Authors
Mateusz Paleczny, Natalia Ślezińska, Daniel Krakowian
Łukasiewicz Research Network – Institute of Industrial Organic Chemistry Branch Pszczyna
e-mail: daniel.krakowian@ipo.lukasiewicz.gov.pl
Aim of the project
Methods for evaluating chemicals for endocrine disruption potential consist of in vitro and in vivo tests. The project aimed to implement methods to identify endocrine disruptors. The following test methods have been selected:
- The trans-activation assay of stably transfected human estrogen receptors for detecting estrogen agonist and antagonist actions using the hERα-HeLa-9903 (ERTA) cell line, according to the OECD guideline 455.
- Uterotrophic Bioassay in Rodents: A short-term screening test for oestrogenic properties according to the OECD guideline 440.
- Hershberger Bioassay in Rats: A Short-term Screening Assay for (Anti)Androgenic Properties, according to the OECD guideline 441).
Short description of the problem addressed by this project
According to the WHO/IPCS (2002) definition, an endocrine disruptor (ED) is an exogenous substance or mixture that affects the function of the endocrine system, producing adverse effects in a healthy organism and its progeny.
The European Union agreed on scientific criteria for assessing substances for ED under the Plant Protection (EC 1107/2009) and Biocidal Products (EU 528/2012) Regulations. To classify a test substance as an ED, test results are required to establish a correlation between adverse effects and their causal relationship with exposure to the substance, indicating its impact on the endocrine system. In addition, ECHA/EFSA guidance has been issued on the identification of EDs in the context of Regulations. Manufacturers of substances were required to identify the risks of possible endocrine-disrupting properties by the scientific criteria set out in the Commission Regulation (EU 2017/2100 – biocidal products and 2018/605 – plant protection products). Due to their serious risk to human and animal health, the identification of these substances is crucial during the registration process of chemicals and their mixtures, plant protection products, medicinal, veterinary, and biocidal products, feed/food additives, industrial waste, household chemical products, and others.
Main results and achievements
- An in vitro (OECD 455) and in vivo (OECD 440/441) test methods were validated.
- OECD 455: 14 reference substances (in 7 concentrations and three replicates) were tested for their effects on the estrogen receptor in the agonist test (substances stimulating the receptor), and 10 reference substances (in 6 concentrations and three replicates) in the antagonist test (substances inhibiting the stimulated receptor). At least two series were performed. All substances were correctly classified.
- OECD 440: five groups of female Wistar rats (6 in each group) were treated with different doses of the reference substance. A test system was obtained from parental pairs. Proficiency in the successful reproduction of rats was confirmed, and a method of marking young rats with a tattoo was developed. The results obtained were correct.
- OECD 441: three groups of male Wistar rats (6 in each group) were treated with two reference substances or vehicles alone. Androgen-dependent organs were excised and weighed. Proficiency in surgery and perioperative care, postoperative analgesia, and the impact of individual analgesics on animal welfare was acquired. The results obtained were correct. OECD 455, 440, and 410 methods were implemented for the Łukasiewicz – IPO offering.
- Historical data were obtained for each method.
Conclusion
Łukasiewicz – Institute of Industrial Organic Chemistry demonstrated proficiency in the use of research methods identifying substances that disrupt the functioning of the endocrine system. Łukasiewicz – IPO offers domestic and foreign clients tests to evaluate chemicals for endocrine disorders through a multi-level testing strategy:
- Level 1 - in silico methods – collecting existing information about a given test material using computational methods, e.g., QSAR, predictive models.
- Level 2 - in vitro methods – providing information on selected endocrine mechanisms or pathways for a given test material (including OECD 455).
- Level 3 - in vivo methods – providing information on selected endocrine mechanisms or pathways for a given test material (OECD 440 and 441 methods).
