OECD/OCDE 403 Acute Inhalation Toxicity

OECD/OCDE 403 Adopted:7 September 2009 @OECD, (2009)

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OECD GUIDELINE FOR THE TESTING OF CHEMICALS

Acute Inhalation Toxicity
INTRODUCTION
1. OECD Guidelines are periodically reviewed in the light of scientific progress, changing
regulatory needs, and animal welfare considerations. The original acute inhalation Test Guideline 403
was adopted in 1981. This revised Test Guideline 403 (TG 403) (1) has been designed to be more
flexible, to reduce animal usage, and to fulfil regulatory needs. The revised TG 403 features two study
types: a Traditional LC50 protocol and a Concentration x Time (C x t) protocol. Primary features of
this Test Guideline are the ability to provide a concentration-response relationship ranging from nonlethal
to lethal outcomes in order to derive a median lethal concentration (LC50), non-lethal threshold
concentration (e.g. LC01), and slope, and to identify possible sex susceptibility. The C x t protocol
should be used when there is a specific regulatory or scientific need that calls for the testing of animals
over multiple time durations, such as for purposes of emergency response planning (e.g. deriving
Acute Exposure Guideline Levels (AEGL), Emergency Response Planning Guidelines (ERPG), or
Acute Exposure Threshold Levels (AETL) values), or for land-use planning.
2. Guidance on the conduct and interpretation of TG 403 studies can be found in the Guidance
Document on Acute Inhalation Toxicity Testing (GD 39) (2).
3. Definitions used in the context of this Guideline are provided in GD 39 (2).
4. This Test Guideline enables test article characterization and quantitative risk assessment, and
allows test articles to be ranked and classified according to the United Nations (UN) Globally
Harmonized System of Classification and Labelling of Chemicals (GHS) (3). GD 39 (2) provides
guidance in the selection of the appropriate Test Guideline for acute testing. When information on
classification and labelling only is required, Test Guideline 436 (4) is generally recommended [see GD
39 (2)]. Test Guideline 403 is not specifically intended for the testing of specialized materials, such as
poorly soluble isometric or fibrous materials or manufactured nanomaterials.
INITIAL CONSIDERATIONS
5. Before considering testing in accordance with this Test Guideline all available information
on the test article, including existing studies (e.g. TG 436)(4) whose data would support not doing
additional testing should be considered by the testing laboratory in order to minimize animal usage.
Information that may assist in the selection of the most appropriate species, strain, sex, mode of
exposure and appropriate test concentrations include the identity, chemical structure, and physicochemical
properties of the test article; results of any in vitro or in vivo toxicity tests; anticipated uses
and potential for human exposure; available (Q)SAR data and toxicological data on structurally related
substances [see GD 39 (2)].
6. Testing corrosive and/or irritating test articles at concentrations that are expected to cause
severe pain and/or distress should be avoided to the extent possible. The corrosive/irritating potential
should be evaluated by expert judgment using such evidence as human and animal experience (e.g.
403 OECD/OCDE
© OCDE, (2009) 2
from repeat dose studies performed at non-corrosive/irritant concentrations), existing in vitro data (e.g.
from TGs 430 (5), 431 (6) or 435 (7)), pH values, information from similar substances or any other
pertinent data, for the purpose of investigating whether further testing can be waived. For specific
regulatory needs (e.g. for emergency planning purposes), the TG 403 may be used for exposing
animals to these materials because it provides the study director or principal investigator with control
over the selection of target concentrations. However, the targeted concentrations should not induce
severe irritation/corrosive effects, yet sufficient to extend the concentration-response curve to levels
that reach the regulatory and scientific objective of the test. These concentrations should be selected
on a case-by-case basis and justification for concentration selection should be provided [see GD 39
(2)].
PRINCIPLE OF THE TEST
7. This revised TG 403 has been designed to obtain sufficient information on the acute toxicity
of a test article to enable its classification and to provide lethality data (e.g. LC50, LC01 and slope) for
one or both sexes as needed for quantitative risk assessments. This Guideline offers two test methods.
The first method is a Traditional protocol in which groups of animals are exposed to a limit
concentration (limit test) or a series of concentrations in a stepwise procedure for a predetermined
duration of usually 4 hours. Other durations of exposure may apply to serve specific regulatory
purposes. The second method is a (C x t) protocol in which groups of animals are exposed to one
(limit concentration) or a series of multiple concentrations over multiple durations.
8. Moribund animals or animals obviously in pain or showing signs of severe and enduring
distress should be humanely killed and are considered in the interpretation of the test result in the same
way as animals that died on test. Criteria for making the decision to kill moribund or severely
suffering animals, and guidance on the recognition of predictable or impending death, are the subject
of an OECD Guidance Document No. 19 on Humane Endpoints (8).
DESCRIPTION OF THE METHOD
Selection of animal species
9. Healthy young adult animals of commonly used laboratory strains should be used. The
preferred species is the rat and justification should be provided if other species are used.
Preparation of animals
10. Females should be nulliparous and nonpregnant. On the exposure day, animals should be
young adults 8 to 12 weeks of age, and body weights should be within ±20% of the mean weight for
each sex of any previously exposed animals of the same age. The animals are randomly selected and
marked for individual identification. The animals are kept in their cages for at least 5 days prior to the
start of the test to allow for acclimatization to laboratory conditions. Animals should also be
acclimatised to the test apparatus for a short period prior to testing, as this will lessen the stress caused
by introduction to the new environment.
Animal husbandry
11. The temperature of the experimental animal maintenance room should be 22±3°C. The
relative humidity should ideally be maintained in the range of 30 to 70%, though this may not be
possible when using water as a vehicle. Before and after exposures, animals generally should be caged
in groups by sex and concentration, but the number of animals per cage should not interfere with clear
observation of each animal and should minimize losses due to cannibalism and fighting. When
animals are to be exposed nose-only, it may be necessary for them to be acclimated to the restraining
tubes. The restraining tubes should not impose undue physical, thermal, or immobilization stress on
the animals. Restraint may affect physiological endpoints such as body temperature (hyperthermia)
and/or respiratory minute volume. If generic data are available to show that no such changes occur to
any appreciable extent, then pre-adaptation to the restraining tubes is not necessary. Animals exposed
whole-body to an aerosol should be housed individually during exposure to prevent them from
filtering the test aerosol through the fur of their cage mates. Conventional and certified laboratory
diets may be used, except during exposure, accompanied with an unlimited supply of municipal
drinking water. Lighting should be artificial, the sequence being 12 hours light/12 hours dark.
Inhalation chambers
12. The nature of the test article and the objective of the test should be considered when
selecting an inhalation chamber. The preferred mode of exposure is nose-only (which term includes
head-only, nose-only or snout-only). Nose-only exposure is generally preferred for studies of liquid or
solid aerosols and for vapours that may condense to form aerosols. Special objectives of the study
may be better achieved by using a whole-body mode of exposure, but this should be justified in the
study report. To ensure atmosphere stability when using a whole-body chamber, the total volume of
the test animals should not exceed 5% of the chamber volume. Principles of the nose-only and whole
body exposure techniques and their particular advantages and disadvantages are described in GD 39
(2).
EXPOSURE CONDITIONS
Administration of concentrations
13. Nose-only exposures may be any duration up to 6 hours in rats. If mice are exposed noseonly,
exposures generally should not exceed 4 hours. Justification should be provided if longer
duration studies are needed [see GD 39 (2)]. Animals exposed to aerosols in whole-body chambers
should be housed individually to prevent ingestion of test article due to grooming of cage mates. Feed
should be withheld during the exposure period. Water may be provided throughout a whole-body
exposure.
14. Animals are exposed to the test article as a gas, vapour, aerosol, or a mixture thereof. The
physical state to be tested depends on the physico-chemical properties of the test article, the selected
concentration, and/or the physical form most likely present during the handling and use of the test
article. Hygroscopic and chemically reactive test articles should be tested under dry air conditions.
Care should be taken to avoid generating explosive concentrations.
Particle-size distribution
15. Particle sizing should be performed for all aerosols and for vapours that may condense to
form aerosols. To allow for exposure of all relevant regions of the respiratory tract, aerosols with
mass median aerodynamic diameters (MMAD) ranging from 1 to 4 μm with a geometric standard
deviation (σg) in the range of 1.5 to 3.0 are recommended (2) (9) (10). Although a reasonable effort
should be made to meet this standard, expert judgment should be provided if it cannot be achieved.
For example, metal fumes may be smaller than this standard, and charged particles, fibres, and
hygroscopic materials (which increase in size in the moist environment of the respiratory tract) may
exceed this standard.
Test article preparation in a vehicle
16. A vehicle may be used to generate an appropriate concentration and particle size of the test
article in the atmosphere. As a rule, water should be given preference. Particulate material may be
subjected to mechanical processes to achieve the required particle size distribution, however, care
should be taken to not decompose or alter the test article. In cases where mechanical processes are
believed to have altered test article composition (e.g. extreme temperatures from excessive milling due
to friction), the composition of the test article should be verified analytically. Adequate care should be
taken to not contaminate the test material. It is not necessary to test non-friable granular materials
which are purposefully formulated to be un-inhalable. An attrition test should be used to demonstrate
that respirable particles are not produced when the granular material is handled. If an attrition test
produces respirable articles, an inhalation toxicity test should be performed.
Control animals
17. A concurrent negative (air) control group is not necessary. When a vehicle other than water
is used to assist in generating the test atmosphere, a vehicle control group should only be used when
historical inhalation toxicity data are not available. If a toxicity study of a test article formulated in a
vehicle reveals no toxicity, it follows that the vehicle is non-toxic at the concentration tested; thus,
there is no need for a vehicle control.
MONITORING OF EXPOSURE CONDITIONS
Chamber airflow
18. The flow of air through the chamber should be carefully controlled, continuously monitored,
and recorded at least hourly during each exposure. The monitoring of test atmosphere concentration
(or stability) is an integral measurement of all dynamic parameters and provides an indirect means to
control all relevant dynamic atmosphere generation parameters. Special consideration should be given
to avoiding re-breathing in nose-only chambers in cases where airflow through the exposure system
are inadequate to provide dynamic flow of test article atmosphere. There are prescribed methodologies
that can be used to demonstrate that re-breathing does not occur under the selected operation
conditions (2) (11). Oxygen concentration should be at least 19% and carbon dioxide concentration
should not exceed 1%. If there is reason to believe that these standards cannot be met, oxygen and
carbon dioxide concentrations should be measured.
Chamber temperature and relative humidity
19. Chamber temperature should be maintained at 22±3°C. Relative humidity in the animals’
breathing zone, for both nose-only and whole-body exposures, should be monitored and recorded at
least three times for durations of up to 4 hrs, and hourly for shorter durations. The relative humidity
should ideally be maintained in the range of 30 to 70%, but this may either be unattainable (e.g. when
testing water based formulations) or not measurable due to test article interference with the test
method.

Test article: Nominal concentration
20. Whenever feasible, the nominal exposure chamber concentration should be calculated and
recorded. The nominal concentration is the mass of generated test article divided by the total volume
of air passed through the chamber system. The nominal concentration is not used to characterize the
animals’ exposure, but a comparison of the nominal concentration and the actual concentration gives
an indication of the generation efficiency of the test system, and thus may be used to discover
generation problems.
Test article: Actual concentration
21. The actual concentration is the test article concentration at the animals’ breathing zone in an
inhalation chamber. Actual concentrations can be obtained by specific methods (e.g. direct sampling,
adsorptive or chemical reactive methods, and subsequent analytical characterisation) or by nonspecific
methods such as gravimetric filter analysis. The use of gravimetric analysis is acceptable only
for single component powder aerosols or aerosols of low volatility liquids and should be supported by
appropriate pre-study test article-specific characterisations. Multi-component powder aerosol
concentration may also be determined by gravimetric analysis. However, this requires analytical data
which demonstrate that the composition of airborne material is similar to the starting material. If this
information is not available, a reanalysis of the test material (ideally in its airborne state) at regular
intervals during the course of the study may be necessary. For aerosolised agents that may evaporate
or sublimate, it should be shown that all phases were collected by the method chosen. The target,
nominal, and actual concentrations should be provided in the study report, but only actual
concentrations are used in statistical analyses to calculate lethal concentration values.
22. One lot of the test article should be used, if possible, and the test sample should be stored
under conditions that maintain its purity, homogeneity, and stability. Prior to the start of the study,
there should be a characterization of the test article, including its purity and, if technically feasible, the
identity, and quantities of identified contaminants and impurities. This can be demonstrated by, but is
not limited to, the following data: retention time and relative peak area, molecular weight from mass
spectroscopy or gas chromatography analyses, or other estimates. Although the test sample’s identity
is not the responsibility of the test laboratory, it may be prudent for the test laboratory to confirm the
sponsor’s characterization at least in a limited way (e.g. colour, physical nature, etc.).
23. The exposure atmosphere shall be held as constant as practicable and monitored
continuously and/or intermittently depending on the method of analysis. When intermittent sampling
is used, chamber atmosphere samples should be taken at least twice in a four hour study. If not
feasible due to limited air flow rates or low concentrations, one sample may be collected over the
entire exposure period. If marked sample-to-sample fluctuations occur, the next concentrations tested
should use four samples per exposure. Individual chamber concentration samples should not deviate
from the mean concentration by more than ±10% for gases and vapours or ±20% for liquid or solid
aerosols. Time to chamber equilibration (t95) should be calculated and recorded. The duration of an
exposure spans the time that the test article is generated and this takes into account the times required
to attain t95. Guidance for estimating t95 can be found in GD 39 (2).
24. For very complex mixtures consisting of gases/vapours, and aerosols (e.g. combustion
atmospheres and test articles propelled from purpose-driven end-use products/devices), each phase
may behave differently in an inhalation chamber so at least one indicator substance (analyte), normally
the principal active substance in the tested product formulation, of each phase (gas/vapour and aerosol)
should be selected. When the test article is a mixture (e.g. a formulation), the analytical concentration
should be reported for the total formulation and not just for the active ingredient or the component
(analyte). Additional information regarding actual concentrations can be found in GD 39 (2).
Test article: Particle size distribution
25. The particle size distribution of aerosols should be determined at least twice during each 4
hour exposure by using a cascade impactor or an alternative instrument such as an aerodynamic
particle sizer. If equivalence of the results obtained by a cascade impactor or an alternative instrument
can be shown, then the alternative instrument may be used throughout the study. A second device,
such as a gravimetric filter or an impinger/gas bubbler, should be used in parallel to the primary
instrument to confirm the collection efficiency of the primary instrument. The mass concentration
obtained by particle size analysis should be within reasonable limits of the mass concentration
obtained by filter analysis [see GD 39 (2)]. If equivalence can be demonstrated in the early phase of
the study, then further confirmatory measurements may be omitted. For animal welfare reasons,
measures should be taken to minimize inconclusive data which may lead to a need to repeat an
exposure. Particle sizing should be performed for vapours if there is any possibility that vapour
condensation may result in the formation of an aerosol, or if particles are detected in a vapour
atmosphere with potential for mixed phases (see paragraph 15).
PROCEDURE
26. Two study types are described below: the Traditional protocol, and the C x t protocol. Both
protocols may include a sighting study, a main study, and/or a limit test (Traditional protocol) or
testing at a limit concentration (C x t). If one sex is known to be more susceptible, the study director
may choose to perform these studies using only the susceptible sex. If rodent species other than rats
are exposed nose-only, maximum exposure durations may be adjusted to minimise species-specific
distress. Before commencing, all available data should be considered in order to minimize animal
usage. For example, data generated using TG 436 (4) may eliminate the need for a sighting study, and
may also demonstrate whether one sex is more susceptible [see GD 39 (2)].
TRADITIONAL PROTOCOL :
General considerations: Traditional protocol
27. In a Traditional study, groups of animals are exposed to a test article for a fixed period of
time (generally 4 hours) in either a nose-only or whole-body exposure chamber. Animals are exposed
to either a limit concentration (limit test), or to at least three concentrations in a stepwise procedure
(main study). A sighting study may precede a main study unless some information about the test
article already exists, such as a previously performed TG 436 study [see GD 39 (2)].
Sighting study: Traditional protocol
28. A sighting study is used to estimate test article potency, identify sex differences in
susceptibility, and assist in selecting exposure concentration levels for the main study or limit test.
When selecting concentration levels for the sighting study, all available information should be used
including available (Q)SAR data and data for similar chemicals. No more than three males and three
females should be exposed at each concentration (3 animals/sex may be needed to establish a sex
difference). A sighting study may consist of a single concentration, but more concentrations may be
tested if necessary. A sighting study should not test so many animals and concentrations that it
resembles a main study. A previously performed TG 436 study (4) may be used instead of a sighting
study [see GD 39 (2)].
Limit test: Traditional protocol
29. A limit test is used when the test article is known or expected to be virtually non-toxic, i.e.
eliciting a toxic response only above the regulatory limit concentration. In a limit test, a single group
of three males and three females is exposed to the test article at a limit concentration. Information
about the toxicity of the test material can be gained from knowledge about similar tested compounds
or similar tested mixtures or products, taking into consideration the identity and percentage of
components known to be of toxicological significance. In those situations where there is little or no
information about its toxicity, or the test material is expected to be toxic, the main test should be
performed.
30. The selection of limit concentrations usually depends on regulatory requirements. When the
GHS Classification System is used, the limit concentrations for gases, vapours, and aerosols are 20000
ppm, 20 mg/L, and 5 mg/L, respectively (or the maximum attainable concentration) (3). It can be
technically challenging to generate limit concentrations of some test articles, especially as vapours and
aerosols. When testing aerosols, the primary goal should be to achieve a respirable particle size
(MMAD of 1-4 μm). This is possible with most test articles at a concentration of 2 mg/L. Aerosol
testing at greater than 2 mg/L should only be attempted if a respirable particle size can be achieved
[see GD 39 (2)]. GHS discourages testing in excess of a limit concentration for animal welfare reasons
(3). The limit concentration should only be considered when there is a strong likelihood that results of
such a test would have direct relevance for protecting human health (3), and justification provided in
the study report. In the case of potentially explosive test articles, care should be taken to avoid
conditions favourable for an explosion. To avoid an unnecessary use of animals, a test run without
animals should be conducted prior to the limit test to ensure that the chamber conditions for a limit test
can be achieved.
31. If mortality or moribundity is observed at the limit concentration, the results of the limit test
can serve as a sighting study for further testing at other concentrations (see main study). If a test
article’s physical or chemical properties make it impossible to attain a limit concentration, the
maximum attainable concentration should be tested. If less than 50% lethality occurs at the maximum
attainable concentration, no further testing is necessary. If the limit concentration could not be
attained, the study report should provide an explanation and supportive data. If the maximum
attainable concentration of a vapour does not elicit toxicity, it may be necessary to generate the test
article as a liquid aerosol.
Main study: Traditional protocol
32. A main study is typically performed using five males and five females (or 5 animals of the
susceptible sex, if known) per concentration level, with at least three concentration levels. Sufficient
concentration levels should be used to obtain a robust statistical analysis. The time interval between
exposure groups is determined by the onset, duration, and severity of toxic signs. Exposure of animals
at the next concentration level should be delayed until there is reasonable confidence of survival for
previously tested animals. This allows the study director to adjust the target concentration for the next
exposure group. Due to the dependence on sophisticated technologies, this may not always be practical
in inhalation studies, so the exposure of animals at the next concentration level should be based on
previous experience and scientific judgement. GD 39 (2) should be consulted when testing mixtures.

CONCE NTRATION X TIME (CXT ) PROTOCOL
General considerations: C x t protocol
33. A step-wise C x t study may be considered as an alternative to a Traditional protocol when
assessing inhalation toxicity (12) (13) (14). This approach allows animals to be exposed to a test
article at several concentration levels and for multiple time durations. All testing is performed in a
nose-only chamber (whole-body chambers are not practical for this protocol). A flow diagram in
Annex 1 illustrates this protocol. A simulation analysis has shown that the Traditional protocol and the
C x t protocol are both capable of yielding robust LC50 values, but the C x t protocol is generally better
at yielding robust LC01 and LC10 values (15).
34. A simulation analysis has demonstrated that using two animals per C x t interval (one per sex
using both sexes, or two of the more susceptible sex) may generally be adequate when testing 4
concentrations and 5 exposure durations in a main study. Under some circumstances, the study
director may elect to use two rats per sex per C x t interval (15). Using 2 animals per sex per
concentration and time point may reduce bias and variability of the estimates, increase the estimation
success rate, and improve confidence interval coverage. However, in case of an insufficient close fit to
the data for estimation (when using one animal per sex or two animals of the more susceptible sex) a
5th exposure concentration may also suffice. Further guidance on the number of animals and
concentrations to be used in a C x t study can be found in GD 39 (2).
Sighting study: C x t protocol
35. A sighting study is used to estimate test article potency and to assist in selecting exposure
concentration levels for the main study. A sighting study using up to three animals/sex/concentration
[for details see Appendix III of GD 39 (2)] may be needed to choose an appropriate starting
concentration for the main study and to minimize the number of animals used. It may be necessary to
use three animals per sex to establish a sex difference. These animals should be exposed for a single
duration, generally 240 min. The feasibility of generating adequate test atmospheres should be
assessed during technical pre-tests without animals. It is generally not necessary to perform a sighting
study if mortality data are available from a TG 436 study (4). When selecting the initial target
concentration in a TG 403 study, the study director should consider the mortality patterns observed in
any available TG 436 studies (4) for both sexes and for all concentrations tested [see GD 39 (2)].
Initial Concentration: C x t protocol
36. The initial concentration (Exposure Session I) (Annex 1) will either be a limit concentration
or a concentration selected by the study director based on the sighting study. Groups of 1 animal/sex
are exposed to this concentration for multiple durations (e.g. 15, 30, 60, 120, or 240 minutes),
resulting in a total number of 10 animals (called Exposure Session I) (Annex 1).
37. The selection of limit concentrations usually depends on regulatory requirements. When the
GHS Classification System is used, the limit concentrations for gases, vapours, and aerosols are 20000
ppm, 20 mg/L and 5 mg/L, respectively (or the maximum attainable concentration) (3). It can be
technically challenging to generate limit concentrations of some test articles, especially as vapours and
aerosols. When testing aerosols, the goal should be to achieve a respirable particle size (i.e. an MMAD
of 1-4 μm) at a limit concentration of 2 mg/L. This is possible with most test articles. Aerosol testing
at greater than 2 mg/L should only be attempted if a respirable particle size can be achieved [see GD
39 (2)]. GHS discourages testing in excess of a limit concentration for animal welfare reasons (3).
Testing in excess of the limit concentration should only be considered when there is a strong
likelihood that results of such a test would have direct relevance for protecting human health (3),
justification should be provided in the study report. In the case of potentially explosive test articles,
care should be taken to avoid conditions favourable for an explosion. To avoid an unnecessary use of
animals, a test run without animals should be conducted prior to testing at the initial concentration to
ensure that the chamber conditions for this concentration can be achieved.
38. If mortality or moribundity is observed at the initial concentration, the results at this
concentration can serve as a starting point for further testing at other concentrations (see main study).
When a test article’s physical or chemical properties make it impossible to attain a limit concentration,
the maximum attainable concentration should be tested. If less than 50% lethality occurs at the
maximum attainable concentration, no further testing is necessary. If the limit concentration could not
be attained, the study report should provide an explanation and supportive data. If the maximum
attainable concentration of a vapour does not elicit toxicity, it may be necessary to generate the test
article as a liquid aerosol.
Main study: C x t protocol
39. The initial concentration (Exposure Session I) (Annex 1) tested in the main study will either
be a limit concentration or a concentration selected by the study director based on the sighting study. If
mortality has been observed during or following Exposure Session I, the minimum exposure (C x t)
which results in mortality will be taken as a guide to establish the concentration and periods of
exposure for Exposure Session II. Each subsequent exposure session will depend on the previous
session (see Annex 1).
40. For many test articles the results obtained at the initial concentration, together with three
additional exposure sessions with a smaller time grid (i.e. the geometric spacing of exposure periods
as indicated by the factor between successive periods, generally √2), will be sufficient to establish the
C x t mortality relationship (16), but there may be some benefit to using a 5th exposure concentration
[see Annex 1 and GD 39 (2)]. For mathematical treatment of results for the C x t protocol, see Annex
1.
OBSERVATIONS
41. The animals should be clinically observed frequently during the exposure period. Following
exposure, clinical observations should be made at least twice on the day of exposure, or more
frequently when indicated by the response of the animals to treatment, and at least once daily
thereafter for a total of 14 days. The length of the observation period is not fixed, but should be
determined by the nature and time of onset of clinical signs and length of the recovery period. The
times at which signs of toxicity appear and disappear are important, especially if there is a tendency
for signs of toxicity to be delayed. All observations are systematically recorded with individual
records being maintained for each animal. Animals found in a moribund condition and animals
showing severe pain and/or enduring signs of severe distress should be humanely killed for animal
welfare reasons. Care should be taken when conducting examinations for clinical signs of toxicity that
initial poor appearance and transient respiratory changes, resulting from the exposure procedure, are
not mistaken for test article-related toxicity that would require premature killing of the animals. The
principles and criteria summarised in the Guidance Document on Humane Endpoints (GD 19) should
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be taken into consideration (8). When animals are killed for humane reasons or found dead, the time of
death should be recorded as precisely as possible.
42. Cage-side observations should include changes in the skin and fur, eyes and mucous
membranes, and also respiratory, circulatory, autonomic and central nervous systems, and
somatomotor activity and behaviour patterns. When possible, any differentiation between local and
systemic effects should be noted. Attention should be directed to observations of tremors, convulsions,
salivation, diarrhoea, lethargy, sleep and coma. The measurement of rectal temperature may provide
supportive evidence of reflex bradypnea or hypo/hyperthermia related to treatment or confinement.
Body weights
43 Individual animal weights should be recorded once during the acclimatization period, on the
day of exposure prior to exposure (day 0), and at least on days 1, 3 and 7 (and weekly thereafter), and
at the time of death or euthanasia if exceeding day 1. Body weight is recognized as a critical indicator
of toxicity so animals exhibiting a sustained decrement of >20%, compared to pre-study values, should
be closely monitored. Surviving animals are weighed and humanely killed at the end of the postexposure
period.
Pathology
44. All test animals, including those which die during the test or are euthanized and removed
from the study for animal welfare reasons, should be subjected to gross necropsy. If necropsy cannot
be performed immediately after a dead animal is discovered, the animal should be refrigerated (not
frozen) at temperatures low enough to minimize autolysis. Necropsies should be performed as soon as
possible, normally within a day or two. All gross pathological changes should be recorded for each
animal with particular attention to any changes in the respiratory tract.
45. Additional examinations included a priori by design may be considered to extend the
interpretive value of the study, such as measuring lung weight of surviving rats, and/or providing
evidence of irritation by microscopic examination of the respiratory tract. Examined organs may also
include those showing evidence of gross pathology in animals surviving 24 or more hours, and organs
known or expected to be affected. Microscopic examination of the entire respiratory tract may provide
useful information for test articles that are reactive with water, such as acids and hygroscopic test
articles.
DATA AND REPORTING
Data
46. Individual animal data on body weights and necropsy findings should be provided. Clinical
observation data should be summarized in tabular form, showing for each test group the number of
animals used, the number of animals displaying specific signs of toxicity, the number of animals found
dead during the test or killed for humane reasons, time of death of individual animals, a description
and time course of toxic effects and reversibility, and necropsy findings.
Test report
47. The test report should include the following information, as appropriate:

Test animals and husbandry
- Description of caging conditions, including: number (or change in number) of animals per
cage, bedding material, ambient temperature and relative humidity, photoperiod, and
identification of diet;

- Species/strain used and justification for using a species other than the rat;

- Number, age and sex of animals;

- Method of randomization;

- Details of food and water quality (including diet type/source, water source);

- Description of any pre-test conditioning including diet, quarantine, and treatment for
disease;

Test article
- Physical nature, purity and, where relevant, physico-chemical properties (including
isomerization);

- Identification data and Chemical Abstract Services (CAS) Registry Number, if known;
Vehicle

- Justification for use of vehicle and justification for choice of vehicle (if other than water);

- Historical or concurrent data demonstrating that the vehicle does not interfere with the
outcome of the study;

Inhalation chamber
- Description of the inhalation chamber including dimensions and volume;

- Source and description of equipment used for the exposure of animals as well as generation of atmosphere;

- Equipment for measuring temperature, humidity, particle-size, and actual concentration;

- Source of air and treatment of air supplied/extracted and system used for conditioning;

- Methods used for calibration of equipment to ensure a homogeneous test atmosphere;

- Pressure difference (positive or negative);

- Exposure ports per chamber (nose-only); location of animals in the system (whole-body);

- Temporal homogeneity/stability of test atmosphere;

- Location of temperature and humidity sensors and sampling of test atmosphere in the
chamber;

- Air flow rates, air flow rate/exposure port (nose-only), or animal load/chamber (wholebody);

- Information about the equipment used to measure oxygen and carbon dioxide, if
applicable;

- Time required to reach inhalation chamber equilibrium (t95)

- Number of volume changes per hour;

- Metering devices (if applicable);

Exposure data

- Rationale for target concentration selection in the main study;

- Nominal concentrations (total mass of test article generated into the inhalation chamber
divided by the volume of air passed through the chamber);

- Actual test article concentrations collected from the animals’ breathing zone; for test
mixtures that produce heterogeneous physical forms (gases, vapours, aerosols), each may
be analysed separately;

- All air concentrations should be reported in units of mass (e.g. mg/L, mg/m3, etc.); units of
volume (e.g. ppm, ppb, etc.) may also be reported parenthetically;

- Particle size distribution, mass median aerodynamic diameter (MMAD), and geometric
standard deviation (σg), including their methods of calculation. Individual particle size
analyses should be reported;
Test conditions

- Details of test article preparation, including details of any procedures used to reduce the
particle size of solid materials or to prepare solutions of the test article. In cases where
mechanical processes may have altered test article composition, include the results of
analyses to verify the composition of the test article;

- A description (preferably including a diagram) of the equipment used to generate the test
atmosphere and to expose the animals to the test atmosphere;

- Details of the chemical analytical method used and method validation (including
efficiency of recovery of test article from the sampling medium);

- The rationale for the selection of test concentrations;
 
Results- Tabulation of chamber temperature, humidity, and airflow;

- Tabulation of chamber nominal and actual concentration data;

- Tabulation of particle size data including analytical sample collection data, particle size
distribution and calculations of the MMAD and σg;

- Tabulation of response data and concentration level for each animal (i.e. animals showing
signs of toxicity including mortality, nature, severity, time of onset and duration of
effects);

- Individual body weights of animals collected on study; date and time of death if prior to
scheduled euthanasia, time course of onset of signs of toxicity and whether these were
reversible for each animal;

- Necropsy findings and histopathological findings for each animal, if available;

- Lethality estimates (e.g. LC50, LD01) including 95% confidence limits, and slope (if
provided by the evaluation method);

- Statistical relation, including estimate for the exponent n (C x t protocol). The name of the
statistical software used should be provided;

Discussion and interpretation of results
- Particular emphasis should be made to the description of methods used to meet this Test
Guideline’s criteria, e.g. the limit concentration or the particle size;

- The respirability of particles in light of the overall findings should be addressed, especially
if the particle-size criteria could not be met;

- An explanation should be provided if there was a need to humanely sacrifice animals in
pain or showing signs of severe and enduring distress, based on the criteria in the OECD
Guidance Document on Humane Endpoints (8);

- If testing with TG 436 (4) was discontinued in favour of TG 403, justifications should be
provided;

- The consistency of methods used to determine nominal and actual concentrations, and the
relation of actual concentration to nominal concentration should be included in the overall
assessment of the study;

- The likely cause of death and predominant mode of action (systemic versus local) should
be addressed.

LITERATURE
1. OECD (2009), Acute Inhalation Toxicity Testing. OECD Guideline for Testing of Chemicals
No. 403, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

2. OECD (2009), Guidance Document on Acute Inhalation Toxicity Testing. Environmental
Health and Safety Monograph Series on Testing and Assessment No. 39, OECD, Paris.
Available at: [http://www.oecd.org/env/testguidelines]

3. UN (2007), United Nations Globally Harmonized System of Classification and Labelling of
Chemicals (GHS), ST/SG/AC.10/30, UN New York and Geneva. Available at:
[http://www.unece.org/trans/danger/publi/ghs/ghs_welcome_e.html]

4. OECD (2009), Acute Inhalation Toxicity – Acute Toxic Class Method. OECD Guideline for
Testing of Chemicals No. 436, OECD, Paris. Available at:
[http://www.oecd.org/env/testguidelines]

5. OECD (2004), In Vitro Skin Corrosion – Transcutaneous Electrical Resistance Test (TER).
OECD Guideline for Testing of Chemicals No. 430, OECD, Paris. Available at:
[http://www.oecd.org/env/testguidelines]

6. OECD (2004), In Vitro Skin Corrosion – Human Skin Model Test. OECD Guideline for Testing
of Chemicals No. 431, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

7. OECD (2005), In Vitro Membrane Barrier Test Method For Skin Corrosion. OECD Guideline
for Testing of Chemicals No. 435, OECD, Paris. Available at:
[http://www.oecd.org/env/testguidelines]

8. OECD (2000), Guidance Document on the Recognition, Assessment and Use of Clinical Signs
as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental
Health and Safety Monograph Series on Testing and Assessment No. 19, OECD, Paris.
Available at: [http://www.oecd.org/env/testguidelines]

9. SOT (1992), Technical Committee of the Inhalation Specialty Section, Society of Toxicology
(SOT). Recommendations for the Conduct of Acute Inhalation Limit Tests. Fund. Appl. Toxicol.
18, 321-327.

10. Phalen R.F. (2009), Inhalation Studies: Foundations and Techniques. (2nd Edition) Informa
Healthcare, New York.

11. Pauluhn, J. and Thiel, A. (2007), A Simple Approach to Validation of Directed-Flow Nose-
Only Inhalation Chambers. J. Appl. Toxicol. 27, 160-167.

12. Zwart, J.H.E., Arts, J.M., ten Berge, W.F. and Appelman, L.M. (1992), Alternative Acute
Inhalation Toxicity Testing by Determination of the Concentration-Time-Mortality
Relationship: Experimental Comparison with Standard LC50 Testing. Reg. Toxicol. Pharmacol.
15, 278-290.

13. Zwart, J.H.E., Arts, J.M., Klokman-Houweling, E.D. and Schoen, E.D. (1990), Determination
of Concentration-Time-Mortality Relationships to Replace LC50 Values. Inhal. Toxicol. 2, 105-
117.

14. Ten Berge, W.F. and Zwart, A. (1989), More Efficient Use of Animals in Acute Inhalation
Toxicity Testing. J. Haz. Mat. 21, 65-71.

15. OECD (2009), Performance Assessment: Comparison of 403 and C x t Protocols via Simulation
and for Selected Real Data Sets. Environmental Health and Safety Monograph Series on Testing
and Assessment No. 104, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

16. Finney D.J. (1977), Probit Analysis, 3rd ed. Cambridge University Press, London/New York.

ANNEX 1
C x t PR OTOCOL

1. A step-wise Concentration x Time (C x t) study may be considered as an alternative to the
Traditional protocol for assessing inhalation toxicity (12) (13) (14). It should be performed
preferentially when there is a specific regulatory or scientific need that calls for the testing of animals
over multiple time durations such as for emergency response planning or land use planning. This
approach usually begins with testing at a limit concentration (Exposure Session I) in which animals
are exposed to a test article for five time durations (e.g. 15, 30, 60, 120 and 240 min) so that multiple
durations of time will be obtained within one exposure session (see Figure 1). When the GHS
Classification System is used, the limit concentrations for gases, vapours, and aerosols are 20000 ppm,
20 mg/L, and 5 mg/L, respectively. These levels may only be exceeded if there is a regulatory or
scientific need for testing at these levels (see paragraph 37 in the TG 403 main text).

2. In situations where there is little or no information about the toxicity of a test article, a
sighting study should be performed in which groups of no more than 3 animals per sex are exposed to
target concentrations selected by the study director, generally for 240 min.

3. If a limit concentration is tested during Exposure Session I and less than 50% mortality is
observed, no additional testing is needed. If there is a regulatory or scientific need to establish the
concentration/time/response relationship at higher levels than the indicated limit concentration, the
next exposure should be carried out at a higher level such as at two times the limit concentration (i.e.
2L in figure 1).

4. If toxicity is observed at the limit concentration, additional testing (main study) is necessary.
These additional exposures are carried out either at lower concentrations (in Figure 1: Exposure
Sessions II, III or IV) or at higher concentrations using shorter durations (in Figure 1: Exposure
Session IV) using durations that are adapted and not as widely spaced.

5. The test (initial concentration and additional concentrations) is carried out using 1
animal/sex per concentration/time point or with 2 animals of the more susceptible sex per
concentration/time point. Under some circumstances, the study director may elect to utilize 2 rats per
sex per concentration/time point (or 4 animals of the susceptible sex per concentration/time point)
(15). Using 2 animals per sex per concentration/time point generally reduces bias and variability of the
estimates, increases the estimation success rate, and improves confidence interval coverage relative to
the protocol as described here. Further details are provided in GD 39 (2).

6. Ideally, each exposure session is carried out on one day. This gives the opportunity to delay
the next exposure until there is reasonable confidence of survival, and it allows the study director to
adjust the target concentration and durations for the next exposure session. It is advised to start each
exposure session with the group that will be exposed the longest, e.g. the 240-min exposure group,
followed by the 120 minute exposure group, and so on. If, for example, animals in the 240 minute
group are dying after 90 minutes or showing severe signs of toxicity (e.g. extreme changes in
breathing pattern such as laboured breathing), it would not make sense to expose a group for 120
minutes because mortality would likely be 100%. Thus the study director should select shorter
exposure durations for that concentration (e.g. 90, 65, 45, 33 and 25 minutes).

7. The chamber concentration should be measured frequently to determine the time-weightedaverage
concentration for each exposure duration. Whenever possible, the time of death for each
animal (rather than the exposure duration) should be used in the statistical analysis.

8. The results of the first four exposure sessions should be examined to identify a data gap in
the concentration-time curve (see Figure 1). In case of an insufficient fit, an additional exposure (5th
concentration) may be performed. Concentration and exposure durations for the 5th exposure should be
chosen to cover this gap.

9. All exposure sessions (including the first Exposure Session) will be used to calculate the
concentration-time-response relationship using Statistical Analysis (16). If possible, for each C x t
interval, the time-weighted average concentration and the duration of exposure until death (if death
occurs during the exposure) should be used.
Figure 1: Hypothetical illustration of a concentration-time-mortality relationship in rats.
Open symbols = survivors; closed symbols = dead animals
Triangles = females; circles = males
Solid line = LC50 values (range 7.5-240 min) for males with n=1
Dashed line = LC50 values (range 7.5-240 min) for females with n=1
Dotted lines = hypothetical LC50 values line for males and females if n had been equal
to 2 (12).

10. Below is an example of the stepwise procedure:
Exposure Session I –Testing at the limit concentration (see Figure 1)
• 1 animal/sex per concentration/time point; 10 animals in total a
• Target concentrationb = limit concentration.
• Expose five groups of animals at this target concentration for durations of
15, 30, 60, 120 and 240 minutes, respectively.
↓
Exposure Session IIc – Main Study
• 1 animal/sex per concentration/time point; 10 animals in total.
• Expose five groups of animals at a lower concentration d (1/2L) with
slightly longer exposure durations (factor √2 spaced; see Figure 1).
↓
Exposure Session III – Main Study
• 1 animal/sex per concentration/time point; 10 animals total.
• Expose five groups of animals at a lower concentration d (1/4L) with
slightly longer exposure durations (factor √2 spaced; see Figure 1).
↓
Exposure Session IV′ – Main Study
• 1 animal/sex per concentration/time point; 10 animals total.
• Expose five groups of animals at a lower concentration d (1/8L) with
slightly longer exposure durations (factor √2 spaced; see Figure 1).
↓ or
Exposure Session IV – Main Study• 1 animal/sex per concentration/time point; 10 animals total.
• Expose five groups of animals at a higher concentration e (2L) with
slightly shorter exposure durations (factor √2 spaced; see Figure 1).

a If no sex susceptibility information is available, rats of both sexes will be used,
i.e. 1 animal/sex per concentration. Based on existing information, or if it
becomes apparent during this exposure session that one sex is more susceptible,
10 animals of the susceptible sex will be used (2 animals per concentration/time
point) at each concentration level during subsequent testing.

b When the GHS Classification System is used, the limit concentrations for gases,
vapours, and aerosols are 20000 ppm, 20 mg/L, and 5 mg/L, respectively. In case
of expected toxicity or based on the results of the sighting study, lower starting
concentrations should be chosen. In case of regulatory or scientific needs, higher
concentrations may be used.

c Ideally, exposure of animals at the next concentration level should be delayed
until there is reasonable confidence of survival for previously treated animals.
This allows the study director to adjust the target concentration and durations for
the next exposure session.

d The minimum dose (concentration x time) which resulted in mortality during
testing at initial concentration (first exposure session) will be taken as a guide to
establish the next combination of concentration and exposure durations.
Typically, the concentration will be decreased two-fold (1/2L) and animals will be
exposed over a new time range with a finer grid using a geometric division of
exposure periods with a factor 1.4 (√2; see reference 12) around the time
according to the minimum lethal dose level (time x concentration) observed
during the first exposure. In this figure (figure 1), mortality in Exposure session I
was first observed at 15 min; the durations during session II are therefore centred
around 30 min, and are 15, 21 30, 42 and 60 min. After the first two exposures, it
is strongly advised to plot the data in a similar figure as indicated above, and to
check whether the relationship between concentration and time has an angle of 45
degrees (n=1) or if the concentration-time-response relationship is less steep (e.g.
n=2) or steeper (e.g. n =0.8). In the latter cases it is strongly advised to adapt the
next concentrations and durations accordingly.

e In certain cases it may be necessary to increase the concentration (2L) over a
new time range with a still finer grid using a geometric division of exposure
periods with a factor 1.4 ( √2) around the time according to the minimum lethal
concentration level observed during the first exposure. The minimum exposure
duration should preferably exceed 5 minutes; the maximum exposure duration
should not exceed 8 hours.

Mathematical treatment of results for the C x t protocol

11. A C x t procedure with 4 or 5 exposure concentrations and five durations will yield 20 or 25
data points, respectively. With these data points, the C x t relationship can be calculated using
statistical analysis (16):
Probit (P) = b0 + b1 ln C + b2 ln t Equation 1
where C = concentration; t = exposure duration, or
Response = f(Cnt) Equation 2
where n = b1 / b2.
Using equation 1, the LC50 value can be calculated for a given time period (e.g. 4 hour, 1 hour, 30
minutes, or any time period within the range of time periods tested) using P = 5 (50% response). Note
that Haber’s rule is only applicable when n = 1. The LC01 can be calculated using P = 2.67.OECD