After successfully completing some of our first studies relating to the extended one generation reproductive toxicity study (EOGRTS) OECD test guideline (TG) 443 we felt it appropriate to share some key findings. Three vitally important factors are experience, communication and planning.
1. Experience - understanding and implementing the changes
The OECD TG 443 study design is considered to impart many enhancements over its predecessor the OECD TG 416, including:
- Enhanced endocrine disruptor related assessment:
- Two to three times more offspring assessed for sexual development
- Addition of ano-genital distance, nipple/areolae counts, thyroid hormone measurements
- Evaluation of developmental neurotoxicity (DNT), where appropriate, such as:
- Auditory startle, motor activity, functional observation battery investigations and experience of perfusion fixation and brain morphometrics
- Evaluation of developmental immunotoxicity (DIT), where appropriate, for example:
- T-cell dependent antibody response assay: keyhole limpet hemocyanin (KLH)
It is therefore vital that laboratories conducting the studies have sufficient experience in these potentially new end-points, but also that sufficient historical control data (HCD) is available for new end-points and to cover potentially different testing ages stipulated by the OECD TG 443.
Under REACH, applicants are now required to conduct a one-generation reproductive toxicity study (OECD test guideline 443) when preparing chemical dossiers discover more in our informative e-book.
To that end, an ultra-high pressure liquid chromatography coupled to a high end tandem mass spectrometry (UHPLC-MS/MS) for detection of thyroid hormones (T4 and T3) in offspring as young as Day 4 of age has been successfully validated, as traditional Immunoassay techniques could only detect T4 and T3 weaned offspring (Day 21 of age) and adult rats. A poster describing this innovative and pioneering work for offspring samples can be found here.
UHPLC-MS/MS for detection of thyroid hormones
(T4 and T3) in offspring as young as Day 4 of age
has been successfully validated
An internal study was also conducted in order to generate sufficient HCD to fully support regulatory EOGRTS, as some of the ages at which evaluations are required in the OECD TG 443 are different to those conducted on routine reproductive toxicology studies. This internal study was conducted in the two most common strains of rats used on reproductive toxicology studies and has provided significant HCD to fully support the EOGRTS.
2. Communication - solving the practical and logistical challenges
The OECD TG 443 study design imparts significant practical and logistical challenges. It is a complex study, which is thought may limit numbers of labs capable of running study due to its sheer size, and it has some unique features, including:
- Evaluation of several offspring per sex per litter in the F1 generation
- “Triggered” F1 mating, potentially reducing animal usage, and time
The conduct of our initial studies have re-enforced the need for excellent communication in the conduct of all toxicology studies, but in particular those of a complex nature.
Frequent and clear communication is vital, especially in the case of the OECD TG 443 which includes the option to trigger mating of the F1 generation based on existing knowledge plus data gathered during the conduct of the study.
There are many triggers within the EOGRTS design:
- Adverse effects upon fertility or fecundity (fruitfulness - litter size) of the parental generation
- Dose related effects on F1 AGD, nipple counts and puberty onset in the absence of effects on body weight
- Equivocal effects on F1 parameters or unusual control data
but only one event is triggered:
- Mating of the F1 generation – specifically cohort 1B offspring
Triggered events on toxicology studies relies on frequent and excellent communication with our customers in order that data and findings are reported in a timely manner to facilitate discussion and decisions, and also internally to communicate clearly the customers’ requirements and to seamlessly incorporate these into the design of the study.
OECD TG 443 study design is considered to impart
many enhancements over its predecessor the OECD TG 416
3. Planning - ensuring an effective study design
The complex nature of the OECD TG 443 study design requires detailed planning, particularly in the following areas:
The study plan - Ensuring the requirements of the study are clear and precise, and that they represent the requirements of the customer
Offspring allocation - With several offspring per sex per litter forming the various cohorts required for the F1 generation, robust offspring selection and allocation is a key component on OECD TG 443 studies.
Timed behavioral and necropsy requirements - Day specific behavioral and necropsy requirements necessitate careful planning to ensure adequate resource
Triggered F1 mating - The triggered nature of F1 mating requires robust planning in advance to ensure adequate resource in terms of rooms, cages and staff.
Looking to discover more?
Under REACH applicants are now required to conduct a one-generation reproductive toxicity study (OECD test guideline 443) when preparing chemical dossiers – but what are they, what are the differences over the traditional two-generation test guideline method and what are the considerations you need to take in to account when planning your study? We’ve put together an informative e-book to help answer these questions and many more.
About the author
Dr David Howes provides high-quality regulatory and scientific advice to customers who wish to achieve regulatory compliance in the industrial chemicals sector. He is also involved with the EU REACH regulation on chemicals; and global regulatory submissions. During his 30 years at Envigo he has been involved in pesticide residue analysis, environmental analysis, physicochemical testing, product chemistry and regulatory affairs.
Other related blogs include: