What is an Inhalation reprotoxicology study – insights, advice, and secrets

What is Inhalation Reprotoxicology and When is it Relevant?

Inhalation reprotoxicology studies are required when a chemical substance or plant protection product (PPP) is suspected of having the potential to cause reproductive or developmental toxicity and there is the possibility that human exposure can occur via the respiratory system.

The triggers for reprotoxicology studies relate to a chemical’s class or in some markets – for example in the EU – its import tonnage band. Triggers for inhalation studies are based on physical properties, e.g., the chemical’s vapor pressure threshold or whether it is a powder or friable pellet that is likely to be accidentally inhaled.

What Makes Inhalation Reprotoxicology Studies So Challenging?

Reprotoxicology studies are already complex, with the inhalation exposure route adding extra challenges and complications. These include how to:

• Ensure aerosolization of the test substance and effective and consistent delivery through an inhalation exposure system
• Maintain high welfare standards for adult animals and offspring
• Manage logistical complications of both inhalation dosing and reprotoxicology studies, which are inherently complex
• Ensure test and analytics quality, given the challenging dynamic nature of inhalation studies

Inhalation Reprotoxicology Studies Need Specialist Insight, Experience and Resources

Expert Insight into Inhalation Reprotoxicology Optimizes Study Planning and Execution

There are many complex elements to balance in inhalation reprotoxicology studies. You can optimize study design and execution by using an expert team with appropriate levels of experience and insight. Study teams should include study directors with experience of the scientific and regulatory complexity of inhalation reprotoxicology studies, aerosol technologists, specialist animal technicians, and specialist analytic groups.

Test System Optimization to Ensure Study Reliability

The principle objective in any type of inhalation toxicology study is the aerosolizing of the test substance and aerosolizing repeatedly over the study duration with a regulatory acceptable particle size. This requires specialist scientific and aerosol expertise, as well as experience of working with a diverse range of chemicals, including those with challenging physical and chemical properties.

Once aerosolization is achieved, it is important to validate the performance of any exposure system in advance of animal dosing. Firstly, to ensure that an appropriate inhaled test dose can be generated. This might be via an atomizer/nebulizer for liquids, a powder generator for powders or dilution system for gases. Secondly, this demonstrates the stability and reproducibility of the test substance concentration and particle size distribution in the exposure system.

The performance of the exposure system will likely change once animals are introduced. This can be predicted based on extensive experience, and the operating system characteristics adjusted accordingly. Ongoing sampling and monitoring of the test environment during dosing allows real-time refinement to maintain optimized and successful dose delivery.

A review of inhalation chamber concentrations and particle size should occur as often as practically possible to ensure study reliability to supplement the continual checks that are undertaken during the dosing period. This should be in real time or least daily depending on the physical state of the aerosol.

Aerosol technology and technical specialists with inhalation reprotoxicology experience are vital in the set-up, optimization and real-time dynamic adjustment of the exposure systems. Ideally, mean chamber concentrations should remain within ±10% for gases and vapours and ±20% for liquid or powder aerosols. Regulators, however, recognize this can be challenging to achieve and supplementary scientific justification may be needed.

Inhalation Dosing Routes and Durations for DART Studies

There are two methods of administration available for dosing inhaled substances for chemical substance and plant protection products:

• Snout only – this is the preferred regulatory route
• Whole body exposure – this is more appropriate, especially for litters.

In general, inhalation studies use considerably more test substance than non-inhalation studies, with whole body exposure using more than the snout only route.

The duration of dosing is considerably longer with inhalation reprotoxicology studies than in non-inhalation reprotoxicology studies. The exposure duration for inhaled non-reprotoxicology studies is 6 hours per day and 5 days per week to mimic potential worker exposure. However, for inhalation reprotoxicology studies, this is 7 days a week for the same duration to ensure that all phases of the female fertility cycle are assessed.

Summary of some key differences between oral, snout-only and whole body administration for inhalation reprotoxicology:

Specific Animal Welfare Considerations with Inhalation Reprotoxicology Studies

There are special welfare considerations needed for pregnant females, dams and pups and timely and regular consultation of a veterinarian with experience in this field is important to ensure that animal welfare aspects are fully considered and adequately managed. For pregnant females, dosing is withdrawn around parturition, with the last day of dosing being gestation Day 16 for mice and Day 20 for rats. If dosing resumes during lactation, it can recommence on lactation Day 2 (rats), although for 6-hour exposure it is more appropriate to start on lactation Day 4. Dosing lactating dams means separating them from their litter, separation times are set by animal welfare regulations (Animal Scientific Procedures Act (ASPA)) and should be adhered to.

Neonate animal dosing can start as young as Day 4 for rats for snout-only administration. But, there should be appropriate consideration of the time away from the dam, whether the eyes are open and whether the respiratory system is sufficiently developed to scientifically justify dosing.

Logistical Complexity of Inhalation Reprotoxicology Studies

Reprotoxicology studies usually require a mating phase, which can complicate study planning. For some studies, such as OECD 443 or Extended One Generation Reproductive Toxicity Study (EOGRTS), there are built in triggers for additional breeding. This makes basic study planning complex, but with inhalation that logistical complexity is exacerbated.

• Challenges from dose timing: 6-hour dosing over 7 days needs a dedicated team who can work flexibly. In some circumstances, behavioral assessments – taking up 4 hours – are required on the same day as dosing, further complicated as all animals in a group are dosed simultaneously.
• Furthermore, as dosing is triggered by mating, a study can start on multiple days and span weekends. Excellent planning of a dedicated, flexible workforce with multiple skills is therefore essential for studies to run efficiently.
• Capacity constraints for whole body chambers: There is a practical limit on the maximum number of animals that can be exposed in a whole-body chamber due to the physical size of the chamber and the regulatory expectations for operation. For larger studies, like OECD 443 or developmental neurotoxicology studies (OECD 426), two chambers per group may be needed for some study periods, e.g., during littering and when the F1 generation is beginning to dose. This could impact on the facility infrastructure and an additional animal room may be needed, even with some whole body chambers being >8ft tall, 6ft wide and 6ft deep utilised.

Inhalation Reprotoxicology Complicates Investigations and Analytics

Reprotoxicology endpoints are generally consistent across inhalation and non-inhalation reprotoxicology studies. However, there are some special considerations, especially for toxicokinetic investigations using inhalation whole body dosing. Inhalation studies have a wider target acceptance range (±15 minutes), as animals cannot be removed individually from the chamber until the concentration in the chamber is <1% of the original target to ensure staff safety. This is unlike other administration routes which use a much tighter (i.e. ±1 minute) acceptance for target toxicokinetic bleed times.

The dose received by an animal for dietary and oral administration routes is normally in terms of mg/kg per day; however, for inhalation studies, it is in terms of exposure level (mg/L) of air. Furthermore, due to the dynamic nature of all inhalation studies, the actual mean concentration of the dosing period is used rather the target aerosol concentration.

Conclusions

Reproductive toxicology studies are complex and expensive, and by adding inhalation dosing the complexity and expense rises exponentially. It is essential, therefore, to design, plan, and execute these studies to extremely high standards to ensure validity, regulatory acceptability, and guarantee cost-effectiveness. Critical success factors include:

• The ability to design and plan studies based on experience with substances with similar chemical or physical properties and built upon regulatory expertise gained from previous study programs and submissions
• Scientific and regulatory insight relevant to both inhalation administration and reproductive toxicology
• Experienced, dedicated, and flexible teams of experts in analytical chemistry, aerosol technology, animal behavior and handling, analytics, and reproductive toxicology
• Logistical excellence and adaptable capacity to manage the complexity of running inhalation reproductive toxicology studies.

Animal Welfare considerations must be included in study design as well as study conduct. Animal care and studies must be conducted in alignment of all applicable animal welfare regulations.