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Acute Inhalation Toxicity






To obtain scientific information regarding health hazards that may result from a single exposure to the agricultural chemical via inhalation which possibly gets into body through respiratory tract. 


1.      Principle of the Test


To provide a concentration-response relationship ranging from non-lethal to lethal outcomes to derive a median lethal concentration (        LC50).  Groups of animals are exposed to a limit concentration (limit test) or a series of concentrations in a stepwise procedure for a duration of 4 hrs. The animals are observed for mortality. Moribund animals or animals in distress should be humanely killed and considered in the same way as animals that died due to exposure.


2.      Test animals


Selection of  animal species : Rat is the preferred species and justification should be provided if other species  are used. Females should be nulliparous and non-pregnant.


3.      Age


On the exposure day, animals should be healthy young adults 8 to 12 weeks of age. 


4.        Weight


On the exposure day, body weights should be within ±20% of the mean weight for each sex of any previously exposed animals of the same age.


5.                  Housing and Feeding Conditions


Temperature - 22±3ºC

Relative Humidity – 50-60% (However, humidity should not be below 30% or exceed                                 70%  at any given point of time).

Lighting  –  12 hours light and dark cycle

Diet and water – Standard laboratory diet specific to the species and  filtered water, free                                         from contamination



6.         Acclimatization           


The animals should be acclimatized to the laboratory conditions for at least 5 days by keeping in individual cages prior to dosing. Animals  should  also  be  acclimatized  to   the   test   apparatus   for   a   short   period  prior  to  testing,  as   this  will  reduce the stress caused by introduction to the new environment.


7.            Exposure method


Expose animals to test substance as a gas, vapour, aerosol or a mixture thereof, for 4 hours using Standard Inhalation Equipment.  The preferred mode of exposure is nose- only ( also include  head only, snout –only). If whole body mode of exposure is used, then the objective to select this mode be justified.  Water may be provided during whole body exposure.  Do not provide food.


The flow of air through the chamber should be carefully controlled, continuously monitored, and recorded at least hourly during each exposure. Particle size should be performed for all aerosols and for vapours that may condense to form aerosols. The  particle size (aerodynamic mass median size) of 1-4 μmis recommended or use the minimum particle size with which it is possible to conduct the test. Temperature of 22±3ºCand 30-70% humidity should be maintained in the chamber.



When a vehicle is used for maintaining the proper concentration and particle size of the test substance in the exposure environment, it is desirable   to use water or  a vehicle that is known to be non-toxic, or that will not affect the test results. When necessary, conduct studies with a vehicle control group. Particulate material may be mechanically processed to achieve the required particle size distribution.


8.               Number of animals


Three animals per sex for each dose level in sighting study and limit test and 5 animals per sex for each dose level for main study.  


9.      Dose Levels and Administration of doses 


i)                    Sighting study


3 male and 3 female animals are exposed to single or more concentrations to determine test article potency, identify test substance susceptibility to particular sex, if any. It will assist in selecting the dose for the main study, limit test.



ii)                  Limit Test


Limit test is undertaken when test substance is known or expected to be non-toxic.

Single group of 3 males and 3 females is exposed to the test article at a limit concentration of 5 mg/L (aerosol with respirable particle size) and 20 mg/L for vapour and gases. If a test article’s physical or chemical properties make it impossible to attain a limit concentration, the maximum attainable concentration should be tested. It is important to maintain the  respirable particle size ( MMAD) of 1-4µm. For aerosols it is possible at the concentration of 2 mg/litre with most of the test substances. Therefore, aerosol testing for concentration more than 2mg/litre should only be done if respirable particle size can be achieved. If no lethality is demonstrated, no further detailed testing for acute inhalation toxicity is needed. If compound-related mortality or moribundity is produced, further study may need to be considered and the results of limit test may be used as sighting study for further testing at other concentrations. 


iii)                Main Test (Number of animals, dose levels etc.)


5 male and 5 female animals (or 5 animals of the susceptible sex, if known) are exposed per concentration level. A minimum of three concentration levels should be used. Alternatively, assign 5 animals, all of the same sex, to each group (at least 3 dosage levels). In addition, administer the substance to at least 1 group of the other sex (preferably at middle or high concentration), to confirm that the other sex does not have a notably high susceptibility.


10.                       Observation period


Animals should be observed for at least 14 days. However, depending on toxic reactions and time of onset and length of recovery period, the period may be extended when considered necessary.



11.                       Observation  and Examination



i)                    Animals should be observed frequently during the exposure period and 

           then at least twice after the exposure on that day. Thereafter, at least

          once daily during the observation period.

ii)                  Observations should include condition of skin and fur, eyes and mucus membrane, respiratory, circulatory, autonomic and central nervous system, somato-motor activity and behaviour pattern. Specific observations should be made for tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.



iii)                Individual animal weights should be recorded once during the acclimatization period, just prior to exposure (day 0), and at least on days 1, 3 and 7 (and weekly thereafter), and at the time of death. Surviving animals are weighed and humanely killed at the end of the post exposure


iv)                All gross pathological changes should be recorded for each animal with particular attention to any changes in the respiratory tract. Histopathology may be considered in the organs showing significant adverse effects.


12.              Monitoring of Exposure Conditions


·         Airflow should be hourly measured during each exposure.

·         Monitoring of test atmosphere concentration: O2 and CO2 concentration in test atmosphere.

·         Monitoring of nominal concentration and actual concentration of test article.

·         Particle size distribution of aerosols should be recorded at least twice during four hours exposure.

·         Relative humidity at least thrice during four hours duration.


13.              Result assessment


                        LC50 to be determined with any recognized statistical method.


14.              Toxic endpoint:


Mortality or moribund animals. Animals humanely killed due to compound related distress and pain should be recorded as compound related deaths.


15.              N.B.


i)                    The protocol is not suitable for testing of specialized materials such as poorly soluble isometric or fibrous materials or manufactured nanomaterials.

ii)                  Testing of concentrations that are expected to cause severe pain or distress in case of test substances having corrosive or irritant properties, should be avoided.

iii)                The granular formulation which is non-friable and specifically made to be un-inhalable need not be tested for acute inhalation toxicity. However, attrition test should be carried out with such formulation to show that respirable particles are not produced when granular material is handled.  If an attrition test produces respirable  particles then the inhalation toxicity test should be undertaken.