**4. Conclusions**

In this study, Ech A degradation products formed during oxidation by O2 in air-equilibrated aqueous solutions were identified, isolated, and structurally characterized. During the oxidation of Ech A, transformation was found to occur only in the quinonoid ring, with the formation of a hydrogenated quinonoid cycle with two carbonyl groups and two pairs of geminal hydroxyl groups. The further oxidation of bis-*gem*-diol occurred with the cleavage of the dihydroquinonoid ring and a chain of subsequent decarboxylations.

The HPLC method with DAD and MS detection was developed and validated to monitor the Ech A degradation process and to identify the appearing compounds. The structural studies and obtained HPLC–MS parameters of the main Ech A oxidation products are of great interest from the point of view of investigation of the chemical properties of drug substances and for developing methods for monitoring the quality of drugs and food additives obtained from sea urchin pigments, in addition to their stability.

In recent years, there has also been a growing interest in the environmental significance of methods to characterize the destruction of pharmaceutical compounds and study their toxic properties in order to avoid the accumulation of these compounds in nature.

The in silico toxicity studies performed using ProTox-II webserver revealed that Ech A oxidative degradation products do not exhibit mutagenic properties, and their toxicity values were much lower than that of Ech A. This means that the spontaneous formation of these degradation products in preparations using Ech A would not be harmful to patients.

**Supplementary Materials:** The following are available online: Figure S1. The UV-Vis spectrum of Histochrome (blue) and Echinochrome A (black) in ethanol solution containing 1 mM HCl; Figure S2. Change in the concentration of echinochrome A during the oxidation of a 1% histochrome solution; Table S1. Accuracy and reproducibility of the quantification of echinochrome A (1) using HPLC method; Figure S3. HRESIMS (negative mode) data for methyl ethers of Ech A oxidation products obtained by methylation with methyl iodide; Table S2. HRESIMS (negative mode) data for methyl ethers of Ech A oxidation products obtained by methylation with methyl iodide; Table S3. ESIMS (negative mode) data for methyl ethers of Ech A oxidation products obtained by methylation with diazomethane; Figures S4 and S5. IR spectrum (CDCl3) of compound 7 dimethyl ether; Figures S6 and S7. IR spectrum (CDCl3) of compound 8 methyl ether; Figures S8 and S9. IR spectrum (CDCl3) of compound 10; Figures S10 and S11. IR spectrum (CDCl3) of compound 11; Figure S12. 1H NMR spectrum (300 MHz, acetone-d6) of 7-ethyl-2,2,3,3,5,6,8-heptahydroxy-2,3-dihydro-1,4-naphthoquinone (2); Figure S13. 13C NMR spectrum (75 MHz, acetone-d6) of 2; Figure S14. HMBC spectrum (300 MHz, acetone-d6) of 2; Figures S15–S17. HMBC correlations of 2 (enlarged); Figure S18. 1H NMR spectrum (300 MHz, CDCl3) of echinolactone (11); Figure S19. 13C NMR spectrum (75 MHz, CD3CN) of echinolactone (11); Figure S20. HMBC spectrum (300 MHz, CD3CN) of echinolactone (11); Table S4. Selected crystal data and refinement parameters for α- and β- forms of C11H8O7•H2O; Table S5. Selected geometric parameters (Å) for α- and β- forms of C11H8O7•H2O; Table S6. Hydrogen-bond geometry (Å, ◦) for α- and β- forms of C11H8O7•H2O; Figure S21. Overall packing for α-C11H8O7•H2O viewed along the a-axis direction; Figure S22. A plot of band for β-C11H8O7•H2O; Figure S23. Overall packing for β-C11H8O7•H2O viewed along the a-axis direction; Figure S24. 1H NMR spectrum (500 MHz, CDCl3) of dimethyl ether of compound 7; Figure S25. 13C NMR spectrum (126 MHz, CDCl3) of dimethyl ether of compound 7; Figure S26. HMBC spectrum (500 MHz, CDCl3) of dimethyl ether of compound 7; Figure S27. 1H NMR spectrum (700 MHz, CDCl3) of methyl ether of 4-ethyl-2-formyl-3,5,6-trihydroxybenzoic acid (8); Figure S28. 13C NMR spectrum (175 MHz, CDCl3) of methyl ether of 8; Figure S29. HMBC spectrum (700 MHz, CDCl3) of methyl ether of 8; Figures S30–S34. HMBC correlations of methyl ether of 8 (enlarged); Figure S35. 1H NMR spectrum (700 MHz, acetone-d6) of 4-ethyl-2,3,5-trihydroxybenzoic acid (9); Figure S36. 13C NMR spectrum (175 MHz, aceton-d6) of 9; Figure S37. HMBC spectrum (700 MHz, aceton-d6) of 9; Figures S38 and S39. HMBC correlations of 9 (enlarged); Figure S40. 1H NMR spectrum (700 MHz, CDCl3) of 3-ethyl-2,5-dihydroxy-1,4-benzoquinone (10); Figure S41. 13C NMR spectrum (175 MHz, CDCl3) of 10; Figure S42. HSQC spectrum (700 MHz, CDCl3) of 10; Figure S43. 1H NMR spectrum (700 MHz, CDCl3) of dimethyl ether of 10; Figure S44. 13C NMR spectrum (175 MHz, CDCl3) of dimethyl ether of 10; Figure S45. HMBC spectrum (700 MHz, CDCl3) of dimethyl ether of 10; Figures S46–S48. HMBC correlations of dimethyl ether of 10; Table S7. Histochrome toxicity values (intraperitoneal administration); Table S8. Accounting for chromosomal aberrations in mammalian bone marrow cells; Table S9. The results of a study of the mutagenic effect of the histochrome drug on indicator strains in the Ames test; Table S10. The results of the study of the ability of the histochrome drug to induce dominant lethal mutations in the germ cells of mice.

**Author Contributions:** Conceptualization, N.P.M.; formal analysis, V.P.G. and P.S.D.; investigation, E.A.V., N.P.M., A.V.G., V.P.G., and P.S.D.; project administration, N.P.M. and P.S.D.; resources, V.P.G.; software, A.V.G.; supervision, S.A.F.; writing—original draft, N.P.M. and E.A.V.; writing—review and editing, S.A.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The study was carried out on the equipment of the Collective Facilities Center "Far Eastern Center for Structural Molecular Research (NMR/MS) PIBOC FEB RAS". The X-ray measurements were performed on the equipment of the Collective Facilities Center "Far Eastern Center of Structural Studies", Institute of Chemistry FEB RAS.

**Conflicts of Interest:** The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
