INHALATION TOXICOLOGY STUDIES PART III DOSING & ANALYSIS

1-. WHAT IS THE DIFFERENCE BETWEEN INHALED OR DELIVERED DOSE AND DEPOSITED DOSE??

The delivered dose is the amount per unit of bodyweight that is presented to the animal. The deposited dose is a fraction of the delivered dose depending on the particle size and species. The FDA assumes 100% deposition in human, 10% in rats and 25% in dogs irrespective of any other information produced by the submitting company. This is based a scientific paper cited by Wolff and Dorato (Wolff and Dorato, Critical Reviews in Toxicology, 23 (4):343-369, 1993). These deposition factors need to be taken into consideration when calculating clinical overages.?

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2-. HOW IS INHALED OR DELIVERED DOSE DETERMINED??

The precise dose is delivered to the animal using syringes, gauges, or needles for oral or parenteral routes and can be measured exactly, based on the bodyweight of the animal and the concentration of the solution being administered.?

With inhalation administration, it is not possible to calculate the dose given to the animal in the same way.?

The animals or cells are presented with a pre-defined concentration of the test item in the atmosphere and spontaneously breathes or are in contact with that aerosol, effectively self-dosing based on the animals own tidal volume and frequency of breathing (in vivo case) or based on the deposition factor and absorption ratios (in vitro case).?

Therefore, the delivered dose needs to be derived based on an estimate of the volume of atmosphere inhaled during the exposure period as well as bodyweight and test atmosphere aerosol concentration. Finally, the proportion of inhaled test item that will enter the lungs is dependent on the particle size.?

The delivery dose is estimated as:?

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3-. WHY DO THE DELIVERED INHALATION DOSES VARY FROM THE TARGET VALUES??

Unlike dispensed volume methods used for other administration routes, inhalation exposure can be affected by several variables. Seemingly minor changes in a system, such as the corner angles in a length of aerosol hose, or the exposure environment, including air temperature, humidity and atmospheric pressure, can impact the achieved aerosol concentration in the breathing zone of the experimental systems. These variations will occur even in the absence of alterations to system settings.??

In addition to this uncertainty, the animals and cells themselves have both a direct effect on dose concentration: they filter the atmospheres they are exposed to; they can vary the moisture levels and can also have highly variable breathing patterns between individuals or the absorption layers in the case of cells. The combination of these variables leads to not only to day-to-day changes in measured drug concentration but also differences between the local and systemic delivered drug profile between animals and cells that have been exposed to the same atmosphere for the same duration. The aim with respiratory studies is to monitor and control as many of the variables as possible in order to minimize the differences between individual delivered doses.?

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4-. WHY CAN PARTICLE SIZE BE DOSE-LIMITING??

Even when the particle size of the input material is within the required range, once generated into a stream of air, particles of many materials can aggregate. The largest of these aggregates are not aero stable and can sediment from the airstream before they reach the exposure chamber. Exposure systems are designed to ensure that only particles of appropriate Mass Median Aerodynamic Diameter (MMAD) reach the breathing zone.?

The proportion of material that is lost in this manner is a major component of the commonly poor “generation efficiency” of a typical particulate aerosol generation system, A poor efficiency system will often be found to suffer from excessive aggregation and a large amount of test item wastage. These are many methods to increase the efficiency of a system and reduce wastage while maintaining a breathable MMAD, e.g. cyclones, micronizers etc. However, increasing the aerosol concentration in an attempt to increase the delivered dose only rarely achieves the desired outcome, due to the rate of sedimentation as represented in the following diagram. In some instances, further reduction in generation efficiency will be seen and achieved concentration will fall.?

5-. WHAT ARE THE TRADITIONAL METHODS OF DOSING NONCLINICAL SPECIES BY THE INHALATION ROUTE??

It is not uncommon for early screening rodent studies to be dosed by intratracheal or intranasal administration as this method only requires a small amount of test item. This type of dosing is then either replaced by nose-only or whole-body exposure when assessing the efficacy in a pharmacology respiratory model. Nose-only exposure is then used a default during pre-clinical toxicology testing to mimic the clinical application. From an ethical point of view, the 3Rs rules -? https://www.dhirubhai.net/pulse/3-rs-rule-animal-experimentation-paradigm-shift-towards-vitro-26xne/?trackingId=9NhkpHgT6aaK1Hqwy2izjQ%3D%3D - tries to develop in vitro research with cellular models that mimic human behaviour when exposed to products. In these cases, exposure using an Air Liquid Interface (ALI) system is the most appropriate, starting with high doses in serial dilutions (>2folds).?

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6-.WHICH LABORATORY SPECIES CAN BE DOSED BY INHALATION??

It is possible to dose virtually all commonly used laboratory species by inhalation. There is much historical data on the use of rats, mice, rabbits, dogs, and monkeys by the inhalation route. In vitro and in vivo experiments need different approaches so you will need a robust partner for specific equipment and expertise in tox or efficacy studies.?

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7-.WHAT STUDY ENDPOINTS ARE USED TO ASSESS POTENTIAL IRRITANCY FOR INHALATED TEST ITEMS??

For in vitro studies, endpoints would include cytotoxicity and viability of the cells including a LC50 on dose-dependent pattern. After that, the cell investigation can be extensive and depends on the aim of the project and the cell line and type that you are investigated. In the in vivo phase, typical endpoints would include airway tissue histopathology, tissue and BALF cellular infiltration, expression of pro-inflammatory cytokines in the BALF and changes in respiratory physiology during the inhalation exposures. Further assessment of the lung mechanics during and after exposure could also be evaluated. These endpoints can be measured during efficacy studies but may be limited to evaluation after acute exposures at efficacious doses. Multiples of the predicated efficacious doses would need to be included in the study protocol to obtain early data on safety margins that could be flags for subsequent toxicology testing.??

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8-. WHAT ARE THE COMPLICATIONS OF ADDING PHARMACOLOGY ENDPOINTS ON TO TOXICOLOGY STUDIES??

This requirement is becoming increasing popular both from a scientific and 3Rs perspective, however, practical consideration needs to be given for its inclusion into the study design as it could impact on the data quality and hinder subsequent interpretation. There are also several logistical complications that need to be considered as they can influence the results that can be obtained.??

Consideration needs to be given to the following, especially recording respiratory parameters:?

• Noise within the dosing room that is either scheduled on non-scheduled as part of the daily routine can influence the data.?

• Movement of staff particularly near the inhalation exposure system can often give elevated respiratory minute volume recordings.?

• Any planned data recordings should be preferentially scheduled on days that do not have other on-going procedures taking place.?

• Pre-dose and post-dose recordings must be scheduled around the toxicology regime, which may mean feeding patterns need to be amended.?

• There are practical limitations to the number of animals that can be recorded simultaneously. This may result in extra test item to cover the additional generation period.?