34 demonstrated slight irritation to the cornea that resolved within 10 minutes. The slug 35 mucosal irritation assay revealed that a low level of mucus was secreted by slugs 36 exposed to eyesalve, indicating mild mucosal irritation. We obtained promising results 37 from mouse wound closure experiments; no visible signs of irritation or inflammation 38 were observed. Our results suggest that Bald's eyesalve could be tested further on 39 human volunteers to assess safety for topical application against bacterial infections. 40 41 Importance: Alternative treatment for bacterial infections are needed to combat the 42 ever increasing repertoire of bacteria resistant to antibiotics. A medieval plant-based 43 remedy, Bald's eyesalve, shows promise as a substitute for the treatment of these 44infections. For any substance to be effective in the treatment of bacterial infections in 45 humans, it is important to consider the safety profile. This is a key consideration in 46 3 order to have the necessary regulatory approval. We demonstrate the safety profile of 47 Bald's eyesalve using a variety of models, including whole-organ and whole-animal 48 models. Our results show that Bald's eyesalve is mildly toxic to cultured human cells, 49 but potentially suitable for patch tests on healthy human volunteers to assess safety 50 for later clinical trials. Our work has the potential to transform the management of 51 diseases caused by bacterial infections, such as diabetic foot ulcers, through topical 52 application of a natural product cocktail based on Bald's eyesalve. 53 54

The rise in antimicrobial resistance has prompted the development of alternatives to combat bacterial infections. Bald’s eyesalve, a remedy used in the Early Medieval period, has previously been shown to have efficacy against Staphylococcus aureus in in vitro and in vivo models of chronic wounds. However, the safety profile of Bald’s eyesalve has not yet been demonstrated, and this is vital before testing in humans. Here, we determined the safety potential of Bald’s eyesalve using in vitro, ex vivo, and in vivo models representative of skin or eye infections. We also confirmed that Bald’s eyesalve is active against an important eye pathogen, Neisseria gonorrhoeae. Low levels of cytotoxicity were observed in eyesalve-treated cell lines representative of skin and immune cells. Results from a bovine corneal opacity and permeability test demonstrated slight irritation to the cornea that resolved within 10 min. The slug mucosal irritation assay revealed that a low level of mucus was secreted by slugs indicating moderate mucosal irritation. We obtained promising results from mouse wound closure experiments; no visible signs of irritation or inflammation were observed. Our results suggest that Bald’s eyesalve could be tested further on human volunteers to assess safety for topical application against bacterial infections.