DMSO Concentrations up to 1% are Safe to be Used in the Zebrafish Embryo Developmental Toxicity Assay

Dimethyl sulfoxide (DMSO) is a popular solvent for developmental toxicity testing of chemicals and pharmaceuticals in zebrafish embryos. In general, it is recommended to keep the final DMSO concentration as low as possible for zebrafish embryos, preferably not exceeding 100 μL/L (0.01%). However, higher concentrations of DMSO are often required to dissolve compounds in an aqueous medium. The aim of this study was to determine the highest concentration of DMSO that can be safely used in our standardized Zebrafish Embryo Developmental Toxicity Assay (ZEDTA). In the first part of this study, zebrafish embryos were exposed to different concentrations (0–2%) of DMSO. No increase in lethality or malformations was observed when using DMSO concentrations up to 1%. In a follow-up experiment, we assessed whether compounds that cause no developmental toxicity in the ZEDTA remain negative when dissolved in 1% DMSO, as false positive results due to physiological disturbances by DMSO should be avoided. To this end, zebrafish embryos were exposed to ascorbic acid and hydrochlorothiazide dissolved in 1% DMSO. Negative control groups were also included. No significant increase in malformations or lethality was observed in any of the groups. In conclusion, DMSO concentrations up to 1% can be safely used to dissolve compounds in the ZEDTA.

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LETHAL CONCENTRATION 50% OF PATCHOULI OIL (Pogostemon cablin) TOWARDS ZEBRAFISH EMBRYO (Danio rerio)

Herb-Medicine Journal ◽

10.30595/hmj.v3i2.6360 ◽

2020 ◽

Vol 3 (2) ◽

pp. 1

Author(s):

Romel Ciptoadi Wijaya

Keyword(s):

Danio Rerio ◽

Zebrafish Embryo ◽

Toxicity Testing ◽

Positive Control ◽

Negative Control ◽

Lethal Concentration ◽

Zebrafish Embryos ◽

Treatment Groups ◽

Precise Prediction ◽

Loss Of Appetite

Preface :Patchouli Oil requires toxicity testing for safety before we can use it widely. It causes side effects such as nausea, vomiting and loss of appetite in some people. Determination of lethal concentration 50% (LC50) in the early stages of zebrafish embryos development will provide an easier, faster and precise prediction of toxicity. At a certain dose, it can cause impairment and death toward organisms. Therefore, the aim of this study was to determine LC50 of Patchouli Oil in carboximethyl cellulose emulsifier towards zebrafish embryo (Danio rerio).Method :Laboratory experimental study was using zebrafish embryos at 2 hours post fertilization. The total of 160 embryos were used and divided into 8 groups, i.e.; Negative control (KN) was given the embryonic fluid, positive control 1 was given 5000 ppm of patchouli oil (KP1), positive control was given 5000 ppm of CMC (KP2), and 5 treatment groups, i.e.; concentration of 10 ppm (P1), 30 ppm (P2), 60 ppm (P3), 90 ppm (P4) and 120 ppm (P5) of Patchouli oil which was emulsified in CMC. The study was conducted with 3 times repetition. Data collection was done by calculating total of embryo’s deaths for each treatment at 24-72 hours of exposure. Data were analyzed using Regression Probit Analysis.Results :Mortality in the group of KN was 1.6%, KP1 was 98.3% and KP2 was 0%. Meanwhile in the treatment group of P1, P2, P3, P4 and P5 respectively were 0%, 5%, 10%, 25%, and 50%.Conclusion :Based on these results, Lethal Concentration 50% of Patchouli Oil in CMC emulsifier towards zebrafish embryo is 120 ppm.

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Validation, Optimization, and Application of the Zebrafish Developmental Toxicity Assay for Pharmaceuticals Under the ICH S5(R3) Guideline

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.721130 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yi-Sheng Song ◽

Ming-Zhu Dai ◽

Chen-Xia Zhu ◽

Yan-Feng Huang ◽

Jing Liu ◽

...

Keyword(s):

Developmental Toxicity ◽

False Negative ◽

Toxicity Testing ◽

The United States ◽

Toxicity Assay ◽

Negative Results ◽

False Negative Results ◽

Developmental Toxicity Testing ◽

Classical Protocol

The zebrafish as an alternative animal model for developmental toxicity testing has been extensively investigated, but its assay protocol was not harmonized yet. This study has validated and optimized the zebrafish developmental toxicity assay previously reported by multiple inter-laboratory studies in the United States and Europe. In this study, using this classical protocol, of 31 ICH-positive compounds, 23 compounds (74.2%) were teratogenic in zebrafish, five had false-negative results, and three were neither teratogenic nor non-teratogenic according to the protocol standard; of 14 ICH-negative compounds, 12 compounds (85.7%) were non-teratogenic in zebrafish and two had false-positive results. After we added an additional TI value in the zebrafish treated with testing compounds at 2 dpf along with the original 5 dpf, proposed a new category as the uncategorized compounds for those TI values smaller than the cutoff both at 2 dpf and 5 dpf but inducing toxic phenotypes, refined the testing concentration ranges, and optimized the TI cut-off value from ≥ 10 to ≥ 3 for compounds with refined testing concentrations, this optimized zebrafish developmental assay reached 90.3% sensitivity (28/31 positive compounds were teratogenic in zebrafish) and 88.9% (40/45) overall predictability. Our results from this study strongly support the use of zebrafish as an alternative in vivo method for screening and assessing the teratogenicity of candidate drugs for regulatory acceptance.

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