Regulation 2014/260 - Commission Regulation 260/2014 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation 1907/2006 on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) - Main contents

This Test Method is equivalent to OECD Test Guideline (TG) 105 (1995). This Test Method is a revised version of the original TG 105 which was adopted in 1981. There is no difference of substance between the current version and that from 1981. Mainly the format has been changed. The revision was based on the EU Test Method “Water Solubility” (1).

The water solubility of a substance can be considerably affected by the presence of impurities. This Test Method addresses the determination of the solubility in water of essentially pure substances which are stable in water and not volatile. Before determining water solubility, it is useful to have some preliminary information on the test substance, like structural formula, vapour pressure, dissociation constant and hydrolysis as a function of pH.

Two methods, the column elution method and the flask method which cover respectively solubilities below and above 10–2 g/l are described in this Test Method. A simple preliminary test is also described. It allows the determination of approximately the appropriate amount of sample to be used in the final test, as well as the time necessary to achieve saturation.

The water solubility of a substance is the saturation mass concentration of the substance in water at a given temperature.

Water solubility is expressed in mass of solute per volume of solution. The SI unit is kg/m3 but g/l may also be used.

Reference chemicals do not need to be employed when investigating a test substance.

The test is preferably run at 20 ± 0,5 °C. The chosen temperature should be kept constant in all relevant parts of the equipment.

In a stepwise procedure, increasing volumes of water are added at room temperature to approximately 0,1 g of the sample (solid test substances must be pulverized) in a 10 ml glass-stoppered measuring cylinder. After each addition of an amount of water, the mixture is shaken for 10 minutes and is visually checked for any undissolved parts of the sample. If, after addition of 10 ml of water, the sample or parts of it remain undissolved, the experiment is continued in a 100 ml measuring cylinder. The approximate solubility is given in Table 1 below under that volume of water in which complete dissolution of the sample occurs. When the solubility is low, a long time may be required to dissolve a test substance and at least 24 hours should be allowed. If, after 24 hours, the test substance is still not dissolved, more time (up to 96 hours) should be allowed or further dilution should be attempted to ascertain whether the column elution method or flask method should be used.

Table 1

This method is based on the elution of a test substance with water from a micro-column which is charged with an inert support material, previously coated with an excess of the test substance (2). The water solubility is given by the mass concentration of the eluate when this has reached a plateau as a function of time.

The apparatus consists of a microcolumn (Figure 1), maintained at constant temperature. It is connected either to a recirculating pump (Figure 2) or to a levelling vessel (Figure 3). The microcolumn contains an inert support held in place by a small plug of glasswool which also serves to filter out particles. Possible materials which can be employed for the support are glass beads, diatomaceous earth, or other inert materials.

The microcolumn shown in Figure 1 is suitable for the set-up with recirculating pump. It has a head space providing for five bed volumes (discarded at the start of the experiment) and the volume of five samples (withdrawn for analysis during the experiment). Alternatively, the size can be reduced if water can be added to the system during the experiment to replace the initial five bed volumes removed with impurities. The column is connected with tubing made of an inert material to the recirculating pump, capable of delivering approximately 25 ml/h. The recirculating pump can be, for example, a peristaltic or membrane pump. Care must be taken that no contamination and/or adsorption occur with the tube material.

A schematic arrangement using a levelling vessel is shown in Figure 3. In this arrangement the microcolumn is fitted with a one way stopcock. The connection to the levelling vessel consists of a ground glass joint and tubing made of an inert material. The flow rate from the levelling vessel should be approximately 25 ml/h.

Figure 1

Dimensions in mm

Figure 2

Figure 3

Approximately 600 mg of support material is transferred to a 50 ml round-bottom flask. A suitable amount of test substance is dissolved in a volatile solvent of analytical reagent quality and an appropriate amount of this solution is added to the support material. The solvent is completely evaporated, e.g. using a rotary evaporator, as otherwise water saturation of the support will not be achieved during the elution step because of partitioning on the surface. The loaded support material is soaked for two hours in approximately 5 ml of water and the suspension is poured into the microcolumn. Alternatively, dry loaded support material may be poured into the water-filled microcolumn and two hours are allowed for equilibrating.

The loading of the support material may cause problems, leading to erroneous results, e.g. when the test substance is deposited as an oil. These problems should be examined and the details reported.

The flow through the column is started. It is recommended that a flow rate of approximately 25 ml/h, corresponding to 10 bed volumes per hour for the column described, be used. At least the first five bed volumes are discarded to remove water soluble impurities. Following this, the pump is allowed to run until equilibrium is established, as defined by five successive samples whose concentrations do not differ by more than ± 30 % in a random fashion. These samples should be separated from each other by time intervals corresponding to the passage of at least ten bed volumes. Depending on the analytical method used, it may be preferable to establish a concentration/time curve to show that equilibrium is reached.

Successive eluate fractions should be collected and analysed by the chosen method. Fractions from the middle eluate range, where the concentrations are constant within ± 30 % in at least five consecutive fractions, are used to determine the solubility.

Double distilled water is the preferred eluent. Deionized water with a resistivity above 10 megohms/cm and total organic carbon content below 0,01 % can also be used.

Under both procedures, a second run is performed at half the flow rate of the first. If the results of the two runs are in agreement, the test is satisfactory. If the measured solubility is higher with the lower flow rate, then the halving of the flow rate must continue until two successive runs give the same solubility.

Under both procedures, the fractions should be checked for the presence of colloidal matter by examination of the Tyndall effect. The presence of particles invalidates the test and the test should be repeated after improvement of the filtering action of the column.

The pH of each sample should be measured, preferably by using special indicator strips.

The test substance (solids must be pulverized) is dissolved in water at a temperature somewhat above the test temperature. When saturation is achieved, the mixture is cooled and kept at the test temperature. Alternatively, and if it is assured by appropriate sampling that the saturation equilibrium is reached, the measurement can be performed directly at the test temperature. Subsequently, the mass concentration of the test substance in the aqueous solution, which must not contain any undissolved particles, is determined by a suitable analytical method (3).

The following materials are needed:

The quantity of test substance necessary to saturate the desired volume of water is estimated from the preliminary test. About five times that quantity is weighed into each of three glass vessels fitted with glass stoppers (e.g. centrifuge tubes, flasks). A volume of water, chosen in function of the analytical method and solubility range, is added to each vessel. The vessels are tightly stoppered and then agitated at 30 °C. A shaking or stirring device capable of operating at constant temperature should be used, e.g. magnetic stirring in a thermostated water bath. After one day, one of the vessels is equilibrated for 24 hours at the test temperature with occasional shaking. The contents of the vessel are then centrifuged at the test temperature and the concentration of the test substance in the clear aqueous phase is determined by a suitable analytical method. The other two flasks are treated similarly after initial equilibration at 30 °C for two and three days respectively. If the concentrations measured in at least the two last vessels do not differ by more than 15 %, the test is satisfactory. If the results from vessels 1, 2 and 3 show a tendency of increasing values, the whole test should be repeated using longer equilibration times.

The test can also be performed without pre-incubation at 30 °C. In order to estimate the rate of establishment of the saturation equilibrium, samples are taken until the stirring time no longer influences the concentrations measured.

The pH of each sample should be measured, preferably by using special indicator strips.

A substance-specific method is preferred since small amounts of soluble impurities can cause large errors in the measured solubility. Examples of such methods are: gas or liquid chromatography, titration, photometry, voltametry.

For each run, the mean value and standard deviation from at least five consecutive samples taken from the saturation plateau should be calculated. The mean values obtained from two tests with different flows should not differ by more than 30 %.

The individual results from each of the three flasks, which should not differ by more than 15 %, are averaged.

The test report must include the following information:

The test report must include the following information:

Commission Directive 92/69/EEC of 31 July 1992 adapting to technical progress for the seventeenth time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances (OJ L 383, 29.12.1992, p. 113).

NF T 20-045 (AFNOR) (September 1985). Chemical products for industrial use — Determination of water solubility of solids and liquids with low solubility — Column elution method.

NF T 20-046 (AFNOR) (September 1985). Chemical products for industrial use — Determination of water solubility of solids and liquids with high solubility — Flask method’.

This Test Method is equivalent to OECD Test Guideline (TG) 123 (2006). 1-octanol/water partition coefficient (POW) values up to a log POW of 8,2 have been accurately determined by the slow-stirring method (1). Therefore it is a suitable experimental approach for the direct determination of POW of highly hydrophobic substances.

Other methods for the determination of the 1-octanol/water partition coefficient (POW) are the “shake-flask” method (2), and the determination of the POW from reversed phase HPLC-retention behaviour (3). The “shake-flask” method is prone to artifacts due to transfer of octanol micro-droplets into the aqueous phase. With increasing values of POW the presence of these droplets in the aqueous phase leads to an increasing overestimation of the concentration of the test substance in the water. Therefore, its use is limited to substances with log POW < 4. The second method relies on solid data of directly determined POW values to calibrate the relationship between HPLC-retention behaviour and measured values of POW. A draft OECD guideline was available for determining 1-octanol/water partition coefficients of ionisable substances (4) but shall no longer be used.

This Test Method has been developed in The Netherlands. The precision of the methods described here has been validated and optimized in a ring-test validation study in which 15 laboratories participated (5).

For inert organic substances highly significant relationships have been found between 1-octanol/water partition coefficients (POW) and their bioaccumulation in fish. Moreover, POW has been demonstrated to be correlated to fish toxicity as well as to sorption of chemicals to solids such as soils and sediments. An extensive overview of the relationships has been given in reference (6).

A wide variety of relationships between the 1-octanol/water partition coefficient and other substance properties of relevance to environmental toxicology and chemistry have been established. As a consequence, the 1-octanol/water partition coefficient has evolved as a key parameter in the assessment of the environmental risk of chemicals as well as in the prediction of fate of chemicals in the environment.

The slow-stirring experiment is thought to reduce the formation of micro-droplets from 1-octanol droplets in the water phase. As a consequence, overestimation of the aqueous concentration due to test substance molecules associated to such droplets does not occur. Therefore, the slow-stirring method is particularly suitable for the determination of POW for substances with expected log POW values of 5 and higher, for which the shake-flask method (2) is prone to yield erroneous results.

The partition coefficient of a substance between water and a lipophilic solvent (1-octanol) characterizes the equilibrium distribution of the chemical between the two phases. The partition coefficient between water and 1-octanol (POW) is defined as the ratio of the equilibrium concentrations of the test substance in 1-octanol saturated with water (CO) and water saturated with 1-octanol (CW).

As a ratio of concentrations it is dimensionless. Most frequently it is given as the logarithm to the base 10 (log POW). POW is temperature dependent and reported data should include the temperature of the measurement.

In order to determine the partitioning coefficient, water, 1-octanol, and the test substance are equilibrated with each other at constant temperature. Then the concentrations of the test substance in the two phases are determined.

The experimental difficulties associated with the formation of micro-droplets during the shake-flask experiment can be reduced in the slow-stirring experiment proposed here. In the slow-stirring experiment, water, 1-octanol and the test substance are equilibrated in a thermostated stirred reactor. Exchange between the phases is accelerated by stirring. The stirring introduces limited turbulence which enhances the exchange between 1-octanol and water without micro-droplets being formed (1).

Since the presence of substances other than the test substance might influence the activity coefficient of the test substance, the test substance should be tested as a pure substance. The highest purity commercially available should be employed for the 1-octanol/water partition experiment.

The present method applies to pure substances that do not dissociate or associate and that do not display significant interfacial activity. It can be applied to determine the 1-octanol/water partition ratio of such substances and of mixtures. When the method is used for mixtures, the 1-octanol/water partition ratios determined are conditional and depend on the chemical composition of the mixture tested and on the electrolyte composition employed as aqueous phase. Provided additional steps are taken, the method is also applicable to dissociating or associating compounds (paragraph 12).

Due to the multiple equilibria in water and 1-octanol involved in the 1-octanol/water partitioning of dissociating substances such as organic acids and phenols, organic bases, and organometallic substances, the 1-octanol/water partition ratio is a conditional constant strongly dependent on electrolyte composition (7)(8). Determination of the 1-octanol/water partition ratio therefore requires that pH and electrolyte composition be controlled in the experiment and reported. Expert judgement has to be employed in the evaluation of these partition ratios. Using the value of dissociation constant(s), suitable pH-values need to be selected, such that a partitioning ratio is determined for each ionization state. Non-complexing buffers must be used when testing organometallic compounds (8). Taking the existing knowledge on the aqueous chemistry (complexation constants, dissociation constants) into account, the experimental conditions should be chosen in such a manner that the speciation of the test substance in the aqueous phase can be estimated. The ionic strength should be identical in all experiments by employing a background electrolyte.

Difficulties in the test may arise in conducting the test for substances with low water solubility or high POW, due to the fact that the concentrations in the water become very low such that their accurate determination is difficult. This Test Method provides guidance on how to deal with this problem.

Chemical reagents should be of analytical grade or of higher purity. The use of non-labelled test substances with known chemical composition and preferably at least 99 % purity, or of radiolabelled test substances with known chemical composition and radiochemical purity, is recommended. In the case of short half-life tracers, decay corrections should be applied. In the case of radiolabelled test substances, a chemical specific analytical method should be employed to ensure that the measured radioactivity is directly related to the test substance.

An estimate of log POW may be obtained by using commercially available software for estimation of log POW, or by using the ratio of the solubilities in both solvents.

Before carrying out a slow-stirring experiment for determination of POW, the following information on the test substance should be available:

Standard laboratory equipment is required, in particular, the following:

Vessels should be made from inert material such that adsorption to vessel surfaces is negligible.

The POW determination should be carried out with the highest purity 1-octanol that is commercially available (at least + 99 %). Purification of 1-octanol by extraction with acid, base and water and subsequent drying is recommended. In addition, distillation can be used to purify 1-octanol. Purified 1-octanol is to be used to prepare standard solutions of the test substances. Water to be used in the POW determination should be glass or quartz distilled, or obtained from a purification system, or HPLC-grade water may be used. Filtration through a 0,22 μm filter is required for distilled water, and blanks should be included to check that no impurities are in the concentrated extracts that may interfere with the test substance. If a glass fibre filter is used, it should be cleaned by baking for at least three hours at 400 °C.

Both solvents are mutually saturated prior to the experiment by equilibrating them in a sufficiently large vessel. This is accomplished by slow-stirring the two-phase system for two days.

An appropriate concentration of test substance is selected and dissolved in 1-octanol (saturated with water). The 1-octanol/water partition coefficient needs to be determined in dilute solutions in 1-octanol and water. Therefore the concentration of the test substance should not exceed 70 % of its solubility with a maximum concentration of 0,1 M in either phase (1). The 1-octanol solutions used for the experiment must be devoid of suspended solid test substance.

The appropriate amount of test substance is dissolved in 1-octanol (saturated with water). If the estimate of log POW exceeds five, care has to be taken that the 1-octanol solutions used for the experiment are devoid of suspended solid test substance. To that end, the following procedure for chemicals with an estimated value of log POW > 5 is followed:

A validated analytical method should be used for the assay of test substance. The investigators have to provide evidence that the concentrations in the water saturated 1-octanol as well as in the 1-octanol saturated water phase during the experiment are above the method limit of quantification of the analytical procedures employed. Analytical recoveries of the test substance from the water phase and from the 1-octanol phase need to be established prior to the experiment in those cases for which extraction methods are necessary. The analytical signal needs to be corrected for blanks and care should be taken that no carry-over of analyte from one sample to another can occur.

Extraction of the water phase with an organic solvent and preconcentration of extract are likely to be required prior to analysis, due to rather low concentrations of hydrophobic test substances in the water phase. For the same reason it is necessary to reduce eventual blank concentrations. To that end, it is necessary to employ high purity solvents, preferably solvents for residue analysis. Moreover, working with carefully pre-cleaned (e.g. solvent washing or baking at elevated temperature) glassware can help to avoid cross-contamination.

An estimate of log POW may be obtained from an estimation program or by expert judgment. If the value is higher than six then blank corrections and analyte carry-over need to be monitored closely. Similarly, if the estimate of log POW exceeds six, the use of a surrogate standard for recovery correction is mandatory, so that high preconcentration factors can be reached. A number of software programs for the estimation of log POW are commercially available (1), e.g. Clog P (16), KOWWIN (17), ProLogP (18) and ACD log P (19). Descriptions of the estimation approaches can be found in references (20-22).

The limits of quantification (LOQ) for determination of the test substance in 1-octanol and water are established using accepted methods. As a rule of thumb, the method limit of quantification can be determined as the concentration in water or 1-octanol that produces a signal to noise ratio of ten. A suitable extraction and pre-concentration method should be selected and analytical recoveries should also be specified. A suitable pre-concentration factor is selected in order to obtain a signal of the required size upon analytical determination.

On the basis of the parameters of the analytical method and the expected concentrations, an approximate sample size required for an accurate determination of the compound concentration is determined. The use of water samples that are too small to obtain a sufficient analytical signal should be avoided. Also, the use of excessively large water samples should be avoided, since otherwise there might be too little water left for the minimum number of analyses required (n = 5). In Appendix 1, the minimum sample volume is indicated as a function of the vessel volume, the LOD of the test substance and the solubility of the test substance.

Quantification of the test substances occurs by comparison with calibration curves of the respective compound. The concentrations in the samples analysed must be bracketed by concentrations of standards.

For test substances with a log POW estimate higher than six a surrogate standard has to be spiked to the water sample prior to extraction in order to register losses occurring during extraction and pre-concentration of the water samples. For accurate recovery correction, the surrogates must have properties that are very close to, or identical with, those of the test substance. Preferably, (stable) isotopically-labelled analogues of the substances of interest (for example, perdeuterated or 13C-labelled) are used for this purpose. If the use of labelled stable isotopes, i.e. 13C or 2H, is not possible it should be demonstrated from reliable data in the LITERATURE that the physical-chemical properties of the surrogate are very close to those of the test substance. During liquid-liquid extraction of the water phase emulsions can form. They can be reduced by addition of salt and allowing the emulsion to settle overnight. Methods used for extracting and pre-concentrating the samples need to be reported.

Samples withdrawn from the 1-octanol phase may, if necessary, be diluted with a suitable solvent prior to analysis. Moreover, the use of surrogate standards for recovery correction is recommended for substances for which the recovery experiments demonstrated a high degree of variation in the recovery experiments (relative standard deviation > 10 %).

The details of the analytical method need to be reported. This includes the method of extraction, pre-concentration and dilution factors, instrument parameters, calibration routine, calibration range, analytical recovery of the test substance from water, addition of surrogate standards for recovery correction, blank values, detection limits and limits of quantification.

When choosing the water and 1-octanol volumes, the LOQ in 1-octanol and water, the pre-concentration factors applied to the water samples, the volumes sampled in 1-octanol and water, and the expected concentrations should be considered. For experimental reasons, the volume of 1-octanol in the slow-stirring system should be chosen such that the 1-octanol layer is sufficiently thick (> 0,5 cm) in order to allow for sampling of the 1-octanol phase without disturbing it.

Typical phase ratios used for the determinations of compounds with log POW of 4,5 and higher are 20 to 50 ml of 1-octanol and 950 to 980 ml of water in a one litre vessel.

During the test the reaction vessel is thermostated to reduce temperature variation to below 1 °C. The assay should be performed at 25 °C.

The experimental system should be protected from daylight by either performing the experiment in a dark room or by covering the reaction vessel with aluminium foil.

The experiment should be performed in a dust-free (as far as possible) environment.

The 1-octanol-water system is stirred until equilibrium is attained. In a pilot experiment the length of the equilibration period is assessed by performing a slow-stirring experiment and sampling water and 1-octanol periodically. The sampling time points should be interspersed by a minimum period of five hours.

Each POW determination has to be performed employing at least three independent slow-stirring experiments.

It is assumed that the equilibrium is achieved when a regression of the 1-octanol/water concentration ratio against time over a time span of four time points yields a slope that is not significantly different from zero at a p-level of 0,05. The minimum equilibration time is one day before sampling can be started. As a rule of thumb, sampling of substances with a log POW estimate of less than five can take place during days two and three. The equilibration might have to be extended for more hydrophobic compounds. For a compound with log POW of 8,23 (decachlorobiphenyl) 144 hours were sufficient for equilibration. Equilibrium is assessed by means of repeated sampling of a single vessel.

At the start of the experiment the reaction vessel is filled with 1-octanol-saturated water. Sufficient time should be allowed to reach the thermostated temperature.

The desired amount of test substance (dissolved in the required volume of 1-octanol saturated with water) is carefully added to the reaction vessel. This is a crucial step in the experiment, since turbulent mixing of the two phases has to be avoided. To that end, the 1-octanol phase can be pipetted slowly against the wall of the experimental vessel, close to the water surface. It will subsequently flow along the glass wall and form a film above the water phase. The decantation of 1-octanol directly into the flask should always be avoided; drops of 1-octanol should not be allowed to fall directly into the water.

After starting the stirring, the stirring rate should be increased slowly. If the stirring motors cannot be appropriately adjusted the use of a transformer should be considered. The stirring rate should be adjusted so that a vortex at the interface between water and 1-octanol of 0,5 to maximally 2,5 cm depth is created. The stirring rate should be reduced if the vortex depth of 2,5 cm is exceeded; otherwise micro-droplets may be formed from 1-octanol droplets in the water phase, leading to an overestimation of the concentration of the test substance in the water. The maximum stirring rate of 2,5 cm is recommended on the basis of the findings in the ring-test validation study (5). It is a compromise between achieving a rapid rate of equilibration, while limiting the formation of 1-octanol micro-droplets.

The stirrer should be turned off prior to sampling and the liquids should be allowed to stop moving. After sampling is completed, the stirrer is started again slowly, as described above, and then the stirring rate is increased gradually.

The water phase is sampled from a stopcock at the bottom of the reaction vessel. Always discard the dead volume of water contained in the taps (approximately 5 ml in the vessel shown in the Appendix 2). The water in the taps is not stirred and therefore not in equilibrium with the bulk. Note the volume of the water samples, and make sure that the amount of test substance present in the discarded water is taken into account when setting up a mass balance. Evaporative losses should be minimized by allowing the water to flow quiescently into the separatory funnel, such that there is no disturbance of the water/1-octanol layer.

1-Octanol samples are obtained by withdrawing a small aliquot (ca. 100 μl) from the 1-octanol layer with a 100 microlitre all glass-metal syringe. Care should be taken not to disturb the boundary. The volume of the sampled liquid is recorded. A small aliquot is sufficient, since the 1-octanol sample will be diluted.

Unnecessary sample transfer steps should be avoided. To that end the sample volume should be determined gravimetrically. In case of water samples this can be achieved by collecting the water sample in a separatory funnel that contains already the required volume of solvent.

According to the present Test Method, POW is determined by performing three slow-stirring experiments (three experimental units) with the compound under investigation employing identical conditions. The regression used to demonstrate attainment of equilibrium should be based on the results of at least four determinations of CO/CW at consecutive time points. This allows for calculating variance as a measure of the uncertainty of the average value obtained by each experimental unit.

The POW can be characterized by the variance in the data obtained for each experimental unit. This information is employed to calculate the POW as the weighted average of the results of the individual experimental units. To do so, the inverse of the variance of the results of the experimental units is employed as weight. As a result, data with a large variation (expressed as the variance) and thus with lower reliability have less influence on the result than data with a low variance.

Analogously, the weighted standard deviation is calculated. It characterizes the repeatability of the POW measurement. A low value of the weighted standard deviation indicates that the POW determination was very repeatable within one laboratory. The formal statistical treatment of the data is outlined below.

The logarithm of the ratio of the concentration of the test substance in 1-octanol and water (log (CO/Cw)) is calculated for each sampling time. Achievement of chemical equilibrium is demonstrated by plotting this ratio against time. A plateau in this plot that is based on at least four consecutive time points indicates that equilibrium has been attained, and that the compound is truly dissolved in 1-octanol. If not, the test needs to be continued until four successive time points yield a slope that is not significantly different from 0 at a p-level of 0,05, indicating that log Co/Cw is independent of time.

The value of log POW of the experimental unit is calculated as the weighted average value of log Co/Cw for the part of the curve of log Co/Cw vs. time, for which equilibrium has been demonstrated. The weighted average is calculated by weighting the data with the inverse of the variance so that the influence of the data on the final result is inversely proportional to the uncertainty in the data.

The average value of log POW of different experimental units is calculated as the average of the results of the individual experimental units weighted with their respective variances.

The calculation is performed as follows:

where:

The reciprocal of the variance of log POW,i is employed as wi ()

The error of the average of log POW is estimated as the repeatability of log Co/Cw determined during the equilibrium phase in the individual experimental units. It is expressed as the weighted standard deviation of log POW,Av (σlog Pow,Av) which in turn is a measure of the error associated with log POW,Av. The weighted standard deviation can be computed from the weighted variance (varlog Pow,Av) as follows:

The symbol n stands for the number of experimental units.

The test report should include the following information:

De Bruijn JHM, Busser F, Seinen W, Hermens J. (1989). Determination of octanol/water partition coefficients with the “slow-stirring” method. Environ. Toxicol. Chem. 8: 499-512.

Chapter A.8 of this Annex, Partition Coefficient.

Chapter A.8 of this Annex, Partition Coefficient.

OECD (2000). OECD Draft Guideline for the Testing of Chemicals: 122 Partition Coefficient (n-Octanol/Water): pH-Metric Method for Ionisable Substances. Paris.

Tolls J (2002). Partition Coefficient 1-Octanol/Water (Pow) Slow-Stirring Method for Highly Hydrophobic Chemicals, Validation Report. RIVM contract-Nrs 602730 M/602700/01.

Boethling RS, Mackay D (eds.) (2000). Handbook of property estimation methods for chemicals. Lewis Publishers Boca Raton, FL, USA.

Schwarzenbach RP, Gschwend PM, Imboden DM (1993). Environmental Organic Chemistry. Wiley, New York, NY.

Arnold CG, Widenhaupt A, David MM, Müller SR, Haderlein SB, Schwarzenbach RP (1997). Aqueous speciation and 1-octanol-water partitioning of tributyl- and triphenyltin: effect of pH and ion composition. Environ. Sci. Technol. 31: 2596-2602.

OECD (1981) OECD Guidelines for the Testing of Chemicals: 112 Dissociation Constants in Water. Paris.

Chapter A.6 of this Annex, Water Solubility.

Chapter C.7 of this Annex, Degradation – Abiotic Degradation Hydrolysis as a Function of pH.

Chapter C.4 — Part II – VII (Method A to F) of this Annex, Determination of “Ready” Biodegradability.

Chapter A.4 of this Annex, Vapour Pressure.

Pinsuwan S, Li A and Yalkowsky S.H. (1995). Correlation of octanol/water solubility ratios and partition coefficients, J. Chem. Eng. Data. 40: 623-626.

Lyman WJ (1990). Solubility in water. In: Handbook of Chemical Property Estimation Methods: Environmental Behavior of Organic Compounds, Lyman WJ, Reehl WF, Rosenblatt DH, Eds. American Chemical Society, Washington, DC, 2-1 to 2-52.

Leo A, Weininger D (1989). Medchem Software Manual. Daylight Chemical Information Systems, Irvine, CA.

Meylan W (1993). SRC-LOGKOW for Windows. SRC, Syracuse, N.Y.

Compudrug L (1992). ProLogP. Compudrug, Ltd, Budapest.

ACD. ACD logP; Advanced Chemistry Development: Toronto, Ontario M5H 3V9, Canada, 2001.

Lyman WJ (1990). Octanol/water partition coefficient. In Lyman WJ, Reehl WF, Rosenblatt DH, eds, Handbook of chemical property estimation, American Chemical Society, Washington, D.C.

Rekker RF, de Kort HM (1979). The hydrophobic fragmental constant: An extension to a 1 000 data point set. Eur. J. Med. Chem. Chim. Ther. 14: 479-488.

Jübermann O (1958). Houben-Weyl, ed, Methoden der Organischen Chemie: 386-390.

Maximum volume of individual aliquots = 10 % of total volume; 5 aliquots = 50 % of total volume.

. In case of lower concentrations, larger volumes would be required.

Volume used for LOD determination = 100 ml.

log Pow vs. log Sw and log Pow vs. SR (Soct/Sw) are reasonable representations of relationships for test substances.

This Test Method is equivalent to OECD Test Guideline 403 (2009) (1). The original acute inhalation Test Guideline 403 (TG 403) was adopted in 1981. This revised Test Method B.2 (as equivalent to the revised TG 403) has been designed to be more flexible, to reduce animal usage, and to fulfil regulatory needs. The revised Test Method features two study types: a Traditional LC50 protocol and a Concentration × Time (C × t) protocol. Primary features of this Test Method are the ability to provide a concentration-response relationship ranging from non-lethal 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 × 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.

Guidance on the conduct and interpretation of this Test Method studies can be found in the Guidance Document on Acute Inhalation Toxicity Testing (GD 39) (2).

Definitions used in the context of this Test Method are provided at the end of this chapter and in GD 39 (2).

This Test Method enables test chemical characterisation and quantitative risk assessment, and allows test chemicals to be ranked and classified according to Regulation (EC) No 1272/2008 (3). GD 39 (2) provides guidance in the selection of the appropriate Test Method for acute testing. When information on classification and labelling only is required, chapter B.52 of this Annex (4) is generally recommended [see GD 39 (2)]. This Test Method B.2 is not specifically intended for the testing of specialised materials, such as poorly soluble isometric or fibrous materials or manufactured nanomaterials.

Before considering testing in accordance with this Test Method all available information on the test chemical, including existing studies (e.g. chapter B.52 of this Annex (4)) whose data would support not doing additional testing should be considered by the testing laboratory in order to minimise 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 physico-chemical properties of the test chemical; 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)].

Testing corrosive and/or irritating test chemicals 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. from repeat dose studies performed at non-corrosive/irritant concentrations), existing in vitro data (e.g. from chapters B.40, (5), B.40bis (6) of this Annex or OECD TG 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), this Test Method 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)].

This revised Test Method B.2 has been designed to obtain sufficient information on the acute toxicity of a test chemical 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 Test Method offers two 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 × t) protocol in which groups of animals are exposed to one (limit concentration) or a series of multiple concentrations over multiple durations.

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).

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.

Females should be nulliparous and non-pregnant. 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 acclimatisation 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.

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 minimise 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 immobilisation 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.

The nature of the test chemical 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).

Nose-only exposures may be any duration up to 6 hours in rats. If mice are exposed nose-only, 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 chemical due to grooming of cage mates. Feed should be withheld during the exposure period. Water may be provided throughout a whole-body exposure.

Animals are exposed to the test chemical as a gas, vapour, aerosol, or a mixture thereof. The physical state to be tested depends on the physico-chemical properties of the test chemical, the selected concentration, and/or the physical form most likely present during the handling and use of the test chemical. Hygroscopic and chemically reactive test chemicals should be tested under dry air conditions. Care should be taken to avoid generating explosive concentrations.

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.

A vehicle may be used to generate an appropriate concentration and particle size of the test chemical 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 chemical. In cases where mechanical processes are believed to have altered test chemical composition (e.g. extreme temperatures from excessive milling due to friction), the composition of the test chemical should be verified analytically. Adequate care should be taken to not contaminate the test chemical. 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 substances, an inhalation toxicity test should be performed.

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 chemical 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.

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 chemical 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 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 mixtures) or not measurable due to test chemical interference with the test method.

Whenever feasible, the nominal exposure chamber concentration should be calculated and recorded. The nominal concentration is the mass of generated test chemical divided by the total volume of air passed through the chamber system. The nominal concentration is not used to characterise 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.

The actual concentration is the test chemical 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 non-specific 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 chemical-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 chemical (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.

One lot of the test chemical 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 characterisation of the test chemical, 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 characterisation at least in a limited way (e.g. colour, physical nature, etc.).

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 chemical is generated and this takes into account the times required to attain t95. Guidance for estimating t95 can be found in GD 39 (2).

For very complex mixtures consisting of gases/vapours, and aerosols (e.g. combustion atmospheres and test chemicals 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 mixture, of each phase (gas/vapour and aerosol) should be selected. When the test chemical is a mixture, the analytical concentration should be reported for the mixture and not just for the active substance or the component (analyte). Additional information regarding actual concentrations can be found in GD 39 (2).

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 minimise 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).

Two study types are described below: the Traditional protocol, and the C × 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 × 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 minimise animal usage. For example, data generated using chapter B.52 of this Annex (4) may eliminate the need for a sighting study, and may also demonstrate whether one sex is more susceptible [see GD 39 (2)].

In a Traditional study, groups of animals are exposed to a test chemical 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 chemical already exists, such as a previously performed B.52 study [see GD 39 (2)].

A sighting study is used to estimate test chemical 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 B.52 study (4) may be used instead of a sighting study [see GD 39 (2)].

A limit test is used when the test chemical 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 chemical at a limit concentration. Information about the toxicity of the test chemical can be gained from knowledge about similar tested chemicals, 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 chemical is expected to be toxic, the main test should be performed.

The selection of limit concentrations usually depends on regulatory requirements. When Regulation (EC) No 1272/2008 is used, the limit concentrations for gases, vapours, and aerosols are 20 000 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 chemicals, 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 chemicals 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)]. Regulation (EC) No 1272/2008 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 chemicals, 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.

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 chemical’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 chemical as a liquid aerosol.

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.

A step-wise C × 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 chemical 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 Appendix 1 illustrates this protocol. A simulation analysis has shown that the Traditional protocol and the C × t protocol are both capable of yielding robust LC50 values, but the C × t protocol is generally better at yielding robust LC01 and LC10 values (15).

A simulation analysis has demonstrated that using two animals per C × 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 × 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 × t study can be found in GD 39 (2).

A sighting study is used to estimate test chemical 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 minimise 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 B.52 study (4). When selecting the initial target concentration in a B.2 study, the study director should consider the mortality patterns observed in any available B.52 studies (4) for both sexes and for all concentrations tested [see GD 39 (2)].

The initial concentration (Exposure Session I) (Appendix 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) (Appendix 1).

The selection of limit concentrations usually depends on regulatory requirements. When Regulation (EC) No 1272/2008 is used, the limit concentrations for gases, vapours, and aerosols are 20 000 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 chemicals, 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 chemicals. Aerosol testing at greater than 2 mg/l should only be attempted if a respirable particle size can be achieved [see GD 39 (2)]. Regulation (EC) No 1272/2008 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 chemicals, 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.

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 chemical’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 chemical as a liquid aerosol.

The initial concentration (Exposure Session I) (Appendix 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 × 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 Appendix 1).

For many test chemicals 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 × t mortality relationship (15), but there may be some benefit to using a 5th exposure concentration [see Appendix 1 and GD 39 (2)]. For mathematical treatment of results for the C × t protocol, see Appendix 1.

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 chemical-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 be taken into consideration (7). When animals are killed for humane reasons or found dead, the time of death should be recorded as precisely as possible.

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.

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 recognised 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 post-exposure period.

All test animals, including those which die during the test or are euthanised 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 minimise 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.

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 chemicals that are reactive with water, such as acids and hygroscopic test chemicals.

Individual animal data on body weights and necropsy findings should be provided. Clinical observation data should be summarised 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.

The test report should include the following information, as appropriate:

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

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]

Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC and amending Regulation (EC) No 1907/2006 (OJ L 353, 31.12.2008, p. 1).

Chapter B.52 of this Annex, Acute Inhalation Toxicity — Acute Toxic Class (ATC) Method.

Chapter B.40 of this Annex, In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER).

Chapter B.40bis of this Annex, In Vitro Skin Corrosion: Human Skin Model Test.

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]

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]

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.

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

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

Zwart JHE, Arts JM, ten Berge WF, Appelman LM (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.

Zwart JHE, Arts JM, Klokman-Houweling ED, Schoen ED (1990). Determination of Concentration-Time-Mortality Relationships to Replace LC50 Values. Inhal. Toxicol. 2: 105-117.

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

OECD (2009). Performance Assessment: Comparison of 403 and C × 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]

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

A step-wise Concentration × Time (C × 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 chemical 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 Regulation (EC) No 1272/2008 is used, the limit concentrations for gases, vapours, and aerosols are 20 000 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 B.2 main text).

In situations where there is little or no information about the toxicity of a test chemical, 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.

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).

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.

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 utilise 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).

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).

The chamber concentration should be measured frequently to determine the time-weighted-average 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.

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.

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 × 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).

Glossary

Concentration:

Time of exposure:

Below is an example of the stepwise procedure:

Exposure Session I —   Testing at the limit concentration (see Figure 1)

↓

Exposure Session II  (4) —   Main Study

↓

Exposure Session III —   Main Study

↓

Exposure Session IV’ —   Main Study

↓ or

Exposure Session IV —   Main Study

A C × 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 × t relationship can be calculated using statistical analysis (16):

Equation 1:

where C = concentration; t = exposure duration, or

Equation 2:

where

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.

This Test Method is equivalent to OECD Test Guideline 407 (2008). The original Test Guideline 407 was adopted in 1981. In 1995 a revised version was adopted, to obtain additional information from the animal used in the study, in particular on neurotoxicity and immunotoxicity.

In 1998, the OECD initiated a high-priority activity, to revise existing Test Guidelines and to develop new Test Guidelines for the screening and testing of potential endocrine disruptors (8). One element of the activity was to update the existing OECD guideline for “repeated dose 28-day oral toxicity study in rodents” (TG 407) by parameters suitable to detect endocrine activity of test chemicals. This procedure underwent an extensive international program to test for the relevance and practicability of the additional parameters, the performance of these parameters for chemicals with (anti)oestrogenic, (anti)androgenic, and (anti)thyroid activity, the intra- and inter-laboratory reproducibility, and the interference of the new parameters with those required by the prior TG 407. The large amount of data thereby obtained has been compiled and evaluated in detail in a comprehensive OECD report (9). This updated Test Method B.7 (as equivalent to TG 407) is the outcome of the experience and results gained during the international test program. This Test Method allows certain endocrine mediated effects to be put into context with other toxicological effects.

In the assessment and evaluation of the toxic characteristics of a chemical, the determination of oral toxicity using repeated doses may be carried out after initial information on toxicity has been obtained by acute toxicity testing. This Test Method is intended to investigate effects on a very broad variety of potential targets of toxicity. It provides information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time, including effects on the nervous, immune and endocrine systems. Regarding these particular endpoints, it should identify chemicals with neurotoxic potential, which may warrant further in-depth investigation of this aspect, and chemicals that interfere with thyroid physiology. It may also provide data on chemicals that affect the male and/or female reproductive organs in young adult animals and may give an indication of immunological effects.

The results from this Test Method B.7 should be used for hazard identification and risk assessment. The results obtained by the endocrine related parameters should be seen in the context of the “OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupting Chemicals” (11). The method comprises the basic repeated dose toxicity study that may be used for chemicals on which a 90-day study is not warranted (e.g. when the production volume does not exceed certain limits) or as a preliminary to a long-term study. The duration of exposure should be 28 days.

The international program conducted on the validation of parameters suitable to potentially detect endocrine activity of a test chemical showed that the quality of data obtained by this Test Method B.7 will depend much on the experience of the test laboratory. This relates specifically to the histopathological determination of cyclic changes in the female reproductive organs and to the weight determination of the small hormone dependent organs which are difficult to dissect. Guidance on histopathology has been developed (19). It is available on the OECD public website on Test Guidelines. It is intended to assist pathologists in their examinations and help increase the sensitivity of the assay. A variety of parameters were found to be indicative of endocrine-related toxicity and have been incorporated in the Test Method. Parameters for which insufficient data were available to prove usefulness or which showed only weak evidence in the validation programme of their ability to help in detection of endocrine disrupters are proposed as optional endpoints (see Appendix 2).

On the basis of data generated in the validation process, it must be emphasised that the sensitivity of this assay is not sufficient to identify all substances with (anti)androgenic or (anti)oestrogenic modes of action (9). The Test Method is not performed in a life-stage that is most sensitive to endocrine disruption. The Test Method nevertheless, during the validation process identified substances weakly and strongly affecting thyroid function, and strong and moderate endocrine active substances acting through oestrogen or androgen receptors, but in most cases failed to identify endocrine active substances that weakly affect oestrogen or androgen receptors. Thus it cannot be described as a screening assay for endocrine activity.

Consequently, the lack of effects related to these modes of action can not be taken as evidence for the lack of effects on the endocrine system. Regarding endocrine mediated effects, substance characterisation should not therefore be based on the results of this Test Method alone but should be used in a weight of evidence approach incorporating all existing data on a chemical to characterise potential endocrine activity. For this reason, regulatory decision making on endocrine activity (substance characterisation) should be a broadly based approach, not solely reliant on results from application of this test method.

It is acknowledged that all animal-based procedures will conform to local standards of animal care; the descriptions of care and treatment set forth below are minimal performance standards, and will be superseded by local regulations where more stringent. Further guidance of the humane treatment of animals is given by the OECD (14).

Definitions used are given in Appendix 1.

The test chemical is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 28 days. During the period of administration the animals are observed closely, each day for signs of toxicity. Animals that die or are euthanised during the test are necropsied and at the conclusion of the test surviving animals are euthanised and necropsied. A 28 day study provides information on the effects of repeated oral exposure and can indicate the need for further longer term studies. It can also provide information on the selection of concentrations for longer term studies. The data derived from using the Test Method should allow for the characterisation of the test chemical toxicity, for an indication of the dose response relationship and the determination of the No-Observed Adverse Effect Level (NOAEL).

The preferred rodent species is the rat, although other rodent species may be used. If the parameters specified within this Test Method B.7 are investigated in another rodent species a detailed justification should be given. Although it is biologically plausible that other species should respond to toxicants in a similar manner to the rat, the use of smaller species may result in increased variability due to technical challenges of dissecting smaller organs. In the international validation program for the detection of endocrine disrupters, the rat was the only species used. Young healthy adult animals of commonly used laboratory strains should be employed. Females should be nulliparous and non pregnant. Dosing should begin as soon as feasible after weaning, and, in any case, before the animals are nine weeks old. At the commencement of the study the weight variation of animals used should be minimal and not exceed ± 20 % of the mean weight of each sex. When a repeated oral dose is conducted as a preliminary to a longer-term study, it is preferable that animals from the same strain and source should be used in both studies.

All procedures should conform to local standards of laboratory animal care. The temperature in the experimental animal room should be 22 °C (± 3 °C). Although the relative humidity should be at least 30 % and preferably not to exceed 70 % other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the photoperiod being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to ensure a suitable admixture of a test chemical when administered by this method. Animals should be group housed in small groups of the same sex; animals may be housed individually if scientifically justified. For group caging, no more than five animals should be housed per cage.

The feed should be regularly analysed for contaminants. A sample of the diet should be retained until finalisation of the report.

Healthy young adult animals are randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. The animals are identified uniquely and kept in their cages for at least five days prior to the start of the treatment study to allow for acclimatisation to the laboratory conditions.

The test chemical is administered by gavage or via the diet or drinking water. The method of oral administration is dependent on the purpose of the study, and the physical/chemical/toxico-kinetic properties of the test chemical.

Where necessary, the test chemical is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/suspension in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of the test chemical in the vehicle should be determined.

At least 10 animals (five female and five male) should be used at each dose level. If interim euthanasia are planned, the number should be increased by the number of animals scheduled to be euthanised before the completion of the study. Consideration should be given to an additional satellite group of ten animals (five per sex) in the control and in the top dose group for observation of reversibility, persistence, or delayed occurrence of toxic effects, for at least 14 days post treatment.

Generally, at least three test groups and a control group should be used, but if from assessment of other data, no effects would be expected at a dose of 1 000 mg/kg bw/d, a limit test may be performed. If there are no suitable data available, a range finding study (animals of the same strain and source) may be performed to aid the determination of the doses to be used. Except for treatment with the test chemical, animals in the control group should be handled in an identical manner to the test group subjects. If a vehicle is used in administering the test chemical, the control group should receive the vehicle in the highest volume used.

Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available for the test chemical or related chemicals. The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and no-observed-adverse effects at the lowest dose level (NOAEL). Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of 10) between dosages.

In the presence of observed general toxicity (e.g. reduced body weight, liver, heart, lung or kidney effects, etc.) or other changes that may not be toxic responses (e.g. reduced food intake, liver enlargement), observed effects on immune, neurological or endocrine sensitive endpoints should be interpreted with caution.

If a test at one dose level of at least 1 000 mg/kg body weight/day or, for dietary or drinking water administration, an equivalent percentage in the diet, or drinking water (based upon body weight determinations), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data from structurally related chemicals, then a full study using three dose levels may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.

The animals are dosed with test chemical daily 7 days each week for a period of 28 days. When the test chemical is administered by gavage, this should be done in a single dose to the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume should not exceed 1 ml/100 g body weight except in the case of aqueous solutions where 2 ml/100 g body weight may be used. Except for irritating or corrosive chemicals, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimized by adjusting the concentration to ensure a constant volume at all dose levels.

For chemicals administered via the diet or drinking water it is important to ensure that the quantities of the test chemical involved do not interfere with normal nutrition or water balance. When the test chemical is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animals’ body weight may be used; the alternative used must be specified. For a chemical administered by gavage, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where a repeated dose study is used as a preliminary to a long term study, a similar diet should be used in both studies.

The observation period should be 28 days. Animals in a satellite group scheduled for follow-up observations should be kept for at least 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

General clinical observations should be made at least once a day, preferably at the same time(s) each day and considering the peak period of anticipated effects after dosing. The health condition of the animals should be recorded. At least twice daily, all animals are observed for morbidity and mortality.

Once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made outside the home cage in a standard arena and preferably at the same time of day on each occasion. They should be carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the test conditions are minimal and that observations are preferably conducted by observers unaware of the treatment. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) should also be recorded (2).

In the fourth exposure week sensory reactivity to stimuli of different types (2) (e.g. auditory, visual and proprioceptive stimuli) (3)(4)(5), assessment of grip strength (6) and motor activity assessment (7) should be conducted. Further details of the procedures that could be followed are given in the respective references. However, alternative procedures than those referenced could be used.

Functional observations conducted in the fourth exposure week may be omitted when the study is conducted as a preliminary study to a subsequent subchronic (90-day) study. In that case, the functional observations should be included in this follow-up study. On the other hand, the availability of data on functional observations from the repeated dose study may enhance the ability to select dose levels for a subsequent subchronic study.

As an exception, functional observations may also be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance.

At necropsy, the oestrus cycle of all females could be determined (optional) by taking vaginal smears. These observations will provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of estrogen sensitive tissues [see guidance on histopathology (19)].

All animals should be weighed at least once a week. Measurements of food consumption should be made at least weekly. If the test chemical is administered via the drinking water, water consumption should also be measured at least weekly.

The following haematological examinations should be made at the end of the test period: haematocrit, haemoglobin concentrations, erythrocyte count, reticulocytes, total and differential leucocyte count, platelet count and a measure of blood clotting time/potential. Other determinations that should be carried out, if the test chemical or its putative metabolites have or are suspected to have oxidising properties include methaemoglobin concentration and Heinz bodies.

Blood samples should be taken from a named site just prior to or as part of the procedure for euthanasia of the animals, and stored under appropriate conditions. Animals should be fasted overnight prior to euthanasia (7).

Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained of all animals just prior to or as part of the procedure for euthanasia of the animals (apart from those found moribund and/or euthanised prior to the termination of the study). Investigations of plasma or serum shall include sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, at least two enzymes indicative of hepatocellular effects (such as alanin aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl trans-peptidase and glutamate dehydrogenase), and bile acids. Measurements of additional enzymes (of hepatic or other origin) and bilirubin may provide useful information under certain circumstances.

Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells.

In addition, studies to investigate plasma or serum markers of general tissue damage should be considered. Other determinations that should be carried out, if the known properties of the test chemical may, or are suspected to, affect related metabolic profiles include calcium, phosphate, triglycerides, specific hormones, and cholinesterase. These need to be identified for chemicals in certain classes or on a case-by-case basis.

Although in the international evaluation of the endocrine related endpoints a clear advantage for the determination of thyroid hormones (T3, T4) and TSH could not be demonstrated, it may be helpful to retain plasma or serum samples to measure T3, T4 and TSH (optional) if there is an indication for an effect on the pituitary-thyroid axis. These samples may be frozen at – 20° for storage. The following factors may influence the variability and the absolute concentrations of the hormone determinations:

Definitive identification of thyroid-active chemicals is more reliable by histopathological analysis rather than hormone levels.

Plasma samples specifically intended for hormone determination should be obtained at a comparable time of the day. It is recommended that consideration should be given to T3, T4 and TSH determinations triggered based upon alterations of thyroid histopathology. The numerical values obtained when analysing hormone concentrations differ with various commercial assay kits. Consequently, it may not be possible to provide performance criteria based upon uniform historical data. Alternatively, laboratories should strive to keep control coefficients of variation below 25 for T3 and T4 and below 35 for TSH. All concentrations are to be recorded in ng/ml.

If historical baseline data are inadequate, consideration should be given to determination of haematological and clinical biochemistry variables before dosing commences or preferably in a set of animals not included in the experimental groups.

All animals in the study shall be subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals, testes, epididymides, prostate + seminal vesicles with coagulating glands as a whole, thymus, spleen, brain and heart of all animals (apart from those found moribund and/or euthanised prior to the termination of the study) should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying. Care must be exercised when trimming the prostate complex to avoid puncture of the fluid filled seminal vesicles. Alternatively, seminal vesicles and prostate may be trimmed and weighed after fixation.

In addition, two other tissues could be optionally weighed as soon as possible after dissection, to avoid drying: paired ovaries (wet weight) and uterus, including cervix (guidance on removal and preparation of the uterine tissues for weight measurement is provided in OECD TG 440 (18)).

The thyroid weight (optional) could be determined after fixation. Trimming should also be done very carefully and only after fixation to avoid tissue damage. Tissue damage could compromise histopathology analysis.

The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination (see paragraph 47): all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, eye, stomach, small and large intestines (including Peyer’s patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by inflation with fixative and then immersion), gonads (testis and ovaries), accessory sex organs (uterus and cervix, epididymides, prostate + seminal vesicles with coagulating glands), vagina, urinary bladder, lymph nodes [besides the most proximal draining node another lymph node should be taken according to the laboratory’s experience (15)], peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, skeletal muscle and bone, with bone marrow (section or, alternatively, a fresh mounted bone marrow aspirate). It is recommended that testes be fixed by immersion in Bouin’s or modified Davidson’s fixative (16) (17). The tunica albuginea must be gently and shallowly punctured at the both poles of the organ with a needle to permit rapid penetration of the fixative. The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test chemical should be preserved.

The following tissues may give valuable indication for endocrine-related effects: Gonads (ovaries and testes), accessory sex organs (uterus including cervix, epididymides, seminal vesicles with coagulation glands, dorsolateral and ventral prostate), vagina, pituitary, male mammary gland, the thyroid and adrenal gland. Changes in male mammary glands have not been sufficiently documented but this parameter may be very sensitive to substances with oestrogenic action. Observation of organs/tissues that are not listed in paragraph 43 is optional (see Appendix 2).

The Guidance on histopathology (19) details extra information on dissection, fixation, sectioning and histopathology of endocrine tissues.

In the international test program some evidence was obtained that subtle endocrine effects by chemicals with a low potency for affecting sex hormone homeostasis may be identified by disturbance of the synchronisation of the oestrus cycle in different tissues and not so much by frank histopathological alterations in female sex organs. Although no definitive proof was obtained for such effects, it is recommended that evidence of possible asynchrony of the oestrus cycle should be taken into account in interpretation of the histopathology of the ovaries (follicular, thecal, and granulosa cells), uterus, cervix and vagina. If assessed, the stage of cycle as determined by vaginal smears could be included in this comparison as well.

Full histopathology should be carried out on the preserved organs and tissues of all animals in the control and high dose groups. These examinations should be extended to animals of all other dosage groups, if treatment-related changes are observed in the high dose group.

All gross lesions shall be examined.

When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.

Individual data should be provided. Additionally, all data should be summarised in tabular form showing for each test group the number of animals at the start of the test, the number of animals found dead during the test or euthanised for humane reasons and the time of any death or euthanasia, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the number of animals showing lesions, the type of lesions, their severity and the percentage of animals displaying each type of lesion.

When possible, numerical results should be evaluated by an appropriate and generally acceptable statistical method. Comparisons of the effect along a dose range should avoid the use of multiple t-tests. The statistical methods should be selected during the design of the study.

For quality control it is proposed that historical control data are collected and that for numerical data coefficients of variation are calculated, especially for the parameters linked with endocrine disrupter detection. These data can be used for comparison purposes when actual studies are evaluated.

The test report must include the following information:

Androgenicity is the capability of a chemical to act like a natural androgenic hormone (e.g. testosterone) in a mammalian organism.

Antiandrogenicity is the capability of a chemical to suppress the action of a natural androgenic hormone (e.g. testosterone) in a mammalian organism.

Antioestrogenicity is the capability of a chemical to suppress the action of a natural oestrogenic hormone (e.g. oestradiol 17ß) in a mammalian organism.

Antithyroid activity is the capability of a chemical to suppress the action of a natural thyroid hormone (e.g. T3) in a mammalian organism.

Dosage is a general term comprising of dose, its frequency and the duration of dosing.

Dose is the amount of test chemical administered. The dose is expressed as weight of test chemical per unit body weight of test animal per day (e.g. mg/kg body weight/day), or as a constant dietary concentration.

Evident toxicity is a general term describing clear signs of toxicity following administration of test chemical. These should be sufficient for hazard assessment and should be such that an increase in the dose administered can be expected to result in the development of severe toxic signs and probable mortality.

NOAEL is the abbreviation for no-observed-adverse-effect level. This is the highest dose level where no adverse treatment-related findings are observed due to treatment.

Oestrogenicity is the capability of a chemical to act like a natural oestrogenic hormone (e.g. oestradiol 17ß) in a mammalian organism.

Test chemical: Any substance or mixture tested using this Test Method.

Thyroid activity is the capability of a chemical to act like a natural thyroid hormone (e.g. T3) in a mammalian organism.

Validation is a scientific process designed to characterise the operational requirements and limitations of a test method and to demonstrate its reliability and relevance for a particular purpose.

OECD (Paris, 1992). Chairman’s Report of the Meeting of the ad hoc Working Group of Experts on Systemic Short-term and (Delayed) Neurotoxicity.

IPCS (1986). Principles and Methods for the Assessment of Neurotoxicity Associated with Exposure to Chemicals. Environmental Health Criteria Document No 60.

Tupper DE, Wallace RB (1980). Utility of the Neurologic Examination in Rats. Acta Neurobiol. Exp. 40: 999-1003.

Gad SC (1982). A Neuromuscular Screen for Use in Industrial Toxicology. J. Toxicol Environ. Health 9: 691-704.

Moser VC, McDaniel KM, Phillips PM (1991). Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol. 108: 267-283.

Meyer OA, Tilson HA, Byrd WC, Riley MT (1979). A Method for the Routine Assessment of Fore- and Hindlimb Grip Strength of Rats and Mice. Neurobehav. Toxicol. 1: 233-236.

Crofton KM, Howard JL, Moser VC, Gill MW, Reiter LW, Tilson HA, MacPhail RC (1991). Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicol. Teratol. 13: 599-609.

OECD (1998). Report of the First Meeting of the OECD Endocrine Disrupter Testing and Assessment (EDTA) Task Force, 10th-11 March 1998, ENV/MC/CHEM/RA(98)5.

OECD. (2006). Report of the Validation of the Updated Test Guideline 407: Repeat Dose 28-day Oral Toxicity Study in Laboratory Rats. Series on Testing and Assessment No 59, ENV/JM/MONO(2006)26.

OECD (2002). Detailed Review Paper on the Appraisal of Test Methods for Sex Hormone Disrupting Chemicals. Series on Testing and Assessment No 21, ENV/JM/MONO(2002)8.

OECD (2012).Conceptual Framework for Testing and Assessment of Endocrine Disrupting Chemicals. http://www.oecd.org/document/58/0,3343,fr_2649_37407_2348794_1_1_1_37407,00.html

OECD (2006). Final Summary report of the meeting of the Validation Management Group for mammalian testing. ENV/JM/TG/EDTA/M(2006)2.

OECD. Draft Summary record of the meeting of the Task Force on Endocrine Disrupters Testing and Assessment. ENV/JM/TG/EDTA/M(2006)3.

OECD (2000). Guidance document on the recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation. Series on Testing and Assessment No 19. ENV/JM/MONO(2000)7.

Haley P, Perry R, Ennulat D, Frame S, Johnson C, Lapointe J-M, Nyska A, Snyder PW, Walker D, Walter G (2005). STP Position Paper: Best Practice Guideline for the Routine Pathology Evaluation of the Immune System. Toxicol Pathol 33: 404-407.

Hess RA, Moore BJ (1993). Histological Methods for the Evaluation of the Testis. In: Methods in Reproductive Toxicology, Chapin RE and Heindel JJ (eds). Academic Press: San Diego, CA, pp. 52-85.

Latendresse JR, Warbrittion AR, Jonassen H, Creasy DM.(2002) Fixation of testes and eyes using a modified Davidson’s fluid: comparison with Bouin’s fluid and conventional Davidson’s fluid. Toxicol. Pathol. 30, 524-533.

OECD (2007). OECD Guideline for Testing of Chemicals No 440: Uterotrophic Bioassay in Rodents: A short-term screening test for oestrogenic properties.

OECD (2009). Guidance Document 106 on Histologic evaluation of Endocrine and Reproductive Tests in Rodents ENV/JM/Mono(2009)11.

This Test Method is equivalent to OECD Test Guideline 412 (2009). The original subacute inhalation Test Guideline 412 (TG 412) was adopted in 1981 (1). This Test Method B.8 (as equivalent to the revised TG 412) has been updated to reflect the state of science and to meet current and future regulatory needs.

This method enables the characterisation of adverse effects following repeated daily inhalation exposure to a test chemical for 28 days. The data derived from 28-day sub-acute inhalation toxicity studies can be used for quantitative risk assessments [if not followed by a 90-day subchronic inhalation toxicity study (Chapter B.29 of this Annex)]. The data can also provide information on the selection of concentrations for longer term studies such as the 90-day subchronic inhalation toxicity study. This test method is not specifically intended for the testing of nanomaterials. Definitions used in the context of this Test Method are provided at the end of this chapter and in the Guidance Document 39 (2).

All available information on the test chemical should be considered by the testing laboratory prior to conducting the study in order to enhance the quality of the study and minimize animal usage. Information that will assist in the selection of appropriate test concentrations might include the identity, chemical structure, and physico-chemical properties of the test chemical; results of any in vitro or in vivo toxicity tests; anticipated use(s) and potential for human exposure; available (Q)SAR data and toxicological data on structurally related chemicals; and data derived from acute inhalation toxicity testing. If neurotoxicity is expected or is observed in the course of the study, the study director may choose to include appropriate evaluations such as a functional observational battery (FOB) and measurement of motor activity. Although the timing of exposures relative to specific examinations may be critical, the performance of these additional activities should not interfere with the basic study design.

Dilutions of corrosive or irritating test chemicals may be tested at concentrations that will yield the desired degree of toxicity [refer to GD 39 (2)]. When exposing animals to these materials, the targeted concentrations should be low enough to not cause marked pain and distress, 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, preferably based upon an adequately designed range-finding study that provides information regarding the critical endpoint, any irritation threshold, and the time of onset (see paragraphs 11-13). The justification for concentration selection should be provided.

Moribund animals or animals obviously in pain or showing signs of severe and enduring distress should be humanely killed. Moribund animals are considered in the same way as animals that die 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 on Humane Endpoints (3).

Healthy young adult rodents of commonly used laboratory strains should be employed. The preferred species is the rat. Justification should be provided if other species are used.

Females should be nulliparous and non-pregnant. On the day of randomisation, animals should be young adults 7 to 9 weeks of age. Body weights should be within ± 20 % of the mean weight for each sex. The animals are randomly selected, marked for individual identification, and kept in their cages for at least 5 days prior to the start of the test to allow for acclimatisation to laboratory conditions.

Animals should be individually identified, if possible with subcutaneous transponders, to facilitate observations and avoid confusion. 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 minimise 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 immobilisation 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.

The nature of the test chemical and the object 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 addressed in GD 39 (2).

Unlike with acute studies, there are no defined limit concentrations in 28-day sub-acute inhalation toxicity studies. The maximum concentration tested should consider: (1) the maximum attainable concentration, (2) the “worst case” human exposure level, (3) the need to maintain an adequate oxygen supply, and/or (4) animal welfare considerations. In the absence of data-based limits, the acute limits of the Regulation (EC) No 1272/2008 (13) may be used (i.e. up to a maximum concentration of 5 mg/l for aerosols, 20 mg/l for vapours and 20 000 ppm for gases); refer to GD 39 (2). Justification should be provided if it is necessary to exceed these limits when testing gases or highly volatile test chemicals (e.g. refrigerants). The limit concentration should elicit unequivocal toxicity without causing undue stress to the animals or affecting their longevity (3).

Before commencing with the main study, it may be necessary to perform a range-finding study. A range-finding study is more comprehensive than a sighting study because it is not limited to concentration selection. Knowledge learned from a range-finding study can lead to a successful main study. A range-finding study may, for example, provide technical information regarding analytical methods, particle sizing, discovery of toxic mechanisms, clinical pathology and histopathological data, and estimations of what may be NOAEL and MTC concentrations in a main study. The study director may choose to use the range-finding study to identify the threshold of respiratory tract irritation (e.g. with histopathology of the respiratory tract, pulmonary function testing, or bronchoalveolar lavage), the upper concentration which is tolerated without undue stress to the animals, and the parameters that will best characterise a test chemical’s toxicity.

A range-finding study may consist of one or more concentration levels. No more than three males and three females should be exposed at each concentration level. A range-finding study should last a minimum of 5 days and generally no more than 14 days. The rationale for the selection of concentrations for the main study should be provided in the study report. The objective of the main study is to demonstrate a concentration-response relationship based on what is anticipated to be the most sensitive endpoint. The low concentration should ideally be a no-observed-adverse effect concentration while the high concentration should elicit unequivocal toxicity without causing undue stress to the animals or affecting their longevity (3).

When selecting concentration levels for the range-finding study, all available information should be considered including structure-activity relationships and data for similar chemicals (see paragraph 3). A range-finding study may verify/refute what are considered to be the most sensitive mechanistically based endpoints, e.g. cholinesterase inhibition by organophosphates, methaemoglobin formation by erythrocytotoxic agents, thyroidal hormones (T3, T4) for thyrotoxicants, protein, LDH, or neutrophils in brochoalveolar lavage for innocuous poorly soluble particles or pulmonary irritant aerosols.

The main sub-acute toxicity study generally consists of three concentration levels, and also concurrent negative (air) and/or vehicle controls as needed (see paragraph 17). All available data should be utilised to aid selection of appropriate exposure levels, including the results of systemic toxicity studies, metabolism and kinetics (particular emphasis should be given to avoiding high concentration levels which saturate kinetic processes). Each test group contains at least 10 rodents (5 male and 5 female) that are exposed to the test chemical for 6 hours per day on a 5 day per week basis for a period of 4 weeks (total study duration of 28 days). Animals may also be exposed 7 days per week (e.g. when testing inhaled pharmaceuticals). If one sex is known to be more susceptible to a given test chemical, the sexes may be exposed at different concentration levels in order to optimise the concentration-response as described in paragraph 15. If rodent species other than rats are exposed nose-only, maximum exposure durations may be adjusted to minimise species-specific distress. A rationale should be provided when using an exposure duration less than 6 hours/day, or when it is necessary to conduct a long duration (e.g. 22 hours/day) whole-body exposure study [refer to GD 39 (2)]. Feed should be withheld during the exposure period unless exposure exceeds 6 hours. Water may be provided throughout a whole-body exposure.

The target concentrations selected should identify the target organ(s) and demonstrate a clear concentration-response:

A satellite (reversibility) study may be used to observe reversibility, persistence, or delayed occurrence of toxicity for a post-treatment period of an appropriate length, but no less than 14 days. Satellite (reversibility) groups consist of five males and five females exposed contemporaneously with the experimental animals in the main study. Satellite (reversibility) study groups should be exposed to the test chemical at the highest concentration level and there should be concurrent air and/or vehicle controls as needed (see paragraph 17).

Concurrent negative (air) control animals should be handled in a manner identical to the test group animals except that they are exposed to filtered air rather than test chemical. When water or another substance is used to assist in generating the test atmosphere, a vehicle control group, instead of a negative (air) control group, should be included in the study. Water should be used as the vehicle whenever possible. When water is used as the vehicle, the control animals should be exposed to air with the same relative humidity as the exposed groups. The selection of a suitable vehicle should be based on an appropriately conducted pre-study or historical data. If a vehicle’s toxicity is not well known, the study director may choose to use both a negative (air) control and a vehicle control, but this is strongly discouraged. If historical data reveal that a vehicle is non-toxic, then there is no need for a negative (air) control group and only a vehicle control should be used. If a pre-study of a test chemical formulated in a vehicle reveals no toxicity, it follows that the vehicle is non-toxic at the concentration tested and this vehicle control should be used.

Animals are exposed to the test chemical as a gas, vapour, aerosol, or a mixture thereof. The physical state to be tested depends on the physico-chemical properties of the test chemical, the selected concentration, and/or the physical form most likely present during the handling and use of the test chemical. Hygroscopic and chemically reactive test chemicals should be tested under dry air conditions. Care should be taken to avoid generating explosive concentrations. Particulate material may be subjected to mechanical processes to decrease the particle size. Further guidance is provided in GD 39 (2).

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 3 μm with a geometric standard deviation (σg) in the range of 1,5 to 3,0 are recommended (4). Although a reasonable effort should be made to meet this standard, expert judgement should be provided if it cannot be achieved. For example, metal fume particles may be smaller than this standard, and charged particles and fibres may exceed it.

Ideally, the test chemical should be tested without a vehicle. If it is necessary to use a vehicle to generate an appropriate test chemical concentration and particle size, water should be given preference. Whenever a test chemical is dissolved in a vehicle, its stability should be demonstrated.

The flow of air through the exposure chamber should be carefully controlled, continuously monitored, and recorded at least hourly during each exposure. The real-time monitoring of the test atmosphere concentration (or temporal stability) is an integral measurement of all dynamic parameters and provides an indirect means to control all relevant dynamic inhalation parameters. If the concentration is monitored real-time, the frequency of measurement of air flows may be reduced to one single measurement per exposure per day. Special consideration should be given to avoiding re-breathing in nose-only chambers. Oxygen concentration should be at least 19 % and carbon dioxide concentration should not exceed 1 %. If there is reason to believe that this standard cannot be met, oxygen and carbon dioxide concentrations should be measured. If measurements on the first day of exposure show that these gases are at proper levels, no further measurements should be necessary.

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 continuously and recorded hourly during each exposure where possible. The relative humidity should preferably be maintained in the range of 30 to 70 %, but this may either be unattainable (e.g. when testing water based mixtures) or not measurable due to test chemical interference with the Test Method.

Whenever feasible, the nominal exposure chamber concentration should be calculated and recorded. The nominal concentration is the mass of generated test chemical divided by the total volume of air passed through the inhalation chamber system. The nominal concentration is not used to characterise 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.

The actual concentration is the test chemical concentration as sampled at the animals’ breathing zone in an inhalation chamber. Actual concentrations can be obtained either by specific methods (e.g. direct sampling, adsorptive or chemical reactive methods, and subsequent analytical characterisation) or by non-specific 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 chemical-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 chemical (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.

One lot of the test chemical should be used throughout the duration of the study, 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 characterisation of the test chemical including its purity and, if technically feasible, the identity, and quantities of identified contaminants and impurities. This can be demonstrated but is not limited by 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 characterisation at least in a limited way (e.g. colour, physical nature, etc.).

The exposure atmosphere should be held as constant as practicable. A real-time monitoring device, such as an aerosol photometer for aerosols or a total hydrocarbon analyser for vapours may be used to demonstrate the stability of the exposure conditions. Actual chamber concentration should be measured at least 3 times during each exposure day for each exposure level. If not feasible due to limited air flow rates or low concentrations, one sample per exposure period is acceptable. Ideally, this sample should then be collected over the entire exposure period. Individual chamber concentration samples should deviate from the mean chamber concentration by no more than ± 10 % for gases and vapours, and by no more than ± 20 % for liquid or solid aerosols. Time to attain chamber equilibration (t95) should be calculated and reported. The duration of an exposure spans the time that the test chemical is generated. This takes into account the times required to attain chamber equilibration (t95) and decay. Guidance for estimating t95 can be found in GD 39 (2).

For very complex mixtures consisting of gases/vapours and aerosols (e.g. combustion atmospheres and test chemicals propelled from purpose-driven end-use products/devices), each phase may behave differently in an inhalation chamber. Therefore, at least one indicator substance (analyte), normally the principal active substance in the mixture, of each phase (gas/vapour and aerosol) should be selected. When the test chemical is a mixture, the analytical concentration should be reported for the total mixture, and not just for the active ingredient or the indicator substance (analyte). Additional information regarding actual concentrations can be found in GD 39 (2).

The particle size distribution of aerosols should be determined at least weekly for each concentration level by using a cascade impactor or an alternative instrument, such as an aerodynamic particle sizer (APS). If equivalence of the results obtained by a cascade impactor and the 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 at all concentrations tested in the early phase of the study, then further confirmatory measurements may be omitted. For the sake of animal welfare, measures should be taken to minimise inconclusive data which may lead to a need to repeat a study.

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.

The animals should be clinically observed before, during and after the exposure period. More frequent observations may be indicated depending on the response of the animals during exposure. When animal observation is hindered by the use of animal restraint tubes, poorly lit whole body chambers, or opaque atmospheres, animals should be carefully observed after exposure. Observations before the next day’s exposure can assess any reversibility or exacerbation of toxic effects.

All observations are recorded with individual records being maintained for each animal. When animals are killed for humane reasons or found dead, the time of death should be recorded as precisely as possible.

Cage-side observations should include changes in the skin and fur, eyes, and mucous membranes; changes in the respiratory and circulatory systems, changes in the nervous system, and changes in somatomotor activity and behaviour patterns. Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep, and coma. The measurement of rectal temperatures may provide supportive evidence of reflex bradypnea or hypo/hyperthermia related to treatment or confinement. Additional assessments may be included in the study protocol such as kinetics, biomonitoring, lung function, retention of poorly soluble materials that accumulate in lung tissue, and behavioural changes.

Individual animal weights should be recorded shortly before the first exposure (day 0), twice weekly thereafter (for example: on Fridays and Mondays to demonstrate recovery over an exposure-free weekend or at a time interval to allow assessment of systemic toxicity), and at the time of death or euthanasia. If there are no effects in the first 2 weeks, body weights may be measured weekly for the remainder of the study. Satellite (reversibility) animals (if used) should continue to be weighed weekly throughout the recovery period. At study termination, all animals should be weighed shortly before sacrifice to allow for an unbiased calculated of organ to body weight ratios.

Food consumption should be measured weekly. Water consumption may also be measured.

Clinical pathology assessments should be made for all animals, including control and satellite (reversibility) animals, when they are sacrificed. The time interval between the end of exposure and blood collection should be recorded, particularly when the reconstitution of the addressed endpoint is rapid. Sampling following the end of exposure is indicated for those parameters with a short plasma half-time (e.g. COHb, CHE, and MetHb).

Table 1 lists the clinical pathology parameters that are generally required for all toxicology studies. Urinalysis is not required on a routine basis, but may be performed when deemed useful based on expected or observed toxicity. The study director may choose to assess additional parameters in order to better characterise a test chemical’s toxicity (e.g. cholinesterase, lipids, hormones, acid/base balance, methaemoglobin or Heinz bodies, creatine kinase, myeloid/erythroid ratio, troponins, arterial blood gases, lactate dehydrogenase, sorbitol dehydrogenase, glutamate dehydrogenase, and gamma glutamyl transpeptidase).

Table 1

Standard Clinical Pathology Parameters

When there is evidence that the lower respiratory tract (i.e., the alveoli) is the primary site of deposition and retention, then bronchoalveolar lavage (BAL) may be the technique of choice to quantitatively analyse hypothesis-based dose-effect parameters focusing on alveolitis, pulmonary inflammation, and phospholipidosis. This allows for dose-response and time-course changes of alveolar injury to be suitably probed. The BAL fluid may be analysed for total and differential leukocyte counts, total protein, and lactate dehydrogenase. Other parameters that may be considered are those indicative of lysosomal injury, phospholipidosis, fibrosis, and irritant or allergic inflammation which may include the determination of pro-inflammatory cytokines/chemokines. BAL measurements generally complement the results from histopathology examinations but cannot replace them. Guidance on how to perform lung lavage can be found in GD 39 (2).

All test animals, including those which die during the test or are removed from the study for animal welfare reasons, should be subjected to complete exsanguination (if feasible) and gross necropsy. The time between the end of each animal’s last exposure and their sacrifice should be recorded. If a necropsy cannot be performed immediately after a dead animal is discovered, the animal should be refrigerated (not frozen) at a temperature low enough to minimise 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.

Table 2 lists the organs and tissues that should be preserved in a suitable medium during gross necropsy for histopathological examination. The preservation of the [bracketed] organs and tissues and any other organs and tissues is at the discretion of the study director. The bolded organs should be trimmed and weighed wet as soon as possible after dissection to avoid drying. The thyroid and epididymides should only be weighed if needed because trimming artefacts may hinder histopathological evaluation. Tissues and organs should be fixed in 10 % buffered formalin or another suitable fixative as soon as necropsy is performed, and no less than 24-48 hours prior to trimming depending on the fixative to be used.

Table 2

Organs and Tissues Preserved During Gross Necropsy

Adrenals

Bone marrow (and/or fresh aspirate)

Brain (including sections of cerebrum, cerebellum, and medulla/pons)

[Eyes (retina, optic nerve) and eyelids]

Heart

Kidneys

Larynx (3 levels, 1 level to include the base of the epiglottis)

Liver

Lung (all lobes at one level, including main bronchi)

Lymph nodes from the hilar region of the lung, especially for poorly soluble particulate test chemicals, For more in depth examinations and/or studies with immunological focus, additional lymph nodes may be considered, e.g. those from the mediastinal, cervical/submandibular and/or auricular regions.

Nasopharyngeal tissues (at least 4 levels; 1 level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue(NALT)

Oesophagus

[Olfactory bulb]

Ovaries

Seminal vesicles

Spinal cord (cervical, mid-thoracic, and lumbar)

Spleen

Stomach

Testes

Thymus

Thyroid

Trachea (at least 2 levels including 1 longitudinal section through the carina and 1 transverse section)

[Urinary bladder]

Uterus

All gross lesions

The lungs should be removed intact, weighed, and instilled with a suitable fixative at a pressure of 20-30 cm of water to ensure that lung structure is maintained (5). Sections should be collected for all lobes at one level, including main bronchi, but if lung lavage is performed, the unlavaged lobe should be sectioned at three levels (not serial sections).

At least 4 levels of the nasopharyngeal tissues should be examined, one of which should include the nasopharyngeal duct, (5, 6, 7, 8, 9) to allow adequate examination of the squamous, transitional (non-ciliated respiratory), respiratory (ciliated respiratory) and olfactory epithelium, and the draining lymphatic tissue (NALT; 10, 11). Three levels of the larynx should be examined, and one of these levels should include the base of the epiglottis (12). At least two levels of the trachea should be examined including one longitudinal section through the carina of the bifurcation of the extrapulmonary bronchi and one transverse section.

A histopathological evaluation of all the organs and tissues listed in Table 2 should be performed for the control and high concentration groups, and for all animals which die or are sacrificed during the study. Particular attention should be paid to the respiratory tract, target organs, and gross lesions. The organs and tissues that have lesions in the high concentration group should be examined in all groups. The study director may choose to perform histopathological evaluations for additional groups to demonstrate a clear concentration response. When a satellite (reversibility) group is used, histopathological evaluation should be performed for all tissues and organs identified as showing effects in the treated groups. If there are excessive early deaths or other problems in the high exposure group that compromise the significance of the data, the next lower concentration should be examined histopathologically. An attempt should be made to correlate gross observations with microscopic findings.

Individual animal data on body weights, food consumption, clinical pathology, gross pathology, organ weights, and histopathology should be provided. Clinical observation data should be summarised 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. All results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used and the statistical methods should be selected during the design of the study.

The test report should include the following information, as appropriate:

OECD (1981). Subchronic Inhalation Toxicity Testing, Original Test Guideline No 412, Environment Directorate, OECD, Paris.

OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing, Environmental Health and Safety Monograph Series on Testing and Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris.

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, ENV/JM/MONO(2000)7, OECD, Paris.

Whalan JE and Redden JC (1994). Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity Studies. Office of Pesticide Programs, United States Environmental Protection Agency.

Dungworth DL, Tyler WS, Plopper CE (1985). Morphological Methods for Gross and Microscopic Pathology (Chapter 9) in Toxicology of Inhaled Material, Witschi, H.P. and Brain, J.D. (eds), Springer Verlag Heidelberg, pp. 229-258.

Young JT (1981). Histopathological examination of the rat nasal cavity. Fundam. Appl. Toxicol. 1: 309-312.

Harkema JR (1990). Comparative pathology of the nasal mucosa in laboratory animals exposed to inhaled irritants. Environ. Health Perspect. 85: 231-238.

Woutersen RA, Garderen-Hoetmer A, van Slootweg PJ, Feron VJ (1994). Upper respiratory tract carcinogenesis in experimental animals and in humans. In: Waalkes MP and Ward JM (eds) Carcinogenesis. Target Organ Toxicology Series, Raven Press, New York, 215-263.

Mery S, Gross EA, Joyner DR, Godo M, Morgan KT (1994). Nasal diagrams: A tool for recording the distribution of nasal lesions in rats and mice. Toxicol. Pathol. 22: 353-372.

Kuper CF, Koornstra PJ, Hameleers DMH, Biewenga J, Spit BJ, Duijvestijn AM, Breda Vriesman van PJC, Sminia T (1992). The role of nasopharyngeal lymphoid tissue. Immunol. Today 13: 219-224.

Kuper CF, Arts JHE, Feron VJ (2003). Toxicity to nasal-associated lymphoid tissue. Toxicol. Lett. 140-141: 281-285.

Lewis DJ (1981). Mitotic Indices of Rat Laryngeal Epithelia. Journal of Anatomy 132(3): 419-428.

Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 (OJ L 353, 31.12.2008, p. 1).

This Test Method is equivalent to OECD Test Guideline 413 (2009). The original subchronic inhalation Test Guideline 413 (TG 413) was adopted in 1981 (1). This Test Method B.29 (as equivalent to the revised TG 413 (2009)) has been updated to reflect the state of the science and to meet current and future regulatory needs.

Subchronic inhalation toxicity studies are primarily used to derive regulatory concentrations for assessing worker risk in occupation settings. They are also used to assess human residential, transportation, and environmental risk. This method enables the characterisation of adverse effects following repeated daily inhalation exposure to a test chemical for 90 days (approximately 10 % of the lifespan of a rat). The data derived from subchronic inhalation toxicity studies can be used for quantitative risk assessments and for the selection of concentrations for chronic studies. This test method is not specifically intended for the testing of nanomaterials. Definitions used in the context of this Test Method are provided at the end of this chapter and in the Guidance Document (GD) 39 (2).

All available information on the test chemical should be considered by the testing laboratory prior to conducting the study in order to enhance the quality of the study and minimise animal usage. Information that will assist in the selection of appropriate test concentrations might include the identity, chemical structure, and physico-chemical properties of the test chemical; results of any in vitro or in vivo toxicity tests; anticipated use(s) and potential for human exposure; available (Q)SAR data and toxicological data on structurally related chemicals; and data derived from other repeated exposure studies. If neurotoxicity is expected or is observed in the course of the study, the study director may choose to include appropriate evaluations such as a functional observational battery (FOB) and measurement of motor activity. Although the timing of exposures relative to specific examinations may be critical, the performance of these additional activities should not interfere with the basic study design.

Dilutions of corrosive or irritating test chemicals may be tested at concentrations that will yield the desired degree of toxicity. Please refer to GD 39 (2) for further information. When exposing animals to these materials, the targeted concentrations should be low enough to not cause marked pain and distress, 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, preferably based upon an adequately designed range-finding study that provides information regarding the critical endpoint, any irritation threshold, and the time of onset (see paragraphs 11-13). The justification for concentration selection should be provided.

Moribund animals or animals obviously in pain or showing signs of severe and enduring distress should be humanely killed. Moribund animals are considered in the same way as animals that die 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 on Humane Endpoints (3).

Healthy young adult rodents of commonly used laboratory strains should be employed. The preferred species is the rat. Justification should be provided if other species are used.

Females should be nulliparous and non-pregnant. On the day of randomisation, animals should be young adults 7 to 9 weeks of age. Body weights should be within ± 20 % of the mean weight for each sex. The animals are randomly selected, marked for individual identification, and 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 be individually identified, preferably with subcutaneous transponders, to facilitate observations and avoid confusion. 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 minimise 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 immobilisation 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.

The nature of the test chemical and the object 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 addressed in GD 39 (2).

Unlike with acute studies, there are no defined limit concentrations in subchronic inhalation toxicity studies. The maximum concentration tested should consider: 1) the maximum attainable concentration, 2) the “worst case” human exposure level, 3) the need to maintain an adequate oxygen supply, and/or 4) animal welfare considerations. In the absence of data-based limits, the acute limits of Regulation (EC) No 1272/2008 (13) may be used (i.e. up to a maximum concentration of 5 mg/l for aerosols, 20 mg/l for vapours, and 20 000 ppm for gases); refer to GD 39 (2). Justification should be provided if it is necessary to exceed these limits when testing gases or highly volatile test chemicals (e.g. refrigerants). The limit concentration should elicit unequivocal toxicity without causing undue stress to the animals or affecting their longevity (3).

Before commencing with the main study, it is generally necessary to perform a range-finding study. A range-finding study is more comprehensive than a sighting study because it is not limited to concentration selection. Knowledge learned from a range-finding study can lead to a successful main study. A range-finding study may, for example, provide technical information regarding analytical methods, particle sizing, discovery of toxic mechanisms, clinical pathology and histopathological data, and estimations of what may be NOAEL and MTC concentrations in a main study. The study director may choose to use the range-finding study to identify the threshold of respiratory tract irritation (e.g. with histopathology of the respiratory tract, pulmonary function testing, or bronchoalveolar lavage), the upper concentration which is tolerated without undue stress to the animals, and the parameters that will best characterise a test chemical’s toxicity.

A range-finding study may consist of one or more concentration levels. Depending on the endpoints chosen, three to six males and three to six females should be exposed at each concentration level. A range-finding study should last a minimum of 5 days and generally no more than 28 days. The rationale for the selection of concentrations for the main study should be provided in the study report. The objective of the main study is to demonstrate a concentration-response relationship based on what is anticipated to be the most sensitive endpoint. The low concentration should ideally be a no-observed-adverse effect concentration while the high concentration should elicit unequivocal toxicity without causing undue stress to the animals or affecting their longevity (3).

When selecting concentration levels for the range-finding study, all available information should be considered including structure-activity relationships and data for similar chemicals (see paragraph 3). A range-finding study may verify/refute what are considered to be the most sensitive mechanistically based endpoints, e.g. cholinesterase inhibition by organophosphates, methaemoglobin formation by erythrocytotoxic agents, thyroidal hormones (T3, T4) for thyrotoxicants, protein, LDH, or neutrophils in bronchoalveolar lavage for innocuous poorly soluble particles or pulmonary irritant aerosols.

The main subchronic toxicity study generally consists of three concentration levels, and also concurrent negative (air) and/or vehicle controls as needed (see paragraph 18). All available data should be utilised to aid selection of appropriate exposure levels, including the results of systemic toxicity studies, metabolism and kinetics (particular emphasis should be given to avoiding high concentration levels which saturate kinetic processes). Each test group contains 10 male and 10 female rodents that are exposed to the test chemical for 6 hours per day on a 5 day per week basis for a period of 13 weeks (total study duration of at least 90 days). Animals may also be exposed 7 days per week (e.g. when testing inhaled pharmaceuticals). If one sex is known to be more susceptible to a given test chemical, the sexes may be exposed at different concentration levels in order to optimise the concentration-response as described in paragraph 15. If rodent species other than rats are exposed nose-only, maximum exposure durations may be adjusted to minimise species-specific distress. A rationale should be provided when using an exposure duration less than 6 hours/day, or when it is necessary to conduct a long duration (e.g. 22 hours/day) whole-body exposure study (refer to GD 39) (2). Feed should be withheld during the exposure period unless exposure exceeds 6 hours. Water may be provided throughout a whole-body exposure.

The target concentrations selected should identify the target organ(s) and demonstrate a clear concentration-response:

If interim sacrifices are planned, the number of animals at each exposure level should be increased by the number to be sacrificed before study completion. The rationale for using interim sacrifices should be provided, and statistical analyses should properly account for them.

A satellite (reversibility) study may be used to observe reversibility, persistence, or delayed occurrence of toxicity for a post-treatment period of an appropriate length, but no less than 14 days. Satellite (reversibility) groups consist of 10 males and 10 females exposed contemporaneously with the experimental animals in the main study. Satellite (reversibility) study groups should be exposed to the test chemical at the highest concentration level and there should be concurrent air and/or vehicle controls as needed (see paragraph 18).

Concurrent negative (air) control animals should be handled in a manner identical to the test group animals except that they are exposed to filtered air rather than test chemical. When water or another substance is used to assist in generating the test atmosphere, a vehicle control group, instead of a negative (air) control group, should be included in the study. Water should be used as the vehicle whenever possible. When water is used as the vehicle, the control animals should be exposed to air with the same relative humidity as the exposed groups. The selection of a suitable vehicle should be based on an appropriately conducted pre-study or historical data. If a vehicle’s toxicity is not well known, the study director may choose to use both a negative (air) control and a vehicle control, but this is strongly discouraged. If historical data reveal that a vehicle is non-toxic, then there is no need for a negative (air) control group and only a vehicle control should be used. If a pre-study of a test chemical formulated in a vehicle reveals no toxicity, it follows that the vehicle is non-toxic at the concentration tested and this vehicle control should be used.

Animals are exposed to the test chemical as a gas, vapour, aerosol, or a mixture thereof. The physical state to be tested depends on the physico-chemical properties of the test chemical, the selected concentrations, and/or the physical form most likely present during the handling and use of the test chemical. Hygroscopic and chemically reactive test chemicals should be tested under dry air conditions. Care should be taken to avoid generating explosive concentrations. Particulate materials may be subjected to mechanical processes to decrease the particle size. Further guidance is provided in GD 39 (2).

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 3 μm with a geometric standard deviation (σg) in the range of 1,5 to 3,0 are recommended (4). Although a reasonable effort should be made to meet this standard, expert judgement should be provided if it cannot be achieved. For example, metal fume particles will be smaller than this standard, and charged particles and fibres may exceed it.

Ideally, the test chemical should be tested without a vehicle. If it is necessary to use a vehicle to generate an appropriate test chemical concentration and particle size, water should be given preference. Whenever a test chemical is dissolved in a vehicle, its stability should be demonstrated.

The flow of air through the exposure chamber should be carefully controlled, continuously monitored, and recorded at least hourly during each exposure. The real-time monitoring of the test atmosphere concentration (or temporal stability) is an integral measurement of all dynamic parameters and provides an indirect means to control all relevant dynamic inhalation parameters. If the concentration is monitored real-time, the frequency of measurement of air flows may be reduced to one single measurement per exposure per day. Special consideration should be given to avoiding rebreathing in nose-only chambers. Oxygen concentration should be at least 19 % and carbon dioxide concentration should not exceed 1 %. If there is reason to believe that this standard cannot be met, oxygen and carbon dioxide concentrations should be measured. If measurements on the first day of exposure show that these gases are at proper levels, no further measurements should be necessary.

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 continuously and recorded hourly during each exposure where possible. The relative humidity should preferably be maintained in the range of 30 to 70 %, but this may either be unattainable (e.g. when testing water based mixtures) or not measurable due to test chemical interference with the Test Method.

Whenever feasible, the nominal exposure chamber concentration should be calculated and recorded. The nominal concentration is the mass of generated test chemical divided by the total volume of air passed through the inhalation chamber system. The nominal concentration is not used to characterise 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.

The actual concentration is the test chemical concentration as sampled at the animals’ breathing zone in an inhalation chamber. Actual concentrations can be obtained either by specific methods (e.g. direct sampling, adsorptive or chemical reactive methods, and subsequent analytical characterisation) or by non-specific 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 chemical-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 chemical (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.

One lot of the test chemical should be used throughout the duration of the study, 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 characterisation of the test chemical, 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 characterisation at least in a limited way (e.g. colour, physical nature, etc.).

The exposure atmosphere should be held as constant as practicable. A real-time monitoring device, such as an aerosol photometer for aerosols or a total hydrocarbon analyser for vapours, may be used to demonstrate the stability of the exposure conditions. Actual chamber concentration should be measured at least 3 times during each exposure day for each exposure level. If not feasible due to limited air flow rates or low concentrations, one sample per exposure period is acceptable. Ideally, this sample should then be collected over the entire exposure period. Individual chamber concentration samples should deviate from the mean chamber concentration by no more than ± 10 % for gases and vapours, and by no more than ± 20 % for liquid or solid aerosols. Time to attain chamber equilibration (t95) should be calculated and reported. The duration of an exposure spans the time that the test chemical is generated. This takes into account the times required to attain chamber equilibration (t95) and decay. Guidance for estimating t95 can be found in GD 39 (2).

For very complex mixtures consisting of gases/vapours and aerosols (e.g. combustion atmospheres and test chemicals propelled from purpose-driven end-use products/devices), each phase may behave differently in an inhalation chamber. Therefore, at least one indicator substance (analyte), normally the principal active ingredient in the mixture, of each phase (gas/vapour and aerosol) should be selected. When the test chemical is a mixture, the analytical concentration should be reported for the total mixture, and not just for the active ingredient or the indicator substance (analyte). Additional information regarding actual concentrations can be found in GD 39 (2).

The particle size distribution of aerosols should be determined at least weekly for each concentration level by using a cascade impactor or an alternative instrument such as an aerodynamic particle sizer (APS). If equivalence of the results obtained by a cascade impactor and the 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 at all concentrations tested in the early phase of the study, then further confirmatory measurements may be omitted. For the sake of animal welfare, measures should be taken to minimise inconclusive data which may lead to a need to repeat a study.

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.

The animals should be clinically observed before, during, and after the exposure period. More frequent observations may be indicated depending on the response of the animals during exposure. When animal observation is hindered by the use of animal restraint tubes, poorly lit whole body chambers, or opaque atmospheres, animals should be carefully observed after exposure. Observations before the next day’s exposure can assess any reversibility or exacerbation of toxic effects.

All observations are recorded with individual records being maintained for each animal. When animals are killed for humane reasons or found dead, the time of death should be recorded as precisely as possible.

Cage-side observations should include changes in the skin and fur, eyes, and mucous membranes; changes in the respiratory and circulatory systems; changes in the nervous system; and changes in somatomotor activity and behaviour patterns. Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep, and coma. The measurement of rectal temperatures may provide supportive evidence of reflex bradypnea or hypo/hyperthermia related to treatment or confinement. Additional assessments may be included in the study protocol such as kinetics, biomonitoring, lung function, retention of poorly soluble materials that accumulate in lung tissue, and behavioural changes.

Individual animal weights should be recorded shortly before the first exposure (day 0), twice weekly thereafter (for example: on Fridays and Mondays to demonstrate recovery over an exposure-free weekend, or at a time interval to allow assessment of systemic toxicity), and at the time of death or euthanasia. If there are no effects in the first 4 weeks, body weights may be measured weekly for the remainder of the study. Satellite (reversibility) animals (if used) should continue to be weighed weekly throughout the recovery period. At study termination, all animals should be weighed shortly before sacrifice to allow for an unbiased calculated of organ to body weight ratios.

Food consumption should be measured weekly. Water consumption may also be measured.

Clinical pathology assessments should be made for all animals, including controls and satellite (reversibility) animals, when they are sacrificed. The time interval between the end of exposure and blood collection should be recorded, particularly when the reconstitution of the addressed endpoint is rapid. Sampling following the end of exposure is indicated for those parameters with a short plasma half-time (e.g. COHb, CHE, and MetHb).

Table 1 lists the clinical pathology parameters that are generally required for all toxicology studies. Urinalysis is not required on a routine basis, but may be performed when deemed useful based on expected or observed toxicity. The study director may choose to assess additional parameters in order to better characterise a test chemical’s toxicity (e.g. cholinesterase, lipids, hormones, acid/base balance, methaemoglobin or Heinz bodies, creatine kinase, myeloid/erythroid ratio, troponins, arterial blood gases, lactate dehydrogenase, sorbital dehydrogenase, glutamate dehydrogenase, and gamma glutamyl transpeptidase).

Table 1

Standard Clinical Pathology Parameters

When there is evidence that the lower respiratory tract (i.e. the alveoli) is the primary site of deposition and retention, then bronchoalveolar lavage (BAL) may be the technique of choice to quantitatively analyse hypothesis-based dose-effect parameters focusing on alveolitis, pulmonary inflammation, and phospholipidosis. This allows for dose-response and time-course changes of alveolar injury to be suitably probed. The BAL fluid may be analysed for total and differential leukocyte counts, total protein, and lactate dehydrogenase. Other parameters that may be considered are those indicative of lysosomal injury, phospholipidosis, fibrosis, and irritant or allergic inflammation which may include the determination of pro-inflammatory cytokines/chemokines. BAL measurements generally complement the results from histopathology examinations but cannot replace them. Guidance on how to perform lung lavage can be found in GD 39 (2).

Using an ophthalmoscope or an equivalent device, ophthalmological examinations of the fundus, refractive media, iris, and conjunctivae should be performed for all animals prior to the administration of the test chemical, and for all high concentration and control groups at termination. If changes in the eyes are detected, all animals in the other groups should be examined including the satellite (reversibility) group.

All test animals, including those which die during the test or are removed from the study for animal welfare reasons, should be subjected to complete exsanguination (if feasible) and gross necropsy. The time between the end of each animal’s last exposure and its sacrifice should be recorded. If a necropsy cannot be performed immediately after a dead animal is discovered, the animal should be refrigerated (not frozen) at a temperature low enough to minimise 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.

Table 2 lists the organs and tissues that should be preserved in a suitable medium during gross necropsy for histopathological examination. The preservation of the [bracketed] organs and tissues and any other organs and tissues is at the discretion of the study director. The bolded organs should be trimmed and weighed wet as soon as possible after dissection to avoid drying. The thyroid and epididymides should only be weighed if needed because trimming artefacts may hinder histopathological evaluation. Tissues and organs should be fixed in 10 % buffered formalin or another suitable fixative as soon as necropsy is performed, and no less than 24-48 hours prior to trimming depending on the fixative to be used.

Table 2

Organs and Tissues Preserved During Gross Necropsy

Adrenals

Aorta

Bone marrow (and/or fresh aspirate)

Brain (including sections of cerebrum, cerebellum, and medulla/pons)

Caecum

Colon

Duodenum

[Epididymides]

[Eyes (retina, optic nerve) and eyelids]

Femur and stifle joint

Gallbladder (where present)

[Harderian glands]

Heart

Ileum

Jejunum

Kidneys

[Lacrimal glands (extraorbital)]

Larynx (3 levels including the base of the epiglottis)

Liver

Lung (all lobes at one level, including main bronchi)

Lymph nodes from the hilar region of the lung, especially for poorly soluble particulate test chemicals. For more in depth examinations and/or studies with immunological focus, additional lymph nodes may be considered, e.g. those from the mediastinal, cervical/submandibular and/or auricular regions.

Lymph nodes (distal from the portal-of-entry)

Mammary gland (female)

Muscle (thigh)

Nasopharyngeal tissues (at least 4 levels; 1 level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT))

Oesophagus

[Olfactory bulb]

Ovaries

Pancreas

Parathyroids

Peripheral nerve (sciatic or tibial, preferably close to muscle)

Pituitary

Prostate

Rectum

Salivary glands

Seminal vesicles

Skin

Spinal cord (cervical, mid-thoracic, and lumbar)

Spleen

Sternum

Stomach

Teeth

Testes

Thymus

Thyroids

[Tongue]

Trachea (at least 2 levels including 1 longitudinal section through the carina and 1 transverse section)

[Ureter]

[Urethra]

Urinary bladder

Uterus

Target organs

All gross lesions and masses

The lungs should be removed intact, weighed, and instilled with a suitable fixative at a pressure of 20-30 cm of water to ensure that lung structure is maintained (5). Sections should be collected for all lobes at one level, including main bronchi, but if lung lavage is performed, the unlavaged lobe should be sectioned at three levels (not serial sections).

At least 4 levels of the nasopharyngeal tissues should be examined, one of which should include the nasopharyngeal duct (5) (6) (7) (8) (9) to allow adequate examination of the squamous, transitional (non-ciliated respiratory), respiratory (ciliated respiratory) and olfactory epithelium, and the draining lymphatic tissue (NALT) (10) (11). Three levels of the larynx should be examined, and one of these levels should include the base of the epiglottis (12). At least two levels of the trachea should be examined including one longitudinal section through the carina of the bifurcation of the extrapulmonary bronchi and one transverse section.

A histopathological evaluation of all the organs and tissues listed in Table 2 should be performed for the control and high concentration groups, and for all animals which die or are sacrificed during the study. Particular attention should be paid to the respiratory tract, target organs, and gross lesions. The organs and tissues that have lesions in the high concentration group should be examined in all groups. The study director may choose to perform histopathological evaluations for additional groups to demonstrate a clear concentration response. When a satellite (reversibility) group is used, histopathological evaluation should be performed for all tissues and organs identified as showing effects in the treated groups. If there are excessive early deaths or other problems in the high exposure group that compromise the significance of the data, the next lower concentration should be examined histopathologically. An attempt should be made to correlate gross observations with microscopic findings.

Individual animal data on body weights, food consumption, clinical pathology, gross pathology, organ weights, and histopathology should be provided. Clinical observation data should be summarised 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. All results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used and the statistical methods should be selected during the design of the study.

The test report should include the following information, as appropriate:

OECD (1981). Subchronic Inhalation Toxicity Testing, Original Test Guideline No 413, Environment Directorate, OECD, Paris.

OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing, Environmental Health and Safety Monograph Series on Testing and Assessment No 39, ENV/JM/MONO(2009)28, OECD, Paris.

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, ENV/JM/MONO(2000)7, OECD, Paris.

Whalan.E and Redden JC (1994). Interim Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity Studies. Office of Pesticide Programs, United States Environmental Protection Agency.

Dungworth DL, Tyler WS, Plopper CE (1985). Morphological Methods for Gross and Microscopic Pathology (Chapter 9) in Toxicology of Inhaled Material, Witschi, H.P. and Brain, J.D. (eds), Springer Verlag Heidelberg, pp. 229-258.

Young JT (1981). Histopathological examination of the rat nasal cavity. Fundam. Appl. Toxicol. 1: 309-312.

Harkema JR (1990). Comparative pathology of the nasal mucosa in laboratory animals exposed to inhaled irritants. Environ. Health Perspect. 85: 231-238.

Woutersen RA, Garderen-Hoetmer A, van Slootweg PJ, Feron VJ (1994). Upper respiratory tract carcinogenesis in experimental animals and in humans. In: Waalkes MP and Ward JM (eds) Carcinogenesis. Target Organ Toxicology Series, Raven Press, New York, 215-263.

Mery S, Gross EA, Joyner DR, Godo M, Morgan KT (1994). Nasal diagrams: A tool for recording the distribution of nasal lesions in rats and mice. Toxicol. Pathol. 22: 353-372.

Kuper CF, Koornstra PJ, Hameleers DMH, Biewenga J, Spit BJ, Duijvestijn AM, Breda Vriesman van PJC, Sminia T (1992). The role of nasopharyngeal lymphoid tissue. Immunol. Today 13: 219-224.

Kuper CF, Arts JHE, Feron VJ (2003). Toxicity to nasal-associated lymphoid tissue. Toxicol. Lett. 140-141: 281-285.

Lewis DJ (1981). Mitotic Indices of Rat Laryngeal Epithelia. Journal of Anatomy 132(3): 419-428.

Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 (OJ L 353, 31.12.2008, p. 1).

This Test Method is equivalent to OECD Test Guideline (TG) 452 (2009). The original TG 452 was adopted in 1981. Development of this revised Test Method B.30 was considered necessary in order to reflect recent developments in the field of animal welfare and regulatory requirements (1) (2) (3) (4). The updating of this Test Method B.30 has been carried out in parallel with revisions of Chapter B.32 of this Annex, Carcinogenicity Studies, and Chapter B.33 of this Annex, Combined Chronic Toxicity/Carcinogenicity studies, with the objective of obtaining additional information from the animals used in the study and providing further detail on dose selection. This Test Method is designed to be used in the testing of a broad range of chemicals, including pesticides and industrial chemicals.

The majority of chronic toxicity studies are carried out in rodent species, and this Test Method is intended therefore to apply primarily to studies carried out in these species. Should such studies be required in non-rodent species, the principles and procedures outlined in this Test Method, together with those outlined in Chapter B.27 of this Annex, Repeated Dose 90-day Oral Toxicity Study in Non-Rodents (5), may also be applied, with appropriate modifications, as outlined in the OECD Guidance Document No 116 on the Design and Conduct of Chronic Toxicity and Carcinogenicity Studies (6).

The three main routes of administration used in chronic toxicity studies are oral, dermal and inhalation. The choice of the route of administration depends on the physical and chemical characteristics of the test chemical and the predominant route of exposure of humans. Additional information on choice of route of exposure is provided in the OECD Guidance Document No 116 (6).

This Test Method focuses on exposure via the oral route, the route most commonly used in chronic toxicity studies. While long–term chronic toxicity studies involving exposure via the dermal or inhalation routes may also be necessary for human health risk assessment and/or may be required under certain regulatory regimes, both routes of exposure involve considerable technical complexity. Such studies will need to be designed on a case-by-case basis, although the Test Method outlined here for the assessment and evaluation of chronic toxicity by oral administration could form the basis of a protocol for inhalation and/or dermal studies, with respect to recommendations for treatment periods, clinical and pathology parameters, etc. OECD Guidance is available on the administration of test chemicals by the inhalation (6) (7) and dermal routes (6). Chapter B.8 of this Annex (8) and Chapter B.29 of this Annex (9), together with the OECD Guidance Document on acute inhalation testing (7), should be specifically consulted in the design of longer term studies involving exposure via the inhalation route. Chapter B.9 of this Annex (10) should be consulted in the case of testing carried out by the dermal route.

The chronic toxicity study provides information on the possible health hazards likely to arise from repeated exposure over a considerable part of the lifespan of the species used. The study will provide information on the toxic effects of the test chemical; indicate target organs and the possibility of accumulation. It can also provide an estimate of the no-observed-adverse effect level which can be used for establishing safety criteria for human exposure. The need for careful clinical observations of the animals, so as to obtain as much information as possible, is also stressed.

The objectives of studies covered by this Test Method include:

In the assessment and evaluation of the toxicological characteristics of a test chemical, all available information on the test chemical should be considered by the testing laboratory prior to conducting the study, in order to focus the design of the study to more efficiently test for chronic toxicity potential and to minimize animal usage. Information that will assist in the study design includes the identity, chemical structure, and physico-chemical properties of the test chemical; any information on the mode of action; results of any in vitro or in vivo toxicity tests; anticipated use(s) and potential for human exposure; available (Q)SAR data and toxicological data on structurally-related chemicals; available toxicokinetic data (single dose and also repeat dose kinetics where available) and data derived from other repeated exposure studies. The determination of chronic toxicity should only be carried out after initial information on toxicity has been obtained from repeated dose 28-day and/or 90-day toxicity tests. A phased testing approach to chronic toxicity testing should be considered as part of the overall assessment of the potential adverse health effects of a particular test chemical (11) (12) (13) (14).

The statistical methods most appropriate for the analysis of results, given the experimental design and objectives, should be established before commencing the study. Issues to consider include whether the statistics should include adjustment for survival and analysis in the event of premature termination of one or more groups. Guidance on the appropriate statistical analyses and key references to internationally accepted statistical methods are given in Guidance Document No 116 (6), and also in Guidance Document No 35 on the analysis and evaluation of chronic toxicity and carcinogenicity studies (15).

In conducting a chronic toxicity study, the guiding principles and considerations outlined in the OECD Guidance Document No 19 on the recognition, assessment, and use of clinical signs as humane endpoints for experimental animals used in safety evaluation (16), in particular paragraph 62 thereof, should always be followed. This paragraph states that “In studies involving repeated dosing, when an animal shows clinical signs that are progressive, leading to further deterioration in condition, an informed decision as to whether or not to humanely kill the animal should be made. The decision should include consideration as to the value of the information to be gained from the continued maintenance of that animal on study relative to its overall condition. If a decision is made to leave the animal on test, the frequency of observations should be increased, as needed. It may also be possible, without adversely affecting the purpose of the test, to temporarily stop dosing if it will relieve the pain or distress, or reduce the test dose.”

Detailed guidance on and discussion of the principles of dose selection for chronic toxicity and carcinogenicity studies can be found in Guidance Document No 116 (6), as well as two International Life Sciences Institute publications (17) (18). The core dose selection strategy is dependent on the primary objective or objectives of the study (paragraph 6). In selecting appropriate dose levels, a balance should be achieved between hazard screening on the one hand and characterisation of low-dose responses and their relevance on the other. This is particularly relevant in the situation where a combined chronic toxicity and carcinogenicity study (Chapter B.33 of this Annex) is to be carried out (paragraph 11).

Consideration should be given to carrying out a combined chronic toxicity and carcinogenicity study (Chapter B.33 of this Annex), rather than separate execution of a chronic toxicity study (this Test Method B.30) and carcinogenicity study (Chapter B.32 of this Annex). The combined test provides greater efficiency in terms of time and cost compared to conducting two separate studies, without compromising the quality of the data in either the chronic phase or the carcinogenicity phase. Careful consideration should however be given to the principles of dose selection (paragraphs 9 and 20-25) when undertaking a combined chronic toxicity and carcinogenicity study (Chapter B.33 of this Annex), and it is also recognised that separate studies may be required under certain regulatory frameworks.

Definitions used in the context of this Test Method can be found at the end of this chapter and in the Guidance Document No 116 (6).

The test chemical is administered daily in graduated doses to several groups of experimental animals, normally for a period of 12 months, although longer or shorter durations may also be chosen depending on regulatory requirements (see paragraph 33). This duration is chosen to be sufficiently long to allow any effects of cumulative toxicity to become manifest, without the confounding effects of geriatric changes. Deviations from exposure duration of 12 months should be justified, particularly in the case of shorter durations. The test chemical is normally administered by the oral route although testing by the inhalation or dermal route may also be appropriate. The study design may also include one or more interim kills, e.g. at 3 and 6 months, and additional groups of animals may be included to accommodate this (see paragraph 19). During the period of administration the animals are observed closely for signs of toxicity. Animals which die or are killed during the test are necropsied and, at the conclusion of the test, surviving animals are killed and necropsied.

This Test Method primarily covers assessment and evaluation of chronic toxicity in rodents (see paragraph 2) although it is recognised that similar studies in non-rodents may be required under certain regulatory regimes. The choice of species should be justified. The design and conduct of chronic toxicity studies in non-rodent species, when required, should be based on the principles outlined in this Test Method together with those in Chapter B.27 of this Annex, Repeated Dose 90-day Oral Toxicity Study in Non-Rodents (5). Additional information on choice of species and strain is provided in Guidance Document No 116 (6).

In this Test Method, the preferred rodent species is the rat, although other rodent species, e.g. the mouse, may be used. Rats and mice have been preferred experimental models because of their relatively short life span, their widespread use in pharmacological and toxicological studies, their susceptibility to tumour induction, and the availability of sufficiently characterised strains. As a consequence of these characteristics, a large amount of information is available on their physiology and pathology. Young healthy adult animals of commonly used laboratory strains should be employed. The chronic toxicity study should be carried out in animals from the same strain and source as those used in preliminary toxicity study(ies) of shorter duration. The females should be nulliparous and non-pregnant.

Animals may be housed individually, or be caged in small groups of the same sex; individual housing should be considered only if scientifically justified (19) (20) (21). Cages should be arranged in such a way that possible effects due to cage placement are minimised. The temperature in the experimental animal room should be 22 °C (± 3 °C). Although the relative humidity should be at least 30 % and preferably not exceed 70 % other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The diet should meet all the nutritional requirements of the species tested and the content of dietary contaminants including but not limited to pesticide residues, persistent organic pollutants, phytoestrogens, heavy metals and mycotoxins, that might influence the outcome of the test, should be as low as possible. Analytical information on the nutrient and dietary contaminant levels should be generated periodically, at least at the beginning of the study and when there is a change in the batch used, and should be included in the final report. Analytical information on the drinking water used in the study should similarly be provided. The choice of diet may be influenced by the need to ensure a suitable admixture of a test chemical and to meet the nutritional requirements of the animals when the test chemical is administered by the dietary route.

Healthy animals, which have been acclimated to laboratory conditions for at least 7 days and have not been subjected to previous experimental procedures, should be used. In the case of rodents, dosing of the animals should begin as soon as possible after weaning and acclimatisation and preferably before the animals are 8 weeks old. The test animals should be characterised as to species, strain, source, sex, weight and age. At the commencement of the study, the weight variation for each sex of animals used should be minimal and not exceed ± 20 % of the mean weight of all the animals within the study, separately for each sex. Animals should be randomly assigned to the control and treatment groups. After randomisation, there should be no significant differences in mean body weights between groups within each sex. If there are statistically significant differences, then the randomisation step should be repeated, if possible. Each animal should be assigned a unique identification number, and permanently marked with this number by tattooing, microchip implant, or other suitable method.

Both sexes should be used. A sufficient number of animals should be used so that at the end of the study enough animals in every group are available for thorough biological and statistical evaluation. For rodents, at least 20 animals per sex per group should normally be used at each dose level, while for non-rodents a minimum of 4 per sex per group is recommended. In studies involving mice, additional animals may be needed in each dose group to conduct all required haematological determinations.

The study may make provision for interim kills (at least 10 animals/sex/group), e.g. at 6 months, to provide information on progression of toxicological changes and mechanistic information, if scientifically justified. Where such information is already available from previous repeat dose toxicity studies on the test chemical, interim kills may not be scientifically justified. Satellite groups may also be included to monitor the reversibility of any toxicological changes induced by the test chemical under investigation; these will normally be restricted to the highest dose level of the study plus control. An additional group of sentinel animals (typically 5 animals per sex) may also be included for monitoring of disease status, if necessary, during the study (22). If interim kills or inclusion of satellite or sentinel groups are planned, the number of animals included in the study design should be increased by the number of animals scheduled to be killed before the completion of the study. These animals should normally undergo the same observations, including body weight, food/water consumption, haematological and clinical biochemistry measurements and pathological investigations as the animals in the chronic toxicity phase of the main study, although provision may also be made (in the interim kill groups) for measurements to be restricted to specific, key measures such as neurotoxicity or immunotoxicity.

Guidance on all aspects of dose selection and dose level spacing is provided in Guidance Document No 116 (6). At least three dose levels and a concurrent control should be used, except where a limit test is conducted (see paragraph 27). Dose levels will generally be based on the results of shorter-term repeated dose or range finding studies and should take into account any existing toxicological and toxicokinetic data available for the test chemical or related chemicals.

Unless limited by the physical-chemical nature or biological effects of the test chemical, the highest dose level should normally be chosen to identify the principal target organs and toxic effects while avoiding suffering, severe toxicity, morbidity, or death. While taking into account the factors outlined in paragraph 22 below, the highest dose level should be chosen to elicit evidence of toxicity, as evidenced by, for example, depression of body weight gain (approximately 10 %).

However, dependent on the objectives of the study (see paragraph 6), a top dose lower than the dose providing evidence of toxicity may be chosen, e.g. if a dose elicits an adverse effect of concern that nonetheless has little impact on lifespan or body weight. The top dose should not exceed 1 000 mg/kg body weight/day (limit dose, see paragraph 27).

Dose levels and dose level spacing may be selected to establish a dose-response and a NOAEL or other intended outcome of the study, e.g. a BMD (see paragraph 25) at the lowest dose level. Factors that should be considered in the placement of lower doses include the expected slope of the dose–response curve, the doses at which important changes may occur in metabolism or mode of toxic action, where a threshold is expected, or where a point of departure for low-dose extrapolation is expected.

The dose level spacing selected will depend on the characteristics of the test chemical, and cannot be prescribed in this Test Method, but two to four fold intervals frequently provide good test performance when used for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of about 6-10) between dosages. In general the use of factors greater than 10 should be avoided, and should be justified if used.

As outlined further in Guidance Document No 116 (6), points to be considered in dose selection include:

The control group shall be an untreated group or a vehicle-control group if a vehicle is used in administering the test chemical. Except for treatment with the test chemical, animals in the control group should be handled in an identical manner to those in the test groups. If a vehicle is used, the control group shall receive the vehicle in the highest volume used among the dose groups. If a test chemical is administered in the diet, and causes significantly reduced dietary intake due to the reduced palatability of the diet, an additional pair-fed control group may be useful, to serve as a more suitable control.

If it can be anticipated, based on information from preliminary studies, that a test at one dose level, equivalent to at least 1 000 mg/kg body weight/day, using the procedures described for this study, is unlikely to produce adverse effects and if toxicity would not be expected based upon data from structurally related chemicals, then a full study using three dose levels may not be considered necessary. A limit of 1 000 mg/kg body weight/day may apply except when human exposure indicates the need for a higher dose level to be used.

The test chemical is normally administered orally, via the diet or drinking water, or by gavage. Additional information on routes and methods of administration is provided in Guidance Document No 116 (6). The route and method of administration is dependent on the purpose of the study, the physical/chemical properties of the test chemical, its bioavailability and the predominant route and method of exposure of humans. A rationale should be provided for the chosen route and method of administration. In the interests of animal welfare, oral gavage should normally be selected only for those agents for which this route and method of administration reasonably represent potential human exposure (e.g. pharmaceuticals). For dietary or environmental chemicals including pesticides, administration is typically via the diet or drinking water. However, for some scenarios, e.g. occupational exposure, administration via other routes may be more appropriate.

Where necessary, the test chemical is dissolved or suspended in a suitable vehicle. Consideration should be given to the following characteristics of the vehicle and other additives, as appropriate: effects on the absorption, distribution, metabolism, or retention of the test chemical; effects on the chemical properties of the test chemical which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water, the toxic characteristics of the vehicle should be known. Information should be available on the stability of the test chemical and the homogeneity of dosing solutions or diets (as appropriate) under the conditions of administration (e.g. diet).

For chemicals administered via the diet or drinking water it is important to ensure that the quantities of the test chemical involved do not interfere with normal nutrition or water balance. In long-term toxicity studies using dietary administration, the concentration of the test chemical in the feed should not normally exceed an upper limit of 5 % of the total diet, in order to avoid nutritional imbalances. When the test chemical is administered in the diet, either a constant dietary concentration (mg/kg diet or ppm) or a constant dose level in terms of the animal’s body weight (mg/kg body weight), calculated on a weekly basis, may be used. The alternative used should be specified.

In the case of oral administration, the animals are dosed with the test chemical daily (seven days each week), normally for a period of 12 months (see also paragraph 33), although a longer duration may be required depending on regulatory requirements. Any other dosing regime, e.g. five days per week, needs to be justified. In the case of dermal administration, animals are normally treated with the test chemical for at least 6 hours per day, 7 days per week, as specified in Chapter B.9 of this Annex (10), for a period of 12 months. Exposure by the inhalation route is carried out for 6 hours per day, 7 days per week, but exposure for 5 days per week may also be used, if justified. The period of exposure will normally be for a period of 12 months. If rodent species other than rats are exposed nose-only, maximum exposure durations may be adjusted to minimise species-specific distress. A rationale should be provided when using an exposure duration of less than 6 hours per day. See also Chapter B.8 of this Annex (8).

When the test chemical is administered by gavage to the animals this should be done using a stomach tube or a suitable intubation cannula, at similar times each day. Normally a single dose will be administered once daily, where for example a chemical is a local irritant, it may be possible to maintain the daily dose-rate by administering it as a split dose (twice a day). The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume should be kept as low as practical, and should not normally exceed 1 ml/100 g body weight for rodents (22). Variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels. Potentially corrosive or irritant chemicals are the exception, and need to be diluted to avoid severe local effects. Testing at concentrations that are likely to be corrosive or irritant to the gastrointestinal tract should be avoided.

While this Test Method primarily is designed as a 12 month chronic toxicity study, the study design also allows for and can be applied to either shorter (e.g. 6 or 9 months) or longer (e.g. 18 or 24 months) duration studies, depending on the requirements of particular regulatory regimes or for specific mechanistic purposes. Deviations from an exposure duration of 12 months should be justified, particularly in the case of shorter durations. Satellite groups included to monitor the reversibility of any toxicological changes induced by the test chemical under investigation should be maintained without dosing for a period not less than 4 weeks and not more than one third of the total study duration after cessation of exposure. Further guidance, including consideration of survival in the study, is provided in Guidance Document No 116 (6).

All animals should be checked for morbidity or mortality, usually at the beginning and end of each day, including at weekends and holidays. General clinical observations should be made at least once a day, preferably at the same time(s) each day, taking into consideration the peak period of anticipated effects after dosing in the case of gavage administration.