Preliminary remark on the avoidance of animal suffering
Nebula Biocides GmbH strives to avoid animal suffering whenever possible and to replace animal testing with in vitro testing. In some cases, in vivo tests are required for the Sporosan® system, as in vitro tests are performed under standardized conditions, such as physiological pH. No ONOOH is formed from H₂O₂ and NO₂- (see explanations of the active substance), which is why the results are not meaningful in most cases.
In order to obtain reliable statements on the safety of the novel active substance, animal experiments are unfortunately unavoidable. The animal experiments are planned and carried out in close cooperation with the test laboratories in strict compliance with ethical and scientific standards.
Mutagenicity and genotoxicity
Oxidative biocidal agents such as hypochlorous acid, hypochlorite, chlorine dioxide and peracetic acid are known in vitro genotoxins [8]. The precursors of ONOOH, NO₂- and H₂O₂, are also considered “false-positive” toxins - substances that show in vitro genotoxicity but are classified as safe based on sufficient in vivo data [1]. In order to exclude a potentially mutagenic effect of H₂O₂ and NO₂- during the Sporosan® reaction, a test product was developed that contains equimolar amounts of both reactants. These reactants are fully converted during the reaction and form the reaction mass of the Sporosan® process. Neither mutagenic nor genotoxic effects were observed in the in vitro tests performed with this test product according to OECD 471 and OECD 487. This test design is considered a suitable standard procedure for the in vitro testing of disinfection products using the Sporosan® process, as it allows the investigation of interactions with other additives such as surfactants or chelating agents, while oxidizing species are excluded from the test.
A direct test of the entire reaction system was carried out in vivo using an alkaline comet assay (OECD 489) with dermal application to detect DNA strand breaks. Both the reactants NO₂- and H₂O₂, as well as ONOOH and its decomposition products NO₂ and OH are known to induce DNA strand breaks in vitro [2-6]. After dermal application with Sporosan®, no DNA-damaging effects were observed either in skin cells or in the liver. This can be explained by the pronounced pH dependence of ONOOH formation (see explanations on the active substance): While the reaction can take place on the skin surface with a pH of 4.5 to 5.5, the pH below the stratum corneum, where living cells are located, increases to 7 to 7.4 [7, 8]. Therefore, the reactants on the skin surface are rapidly consumed, and even reactants that can potentially diffuse into deeper skin layers do not contribute to the formation of ONOOH due to the higher pH in these layers. Both reactants occur naturally in the skin: Thus, it is assumed that acidified NO₂-, which is contained in sweat, contributes to the non-specific defense mechanism against microorganisms. H₂O₂, on the other hand, which comes from both endogenous and exogenous sources, is regulated by the high concentration of catalase in the skin [9, 10].
Conclusion:
As with other oxidative active substances, the Sporosan® process shows genotoxic potential in in-vitro tests. In contrast, the reaction mass of NO₂- and H₂O₂ has no genotoxic potential. The practical in vivo tests confirm that the Sporosan® system has no genotoxic potential.
Table 1: Overview of the results in the categories mutagenicity and genotoxicity | ||||
Test specification | Test type | Date | Description of results | Laboratory |
OECD 489 | in vivo: Alkaline Comet Assay on Rat Skin and Liver | 11.12.2019 | The test substance showed no genotoxic or DNA-damaging effect at any of the concentrations tested. Furthermore, no toxic symptoms or clinically relevant changes (including erythema formation on the treated skin) were detected. Sporosan® and Sporosan® in combination with isopropanol were tested. | TOXI-COOP (Hungary) |
OECD 471 | in vitro: Ames Test on reaction products (equimolar) | 18.09.2023 | No genotoxic activity of the test material was observed. Tests were performed using reaction product with equimolar H2O2 and NO2- concentrations. | Bioserv (Germany) |
OECD 487 | in vitro: Mammalian Cell Micronucleus Test on reaction producs (equimolar) | 30.05.2024 | No genotoxic effects could be observed. Tests were performed using reaction product with equimolar H2O2 and NO2-concentrations. | Bioserv (Germany) |
Cytotoxicity, skin reactions and systemic toxicity
The toxicity of the Sporosan® system was investigated in various in vitro and in vivo studies (see Table 2).
The LD50 cut-off value for ONOOH formed from NO₂- and H₂O₂ was determined in an in vivo study according to OECD 423 (cut-off value for GHS classification) at 500 mg/kg body weight after a single oral administration. No animals died at this dose. This seems surprising, as sodium nitrite has an LD50 of only 85 mg/kg. However, the difference can be explained by the accelerated conversion of reactants to ONOOH under acidic conditions in the stomach, with ONOOH decomposing directly to NO₃-. The LD50 value of sodium nitrate, which is given as 1267 mg/kg, is consistent with our results. To put these values into perspective: A 50 kg person would need to consume 50 ml of Sporosan® SD to reach the acceptable daily nitrate intake of 185 mg set by EFSA [11]. In comparison, 8 liters would be required to reach the LD50 limit determined for ONOOH.
Repeated dose toxicity and the potential effects of Sporosan® on reproduction and development were evaluated in an in vivo study according to OECD 422. The dermal application of the Sporosan® system in rats showed no evidence of major systemic or reproductive/developmental toxic effects. However, inflammatory skin lesions were observed at the application site, which were most likely caused by mechanical irritation (itching) as a result of the test substance applied. Such adverse effects only occurred at concentrations exceeding twelve times the amounts used in Sporosan® SD, but not at lower concentrations (four times the ONOOH concentration). At these lower concentrations, ONOOH, which is produced from H₂O₂ and NO₂-, was not classified as a skin or eye irritant in in vitro studies according to OECD 439 and OECD 492 guidelines.
An in vitro cytotoxicity test was carried out in accordance with DIN EN ISO 10993-5 to rule out the formation of cytotoxic compounds from the residues of the Sporosan® disinfection system and the materials of the wash ware (stainless steel and polypropylene) after a washing and disinfection cycle. The test on stainless steel, white and transparent polypropylene samples showed that no cytotoxic residues remained on the treated surfaces during the process. Patient safety is guaranteed at all times.
Conclusion:
Experiments with dermal application of the active ingredient system show that systemic availability of ONOOH is not to be expected. The toxicity of Sporosan® after oral intake is lower than that of the reactant NO₂-, which is due to the direct conversion to NO₃- under acidic conditions.
Table 2: Overview of the results in the categories cytotoxicity, skin reactions and systemic toxicity | ||||
Test specification | Test type | Date | Description of results | Laboratory |
OECD 439 & EU-Method B.46 | in vitro: Skin Irritation Potential | 23.08.2022 | The test item B001_N+H is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method | LAUS (Germany) |
OECD 492 | in vitro: Eye Hazard Potential | 26.08.2022 | Under the conditions of the test, B001_N+H is considered non-eye irritant (GHS No Category) in the EpiOcular Eye irritation Test | LAUS (Germany) |
OECD 429 | in vivo: Local Lymph Node Assay | 29.09.2022 | Based on the results of this study, PEROXINITROUS ACID, generated form SODIUM nitrite ans Hydrogen peroxide is not considered a skin sensitizer under the conditions of this LLNA study. | Slovak Medical University |
OECD 422 | in vivo: repeated Dose Toxicity with Reproduction Screening | 28.08.2023 | dermal application of the test material in different doses did not cause evidence of major systemic toxicity or reproduction/developmental toxicity according to OECD 422 | Bioserv (Germany) |
OECD 423 | in vivo: Acute oral toxicity | 20.06.2024 | the test item Peroxynitrous Acid is classified in GHS Category 4 with a LD50 cut off value 500 mg/kg body weight, after single oral administration to Wistar rats ( for comparison: LD50 value for NaNO2 is 85 mg/kg) | Slovak Medical University |
DIN EN ISO 10993-5 | in vitro: Cytotoxicity | 04.07.2024 | Possible residues on the wash ware after cleaning and disinfection in the washer-disinfector do not trigger any cytotoxic effects. Patient safety can be regarded as given. | Hygiene Nord (Germany) |
Literature:
[1] M. J. Prival, V. F. Simmon, and K. E. Mortelmans, "Bacterial mutagenicity testing of 49 food ingredients gives very few positive results," Mutation research, vol. 260, no. 4, pp. 321–329, 1991. doi: 10.1016/0165-1218(91)90017-G. [Online]. Available: https://www.sciencedirect.com/science/article/pii/016512189190017g
[2] J. Cadet et al., "Hydroxyl radicals and DNA base damage," Mutation research, vol. 424, 1-2, pp. 9–21, 1999. doi: 10.1016/S0027-5107(99)00004-4. [Online]. Available: https://www.sciencedirect.com/science/article/pii/s0027510799000044
[3] D. Schrenk et al., "Risk assessment of nitrate and nitrite in feed," EFSA Journal, vol. 18, no. 11, 2020, doi: 10.2903/j.efsa.2020.6290.
[4] Radiation and hydrogen peroxide induced free radical damage to DNA, 1987. [Online]. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2149485/
[5] E. Bermúdez, S.-F. Ferng, C. Castro, and M. G. Mustafa, "DNA Strand Breaks Caused by Exposure to Ozone and Nitrogen Dioxide," Environmental Research, vol. 81, no. 1, pp. 72–80, 1999. doi: 10.1006/enrs.1999.3955. [Online]. Available: https://www.sciencedirect.com/science/article/pii/s0013935199939555
[6] V. Yermilov, Y. Yoshie, J. Rubio, and H. Ohshima, "Effects of carbon dioxide/bicarbonate on induction of DNA single-strand breaks and formation of 8-nitroguanine, 8-oxoguanine and base-propenal mediated by peroxynitrite," 0014-5793, vol. 399, 1-2, pp. 67–70, 1996. doi: 10.1016/S0014-5793(96)01288-4. [Online]. Available: https://www.sciencedirect.com/science/article/pii/s0014579396012884
[7] H. Lambers, S. Piessens, A. Bloem, H. Pronk, and P. Finkel, "Natural skin surface pH is on average below 5, which is beneficial for its resident flora," International Journal of Cosmetic Science, vol. 28, no. 5, pp. 359–370, 2006, doi: 10.1111/j.1467-2494.2006.00344.x.
[8] H. Ohman and A. Vahlquist, "In vivo studies concerning a pH gradient in human stratum corneum and upper epidermis," Acta Derm Venereol, vol. 74, no. 5, pp. 375–379, 1994. doi: 10.2340/0001555574375379. [Online]. Available: https://medicaljournalssweden.se/actadv/article/view/16406
[9] R. Weller, R. J. Price, A. D. Ormerod, N. Benjamin, and C. Leifert, "Antimicrobial effect of acidified nitrite on dermatophyte fungi, Candida and bacterial skin pathogens," J Appl Microbiol, vol. 90, no. 4, pp. 648–652, 2001, doi: 10.1046/j.1365-2672.2001.01291.x.
[10] M. Szczepanczyk, T. Ruzgas, F. Gullfot, A. Gustafsson, and S. Björklund, "Catalase Activity in Keratinocytes, Stratum Corneum, and Defatted Algae Biomass as a Potential Skin Care Ingredient," Biomedicines, early access. doi: 10.3390/biomedicines9121868.
[11] A. Mortensen et al., "Re-evaluation of potassium nitrite (E 249) and sodium nitrite (E 250) as food additives," vol. 15, no. 6, 2017, doi: 10.2903/j.efsa.2017.4786.