Did you make it along to this year’s Society of Toxicology (SOT) Annual Meeting? We did, and it proved to be just as interesting and inspiring as previous years. But if you weren’t able to come along - don’t worry.
Here, you’ll find a summary of one of the most popular sessions we hosted. John Carter, Head of Cell and Molecular Sciences and David Myers, Senior Toxicologist at Envigo gave a presentation entitled “Endocrine Disruptor (ED) assessment in the EU: From conceptual framework to reality.” Below you’ll discover key insights and advice they shared with their appreciative audience.
John, David and their teams will be presenting the session in a live webinar on May 15, 2019, in case you missed it. They'll also share some additional informative and thought-provoking case studies. Register for the endocrine disrupting chemicals live webinar.
A packed and productive SOT 2019
Baltimore, Maryland was the venue for this year’s packed five-day event. Last month, attendees from over 50 countries gathered together to share their expertise and research. They included leading toxicologists, thought leaders and major industry figures.
The SOT agenda packed in over 100 featured and scientific sessions. They allowed attendees to discuss cutting-edge science, mechanisms, regulatory considerations - along with many other pressing industry issues.
Along with presenting four posters, Envigo hosted three sessions at SOT. They included John and David's presentation on Endocrine Disrupting chemicals.
New guidance on identification of EDs was published in June 2018
and took effect from 10 November 2018
Introduction to ED assessment in the EU - conceptual framework
John began the presentation with an introduction to the Organization for Economic Cooperation and Development (OECD)'s 'Conceptual Framework (CF).' The OECD's CF for Endocrine Assessment has, John said, 'now come of age.' Endocrine disruptor assessment deadlines for plant protection product and biocidal product AI submissions, John told the audience, came into force from November 10, 2018.
John explained the OECD hosts the Adverse Outcome Pathway Knowledge-Base (AOP-KB). It includes over 40 listed AOPs:
- 6 estrogen AOPs
- 4 androgen AOPs
- 9 thyroid AOPs
- 21+ other endocrine AOPs
Development and divergence in ED testing
John set out for the audience a list of OECD test guidelines that support the CF. He proceeded to contrast the development and divergence of testing strategies between the EU and the US, and the models that are used in each area.
The OECD Conceptual Framework, he explained, has 5 levels of mammalian and mammalian-toxicology. The top levels are:
- Level 4: in vivo assays providing data on adverse effects of endocrine relevant endpoints
- Level 5: in vivo assays providing more comprehensive data on adverse effects of endocrine relevant endpoints over extensive parts of the life cycle of the organism
John highlighted the importance of endocrine disruptor endpoints on OECD level 4 and level 5 studies, and in vivo capabilities and mass spectrometry analytical support for ED endpoints.
In conclusion to his part of the presentation, John gave the audience a background on European Union Network of Validation Laboratories for Alternative Methods (EU-NETVAL). “EU-NETVAL,” he said, “was established in 2014 by EURL ECVAM. It was tasked with prioritizing development of in vitro Endocrine Disruptor assays.”
Endocrine disruptor endpoints on OECD CF level 4 and level 5 studies
Next up was David Myers. He began by giving attendees an overview of endocrine disruptor endpoints on OECD CF level 4 and level 5 studies.
For OECD CF Level 4 in vivo mammalian toxicity, David looked at:
- ECD 407 - 28-day repeat dose oral study (rats)
- OECD 408 - 90-day repeat dose oral study (rats)
- OECD 414 - pre-natal developmental toxicity study in rats
- OECD 421/422 - reproduction/developmental toxicity screening tests
Next David talked about OECD CF level 5 in vivo mammalian reproduction toxicity, namely:
- OECD 416 – two-generation reproduction toxicity study
- OECD 443 – extended one generation reproduction toxicity study (EOGTRS)
Endocrine disruptor endpoints on OECD CF level 4 and level 5 studies
Next, David spoke about “T3 and T4 Analysis by Liquid Chromatography Coupled to Tandem Mass Spectrometry Detector (LC-MS/MS)”.
He highlighted the failure of standard T3/T4 analysis to meet new OECD regulatory requirements. “Currently published immunoassay and LC MS/MS techniques are,” he said, “sufficient for the determination of T3 and T4 levels in adult rats. But they are generally not sufficient for T3 and T4 determination in fetuses and juveniles on reproductive toxicology studies.”
“This,” he continued, “is due to lack of sensitivity, low selectivity and large sample volume requirements.”
To help bring this point to life for the audience, and to show them ways around this pressing commercial problem, David described a successful positive control pre and post natal developmental thyroid study in Sprague-Dawley rats by oral administration of PTU (Propylthiouracil).
“Quantifiable levels of total T3 and T4 hormones were detected in pregnant and lactating females and weanlings,” he said. “However, the validated immunoassay technique was, to our surprise, unable to detect T3 and T4 in fetuses or in F1 offspring at Days 4 and 13 of age.”
He followed the study through, explaining:
- How the modifications to the LC MS-MS method were developed and validated
- The GLP Validation focus adopted
David outlined to his audience the study’s challenges and best practice, including:
- Integrity of method – selectivity
- Different collection tubes – and which proved the most effective
- Triggered thyroid hormone analysis – stability requirements
- How historical control data is steadily being generated
Challenges and best practice
In conclusion, David said: “a LC-MS/MS method was validated to be selective, sufficiently sensitive, and accurate. It was robust irrespective of sample collection method,” he continued, “and successful in establishing sufficient stability of T3 and T4 in serum samples.”
David went on: “the range validated for total T3 and T4 only utilizes 50 μL sample. It achieved the required detection/quantification in control samples for fetuses”.
In addition, he said:
- The validated method allows for more efficient sampling times within the animal unit (30 min clotting period instead of traditional 1 hour for plain tubes)
- Test item related increases or reductions in hormone levels were quantifiable in the range validated. At the same time they minimized the need to perform repeat analysis arising from high concentration samples
Regulatory framework and support
From working closely with many crop and chemical companies, John and David highlighted having a grip on the science and the history of this area is only part of the story. It’s also vital to understand the regulatory framework and support that it is available. No business wants to fall foul of the regulators, especially in such a high-profile area such as endocrine disruptors.
'Guilty until proven innocent'
John and David were keen to make the point it's not just active substances that are covered by the EU regulations. They also stressed when it comes to the applicability of Biocidal Product (BP) regulations and Plant Protection Product (PPP) regulations, the regulators take a “guilty until proven innocent” approach.
“A substance of concern under the European BP or PPP legislation,” he explained, “is to be regarded as having ED properties, as long as it is not demonstrated that the substance is not an endocrine active substance.”
John and David then went on to look at:
- the three conditions stipulated in ED assessment criteria
- OECD CF Level 1 workflow tasks
- ECHA/EFSA Guidance on generating further information for OECD CF Level 2/3: in vitro/ in vivo mechanistic testing
- MoA(s): adversity and/or endocrine activity (ECHA/EFSA)
Deadlines to be aware of
The presenters concluded by reminding the audience the EU’s ED criteria have been applicable from November 10, 2018 for plant protection and biocidal product registrations. They advised understanding what the new criteria is would be critical to ensuring requirements are met.
Couldn't make SOT this year? There's no need to miss out. Join John, David and their team of experts as they relay the session in a live webinar on May 15. 'Endocrine disruptor (ED) assessment in the EU' - secure your spot today!