EXPOSURE CHAMBERS Yves Alarie, Ph.D Professor Emeritus U niversity of Pittsburgh,USA

A.BEST DESIGN There is no such thing as “best design” chamber to answer all the needs of inhalation toxicology studies. The “best design” is a chamber that works according to the following criteria: Fairly uniform distribution of contaminants, variation of 10-15% between sampling ports is quite acceptable.

Ammonia level should be verified with high animal load particularly when reaction with pollutants is possible, CI 2, SO 2, NO 2, etc. If this airflow presents a problem (high cost of pollutant, availability of pollutant, cleaning, etc.) it can be reduced, but probably not below 0.2  the total volume of the chamber without creating problems with temperature, ammonia, CO 2, humidity, etc.

Animal load should be lower than 1% to 5% of chamber volume. Calculate as follows: take body weight of each animal to be same as its volume (i.e., 200 grams rat = 0.2 Liter) and multiply by number of animals. Thus to expose 100 rats of 200 grams (20 liters of rats) you need a minimum chamber volume of 2,000 liters and preferably there will be only one layer of animals. This can be reduced to a 400 liter chamber (5%) but likely to need 2 layers. One layer is preferable if working with highly reactive gases which will react with fur of animals or when working with large particles, but is not necessary with aerosol around 1 μ m in size and with non reactive gases such as CO etc. or vapors such as benzene, carbon tetrachloride etc.

Do not use the chamber for mixing. The added pollutant should be mixed with the incoming diluting air prior to entering the chamber or mixed at the top or entrance of the chamber. The idea of placing baffles etc. in a chamber for better mixing is nonsense. This simply provides more surface area for interaction with gases or aerosols. A baffle can be used to prevent large particles to enter the chamber or at the entrance of the chamber for proper mixing but never within a chamber.

Do not use a fan in a chamber for mixing. A fan will increase turbulence which will cause aerosol coagulation, stratification and deposition on various surfaces.

With reactive gases, “conditioning” of the chamber should be done prior to loading animals in the chamber. Once this is done addition of animals may drop the concentration by at least 50% and sometimes by as much as 95%. This is not serious for a long-term chronic study. Indeed during the first week of such a study adjustment can be made to the delivery system to bring the concentration upward to the desired level. In cases of acute studies, if you want to know what the animals are likely to “soak” collect dead animals used for other experiments, keep them refrigerated and then place in your chamber.

Permitted daily variation in exposure concentration for chronic studies Reactive gases  20% of desired Non-reactive gases  15% of desired Aerosol 5 μ m: Don’t use them in large chamber, nose or head only exposure is more appropriate. However, relevance is in question, rats do not inhale rocks!

The only solution to this problem is to set up elutriation systems to remove the large particles or to reformulate the aerosol delivery system so that the particle size is appropriate for an inhalation study. Obviously this will not be exactly the same as what consumers will be exposed to. However, without doing this you may end up with an LC 50 of 1 ton/m 3. While this may be reassuring, it is not appropriate, since none of the particles may have been inspirable or respirable for the exposed animals.

B.DESIGN FOR VERY HIGH LEVEL OF AEROSOL EXPOSURE The decision to use a chamber or head only exposure is impossible to make without some preliminary work with the chemical. This work can be done using a small chamber and a few animals. In general concentrations higher than 500 mg/m 3 are difficult to work with unless the particle size is small. Head-only or nose- only exposure is indicated.

There is an EPA recommended level of 5 gram/m 3 of aerosol as a level at which if no deaths occur the chemical would be considered “non toxic”. This is impossible to achieve for most solid or liquid and keeping the particle size reasonably small for animals to inhale and at the same time using an inhalation chamber with the particle size and exposure concentration remaining fairly uniform from top to bottom.

<50 mg/m 3 : small particles: O.K. larger particles: O.K. >50 mg/m 3 : small particles: O.K. larger particles: more difficult 500 mg/m 3 : small particles: O.K. larger particles:large particles will deposit 5,000 mg/m 3 : small particles: O.K. larger particles:too much deposition 1-3 μ m MMD and below.

C.CHEAP CHAMBER, GOOD RESULTS For acute, sub-acute or chronic work using rats or mice an all-glass 100-liter aquarium is just perfect. A cover can be made out of plywood or plexiglass with the inside of the cover lined with teflon or polyethylene film. Such a chamber costs about $100 with all fittings.

Operated at 100 liters/min, equilibrium concentration will be reached in about 5 minutes with uniform distribution of contaminants, gases or aerosols.(6,7) It is not necessary to use expensive stainless steel chambers with pyramidal tops and bottoms and a glass door to observe the animals.

Murphy’s Law says you will break the glass aquarium. So what? Just have a spare one on hand. However, if you are exposing a large number of animals for a chronic study it obviously makes sense to have a stainless steel chamber. However, you will break the glass door!

D.EXPOSURE SYSTEMS FOR CALCULATION OF DOSE In order to calculate the net dose received during exposure to aerosols and reactive gases the exposure concentration and duration of exposure are needed. Two other variables are also needed: tidal volume (VT) and respiratory frequency (f or BPM). These cannot be obtained with exposure systems described above. They can be obtained with “head” only exposure system with the body of the animal held in an enclosure with a seal at the neck, such a device is called a “body plethysmograph” or a “head-out body plethysmograph”.

A pneumotachograph is a device for measuring the rate at which air flows in and out of the plethysmograph due to thoracic displacement with each breath. The rate of airflow (ml/sec) when integrated with time yields volume (ml) and a record of tidal volume is obtained. Another way of obtaining VT and f is to use a “whole body plethysmograph”. With this device, the whole animal is within an airtight enclosure and again the pressure created with each breath is measured. The pressure created with each breath is more complex in its origin than with the body plethysmograph. However, there is no need for restraint of the animal.

E.DO NOT EXPOSE ANIMALS UNLESS YOU ARE SURE Prior to exposing animals you should have good experience with an empty chamber, including the housing cages and the contaminant delivery system as well as the method for analysis of the contaminant, and if you are dealing with an aerosol, determinations of the particle size need to be done. You should know how large a difference there is between the “nominal concentration” and “actual concentration”. If there is a large discrepancy between the two you should know why.

F.RAPID GUIDE FOR EQUILIBRATION TIME AND ITS USE As a gas or aerosol is introduced at a uniform rate in an exposure chamber maintained at a continuous airflow, the concentration within the chamber increases until it is practically constant. Assuming perfect mixing, from Silver, (see reference above):

F.RAPID GUIDE FOR EQUILIBRATION TIME AND ITS USE As a gas or aerosol is introduced at a uniform rate in an exposure chamber maintained at a continuous airflow, the concentration within the chamber increases until it is practically constant. Assuming perfect mixing, from Silver, (see reference above):

Thus, the % of the desired concentration w/b obtained in time t is: The time required for equilibration of the chamber to 99% can be calculated by setting equation 2 equal to 99.

or Transformed into logarithm form

and t99 should be calculated for each situation so that the time (min) to reach equilibration is very short compared to the duration of exposure.

G.AIR CHANGES An air change is said to occur when a volume of air equal to the volume of the chamber has passed through the chamber (i.e., a = b ). This is a convenient term used by ventilation engineers, but is not, strictly speaking, correct. When such occurs, from Eq (2), only 63% of the air has been “changed”. From Eq (5), the time for one “air change” must be multiplied by a factor of 4.6 before 99% of the expected “change” in air can be accomplished.

Therefore, if a and b are equal, there is one “air change” every 4.6 minutes or 12/hour, not 60/hour as one would think from the ventilation engineers’ terminology given in the first sentence above. The best thing to do is to forget about “air change” and give a and b. Then we know what you are doing.