(5)
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Chapters B.29 and B.30 are replaced by the following:
‘B.29. SUBCHRONIC INHALATION TOXICITY: 90-DAY STUDY
SUMMARY
This revised Test Method B.29 has been designed to fully characterise test chemical toxicity by the inhalation route for a subchronic duration (90 days), and to provide robust data for quantitative inhalation risk assessments. Groups of 10 male and 10 female rodents are exposed 6 hours per day during a 90 day (13 week) period to a) the test chemical at three or more concentration levels, b) filtered air (negative control), and/or c) the vehicle (vehicle control). Animals are generally exposed 5 days per week but exposure for 7 days per week is also allowed. Males and females are always tested, but they may be exposed at different concentration levels if it is known that one sex is more susceptible to a given test chemical. This method allows the study director the flexibility to include satellite (reversibility) groups, interim sacrifices, bronchoalveolar lavage (BAL), neurologic tests, and additional clinical pathology and histopathological evaluations in order to better characterise the toxicity of a test chemical.
INTRODUCTION
1.
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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.
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2.
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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).
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INITIAL CONSIDERATIONS
3.
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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.
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4.
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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.
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5.
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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).
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DESCRIPTION OF THE METHOD
Selection of Animal Species
6.
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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.
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Preparation of Animals
7.
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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.
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Animal Husbandry
8.
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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.
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Inhalation Chambers
9.
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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).
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TOXICITY STUDIES
Limit Concentrations
10.
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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).
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Range-Finding Study
11.
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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.
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12.
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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).
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13.
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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.
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Main Study
14.
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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.
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15.
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The target concentrations selected should identify the target organ(s) and demonstrate a clear concentration-response:
—
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The high concentration level should result in toxic effects but not cause lingering signs or lethality which would prevent a meaningful evaluation.
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—
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The intermediate concentration level(s) should be spaced to produce a gradation of toxic effects between that of the low and high concentration.
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—
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The low concentration level should produce little or no evidence of toxicity.
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Interim Sacrifices
16.
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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.
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Satellite (Reversibility) Study
17.
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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).
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Control Animals
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.
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EXPOSURE CONDITIONS
Administration of Concentrations
19.
|
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).
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Particle-Size Distribution
20.
|
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.
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Test chemical Preparation in a Vehicle
21.
|
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.
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MONITORING OF EXPOSURE CONDITIONS
Chamber Airflow
22.
|
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.
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Chamber Temperature and Relative Humidity
23.
|
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.
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Test chemical: Nominal Concentration
24.
|
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.
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Test chemical: Actual Concentration
25.
|
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.
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26.
|
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.).
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27.
|
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).
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28.
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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).
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Test chemical: Particle Size Distribution
29.
|
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.
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30.
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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.
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31.
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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.
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OBSERVATIONS
32.
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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.
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33.
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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.
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34.
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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.
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BODY WEIGHTS
35.
|
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.
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FOOD AND WATER CONSUMPTION
36.
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Food consumption should be measured weekly. Water consumption may also be measured.
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CLINICAL PATHOLOGY
37.
|
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).
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38.
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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
Haematology
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Erythrocyte count
Haematocrit
Haemoglobin concentration
Mean corpuscular haemoglobin
Mean corpuscular volume
Mean corpuscular haemoglobin concentration
Reticulocytes
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Total leukocyte count
Differential leukocyte count
Platelet count
Clotting potential (select one):
—
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Partial thromboplastin time
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Clinical Chemistry
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Glucose (9)
Total cholesterol
Triglycerides
Blood urea nitrogen
Total bilirubin
Creatinine
Total protein
Albumin
Globulin
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Alanine aminotransferase
Aspartate aminotransferase
Alkaline phosphatase
Potassium
Sodium
Calcium
Phosphorus
Chloride
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Urinalysis (optional)
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Appearance (colour and turbidity)
Volume
Specific gravity or osmolality
pH
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Total protein
Glucose
Blood/blood cells
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39.
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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).
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OPHTHALMOLOGICAL EXAMINATION
40.
|
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.
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GROSS PATHOLOGY AND ORGAN WEIGHTS
41.
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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.
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42.
|
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
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43.
|
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).
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44.
|
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.
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HISTOPATHOLOGY
45.
|
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.
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DATA AND REPORTING
Data
46.
|
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.
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Test Report
47.
|
The test report should include the following information, as appropriate:
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Test animals and husbandry
—
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Description of caging conditions, including: number (or change in number) of animals per cage, bedding material, ambient temperature and relative humidity, photoperiod, and identification of diet.
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—
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Species/strain used and justification for using a species other than the rat. Source and historical data may be provided, if they are for animals exposed under similar exposure, housing, and fasting conditions.
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—
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Number, age, and sex of animals.
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—
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Method of randomisation.
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—
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Description of any pre-test conditioning including diet, quarantine, and treatment for disease.
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Test chemical
—
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Physical nature, purity, and, where relevant, physico-chemical properties (including isomerisation).
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—
|
Identification data and Chemical Abstract Services (CAS) Registry Number, if known.
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|
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Vehicle
—
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Justification for use of vehicle and justification for choice of vehicle (if other than water).
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—
|
Historical or concurrent data demonstrating that the vehicle does not interfere with the outcome of the study.
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|
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Inhalation chamber
—
|
Detailed description of the inhalation chamber including volume and a diagram.
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—
|
Source and description of equipment used for the exposure of animals as well as generation of atmosphere.
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—
|
Equipment for measuring temperature, humidity, particle-size, and actual concentration.
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—
|
Source of air and system used for conditioning.
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—
|
Methods used for calibration of equipment to ensure a homogeneous test atmosphere.
|
—
|
Pressure difference (positive or negative).
|
—
|
Exposure ports per chamber (nose-only); location of animals in the chamber (whole-body).
|
—
|
Stability of the test atmosphere.
|
—
|
Location of temperature and humidity sensors and sampling of test atmosphere in the chamber.
|
—
|
Treatment of air supplied/extracted.
|
—
|
Air flow rates, air flow rate/exposure port (nose-only), or animal load/chamber (whole-body).
|
—
|
Time to inhalation chamber equilibrium (t95).
|
—
|
Number of volume changes per hour.
|
—
|
Metering devices (if applicable).
|
|
|
Exposure data
—
|
Rationale for target concentration selection in the main study.
|
—
|
Nominal concentrations (total mass of test chemical generated into the inhalation chamber divided by the volume of air passed through the chamber).
|
—
|
Actual test chemical concentrations collected from the animals’ breathing zone; for mixtures that produce heterogeneous physical forms (gases, vapours, aerosols), each may be analysed separately.
|
—
|
All air concentrations should be reported in units of mass (mg/l, mg/m3, etc.) rather than in units of volume (ppm, ppb, etc.).
|
—
|
Particle size distribution, mass median aerodynamic diameter (MMAD), and geometric standard deviation (σg), including their methods of calculation. Individual particle size analyses should be reported.
|
|
|
Test conditions
—
|
Details of test chemical preparation, including details of any procedures used to reduce the particle size of solid materials or to prepare solutions of the test chemical.
|
—
|
A description (preferably including a diagram) of the equipment used to generate the test atmosphere and to expose the animals to the test atmosphere.
|
—
|
Details of the equipment used to monitor chamber temperature, humidity, and chamber airflow (i.e. development of a calibration curve).
|
—
|
Details of the equipment used to collect samples for determination of chamber concentration and particle size distribution.
|
—
|
Details of the chemical analytical method used and method validation (including efficiency of recovery of test chemical from the sampling medium).
|
—
|
Method of randomisation in assigning animals to test and control groups.
|
—
|
Details of food and water quality (including diet type/source, water source).
|
—
|
The rationale for the selection of test concentrations.
|
|
|
Results
—
|
Tabulation of chamber temperature, humidity, and airflow.
|
—
|
Tabulation of chamber nominal and actual concentration data.
|
—
|
Tabulation of particle size data including analytical sample collection data, particle size distribution, and calculations of the MMAD and σg.
|
—
|
Tabulation of response data and concentration level for each animal (i.e. animals showing signs of toxicity including mortality, nature, severity, time of onset, and duration of effects).
|
—
|
Tabulation of individual animal weights.
|
—
|
Tabulation of food consumption
|
—
|
Tabulation of clinical pathology data
|
—
|
Necropsy findings and histopathological findings for each animal, if available.
|
|
|
Discussion and interpretation of results
—
|
Particular emphasis should be made to the description of methods used to meet the criteria of this Test Method, e.g. the limit concentration or the particle size.
|
—
|
The respirability of particles in light of the overall findings should be addressed, especially if the particle-size criteria could not be met.
|
—
|
The consistency of methods used to determine nominal and actual concentrations, and the relation of actual concentration to nominal concentration should be included in the overall assessment of the study.
|
—
|
The likely cause of death and predominant mode of action (systemic versus local) should be addressed.
|
—
|
An explanation should be provided if there was a need to humanely sacrifice animals in pain or showing signs of severe and enduring distress, based on the criteria in the OECD Guidance Document on Humane Endpoints (3).
|
—
|
The target organ(s) should be identified.
|
—
|
The NOAEL and LOAEL should be determined.
|
|
|
LITERATURE:
(1)
|
OECD (1981). Subchronic Inhalation Toxicity Testing, Original Test Guideline No 413, Environment Directorate, OECD, Paris.
|
(2)
|
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.
|
(3)
|
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.
|
(4)
|
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.
|
(5)
|
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.
|
(6)
|
Young JT (1981). Histopathological examination of the rat nasal cavity. Fundam. Appl. Toxicol. 1: 309-312.
|
(7)
|
Harkema JR (1990). Comparative pathology of the nasal mucosa in laboratory animals exposed to inhaled irritants. Environ. Health Perspect. 85: 231-238.
|
(8)
|
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.
|
(9)
|
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.
|
(10)
|
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.
|
(11)
|
Kuper CF, Arts JHE, Feron VJ (2003). Toxicity to nasal-associated lymphoid tissue. Toxicol. Lett. 140-141: 281-285.
|
(12)
|
Lewis DJ (1981). Mitotic Indices of Rat Laryngeal Epithelia. Journal of Anatomy 132(3): 419-428.
|
(13)
|
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).
|
Appendix 1
DEFINITION
Test chemical: Any substance or mixture tested using this Test Method.
B.30. CHRONIC TOXICITY STUDIES
INTRODUCTION
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.
|
2.
|
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).
|
3.
|
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).
|
4.
|
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.
|
5.
|
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.
|
6.
|
The objectives of studies covered by this Test Method include:
—
|
The identification of the chronic toxicity of a test chemical;
|
—
|
The identification of target organs;
|
—
|
Characterisation of the dose-response relationship;
|
—
|
Identification of a no-observed-adverse-effect level (NOAEL) or point of departure for establishment of a Benchmark Dose (BMD);
|
—
|
The prediction of chronic toxicity effects at human exposure levels;
|
—
|
Provision of data to test hypotheses regarding mode of action (6).
|
|
INITIAL CONSIDERATIONS
7.
|
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).
|
8.
|
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).
|
9.
|
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.”
|
10.
|
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).
|
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.
|
12.
|
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).
|
PRINCIPLE OF THE TEST
13.
|
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.
|
DESCRIPTION OF THE METHOD
Selection of animal species
14.
|
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).
|
15.
|
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.
|
Housing and feeding conditions
16.
|
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.
|
Preparation of animals
17.
|
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.
|
PROCEDURE
Number and sex of animals
18.
|
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.
|
Provision for interim kills, satellite groups and sentinel animals
19.
|
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.
|
Dose groups and dosage
20.
|
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.
|
21.
|
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 %).
|
22.
|
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).
|
23.
|
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.
|
24.
|
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.
|
25.
|
As outlined further in Guidance Document No 116 (6), points to be considered in dose selection include:
—
|
Known or suspected nonlinearities or inflection points in the dose–response;
|
—
|
Toxicokinetics, and dose ranges where metabolic induction, saturation, or nonlinearity between external and internal doses does or does not occur;
|
—
|
Precursor lesions, markers of effect, or indicators of the operation of key underlying biological processes;
|
—
|
Key (or suspected) aspects of mode of action, such as doses at which cytotoxicity begins to arise, hormone levels are perturbed, homeostatic mechanisms are overwhelmed, etc.;
|
—
|
Regions of the dose–response curve where particularly robust estimation is needed, e.g. in the range of the anticipated BMD or a suspected threshold;
|
—
|
Consideration of anticipated human exposure levels.
|
|
26.
|
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.
|
27.
|
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.
|
Preparation of doses and administration of test chemical
28.
|
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.
|
29.
|
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).
|
30.
|
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.
|
31.
|
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).
|
32.
|
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.
|
Duration of study
33.
|
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).
|
OBSERVATIONS
34.
|
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.
|
35.
|
Detailed clinical observations should be made on all animals at least once prior to the first exposure (to allow for within-subject comparisons), at the end of the first week of the study and monthly thereafter. The protocol for observations should be arranged such that variations between individual observers are minimised and independent of test group. These observations should be made outside the home cage, preferably in a standard arena and at similar times on each occasion. They should be carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Efforts should be made to ensure that variations in the observation conditions are minimal. 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, and 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 (24).
|
36.
|
Ophthalmological examination, using an ophthalmoscope or other suitable equipment, should be carried out on all animals prior to the first administration of the test chemical. At the termination of the study, this examination should be preferably conducted in all animals but at least in the high dose and control groups. If treatment-related changes in the eyes are detected, all animals should be examined. If structural analysis or other information suggests ocular toxicity, then the frequency of ocular examination should be increased.
|
37.
|
For chemicals where previous repeated dose 28-day and/or 90-day toxicity tests indicated the potential to cause neurotoxic effects, sensory reactivity to stimuli of different types (24) (e.g. auditory, visual and proprioceptive stimuli) (25), (26), (27), assessment of grip strength (28) and motor activity assessment (29) may optionally be conducted before commencement of the study and at 3 month periods after study initiation up to and including 12 months, as well as at study termination (if longer than 12 months). Further details of the procedures that could be followed are given in the respective references. However, alternative procedures than those referenced could also be used.
|
38.
|
For chemicals where previous repeated dose 28-day and/or 90-day toxicity tests indicated the potential to cause immunotoxic effects, further investigations of this endpoint may optionally be conducted at termination.
|
Body weight, food/water consumption and food efficiency
39.
|
All animals should be weighed at the start of treatment, at least once a week for the first 13 weeks, and at least monthly thereafter. Measurements of food consumption and food efficiency should be made at least weekly for the first 13 weeks and at least monthly thereafter. Water consumption should be measured at least weekly for the first 13 weeks and at least monthly thereafter when the chemical is administered in drinking water. Water consumption measurements should also be considered for studies in which drinking activity is altered.
|
Haematology and clinical biochemistry
40.
|
In studies involving rodents, haematological examinations should be carried out in at least 10 male and 10 female animals per group, at 3, 6, and 12 months, as well as at study termination (if longer than 12 months), using the same animals throughout. In mice, satellite animals may be needed in order to conduct all required haematological determinations (see paragraph 18). In non-rodent studies, samples will be taken from smaller numbers of animals (e.g. 4 animals per sex and per group in dog studies), at interim sampling times and at termination as described for rodents. Measurements at 3 months, either in rodents or non-rodents, need not be conducted if no effect was seen on haematological parameters in a previous 90 day study carried out at comparable dose levels. Blood samples should be taken from a named site, for example by cardiac puncture or from the retro-orbital sinus, under anaesthesia.
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41.
|
The following list of parameters should be investigated (30): Total and differential leukocyte count, erythrocyte count, platelet count, haemoglobin concentration, haematocrit (packed cell volume), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), prothrombin time, and activated partial thromboplastin time. Other hematology parameters such as Heinz bodies or other atypical erythrocyte morphology or methaemoglobin may be measured as appropriate depending on the toxicity of the test chemical. Overall, a flexible approach should be adopted, depending on the observed and/or expected effect from a given test chemical. If the test chemical has an effect on the haematopoietic system, reticulocyte counts and bone marrow cytology may also be indicated, although these need not be routinely conducted.
|
42.
|
Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained from at least 10 male and 10 female animals per group at the same time intervals as specified for the haematological investigations, using the same animals throughout. In mice, satellite animals may be needed in order to conduct all required clinical biochemistry determinations. In non-rodent studies, samples will be taken from smaller numbers of animals (e.g. 4 animals per sex and per group in dog studies), at interim sampling times and at termination as described for rodents. Measurements at 3 months, either in rodents or non-rodents, need not be conducted if no effect was seen on clinical biochemistry parameters in a previous 90 day study carried out at comparable dose levels. Overnight fasting of the animals (with the exception of mice) prior to blood sampling is recommended The following list of parameters should be investigated (30): glucose, urea (urea nitrogen), creatinine, total protein, albumin, calcium, sodium, potassium, total cholesterol, at least two appropriate tests for hepatocellular evaluation (alanine aminotransferase, aspartate aminotransferase, glutamate dehydrogenase, total bile acids) (31), and at least two appropriate tests for hepatobiliary evaluation (alkaline phosphatase, gamma glutamyl transferase, 5’-nucleotidase, total bilirubin, total bile acids) (31). Other clinical chemistry parameters such as fasting triglycerides, specific hormones and cholinesterase may be measured as appropriate, depending on the toxicity of the test chemical. Overall, there is a need for a flexible approach, depending on the observed and/or expected effect from a given test chemical.
|
43.
|
Urinalysis determinations should be performed on at least 10 male and 10 female animals per group on samples collected at the same intervals as for haematology and clinical chemistry. Measurements at 3 months need not be conducted if no effect was seen on urinalysis in a previous 90 day study carried out at comparable dose levels. The following list of parameters was included in an expert recommendation on clinical pathology studies (30): appearance, volume, osmolality or specific gravity, pH, total protein, and glucose. Other determinations include ketone, urobilinogen, bilirubin, and occult blood. Further parameters may be employed where necessary to extend the investigation of observed effect(s).
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44.
|
It is generally considered that baseline haematological and clinical biochemistry variables are needed before treatment for dog studies, but need not be determined in rodent studies (30). However, if historical baseline data (see paragraph 50) are inadequate, consideration should be given to generating such data.
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Pathology
Gross necropsy
45.
|
All animals in the study shall normally 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. However provision may also be made (in the interim kill or satellite groups) for measurements to be restricted to specific, key measures such as neurotoxicity or immunotoxicity (see paragraph 19). These animals need not be subjected to necropsy and the subsequent procedures described in the following paragraphs. Sentinel animals may require necropsy on a case-by-case basis, at the discretion of the study director.
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46.
|
Organ weights should be collected from all animals, other than those excluded by the latter part of paragraph 45. The adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thyroid (weighed post-fixation, with parathyroids), and uterus of all animals (apart from those found moribund and/or intercurrently killed) should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to prevent drying. In a study using mice, weighing of the adrenal glands is optional.
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47.
|
The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination (32) (tissues in square brackets are optional):
all gross lesions
|
heart
|
pancreas
|
stomach (forestomach, glandular stomach)
|
adrenal gland
|
ileum
|
parathyroid gland
|
[teeth]
|
aorta
|
jejunum
|
peripheral nerve
|
testis
|
brain (including sections of cerebrum, cerebellum, and medulla/pons)
|
kidney
|
pituitary
|
thymus
|
caecum
|
lacrimal gland (exorbital)
|
prostate
|
thyroid
|
cervix
|
liver
|
rectum
|
[tongue]
|
coagulating gland
|
lung
|
salivary gland
|
trachea
|
colon
|
lymph nodes (both superficial and deep)
|
seminal vesicle
|
urinary bladder
|
duodenum
|
mammary gland (obligatory for females and, if visibly dissectable, from males)
|
skeletal muscle
|
uterus (including cervix)
|
epididymis
|
[upper respiratory tract, including nose, turbinates, and paranasal sinuses]
|
skin
|
[ureter]
|
eye (including retina)
|
oesophagus
|
spinal cord (at three levels: cervical, mid-thoracic, and lumbar)
|
[urethra]
|
[femur with joint]
|
[olfactory bulb]
|
spleen
|
vagina
|
gall bladder (for species other than rat)
|
ovary
|
[sternum],
|
section of bone marrow and/or a fresh bone marrow aspirate
|
Harderian gland
|
|
|
|
In the case of paired organs, e.g. kidney, adrenal, both organs should be preserved. 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. In studies involving the dermal route of administration, the list of organs as set out for the oral route should be preserved, and specific sampling and preservation of the skin from the site of application is essential. In inhalation studies, the list of preserved and examined tissues from the respiratory tract should follow the recommendations of Chapters B.8 of this Annex (8) and Chapter B.29 of this Annex (9). For other organs/tissues (and in addition to the specifically preserved tissues from the respiratory tract) the list of organs as set out for the oral route should be examined.
|
Histopathology
48.
|
Guidance is available on best practices in the conduct of toxicological pathology studies (32). The minimum histopathological examinations should be:
—
|
all tissues from the high dose and control groups;
|
—
|
all tissues from animals dying or killed during the study;
|
—
|
all tissues showing macroscopic abnormalities;
|
—
|
target tissues, or tissues which showed treatment-related changes in the high dose group, from all animals in all other dose groups;
|
—
|
in the case of paired organs, e.g. kidney, adrenal, both organs should be examined.
|
|
DATA AND REPORTING
Data
49.
|
Individual animal data should be provided for all parameters evaluated. 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 killed for humane reasons and the time of any death or humane kill, 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 and the percentage of animals displaying each type of lesion. Summary data tables should provide the means and standard deviations (for continuous test data) of animals showing toxic effects or lesions, in addition to the grading of lesions.
|
50.
|
Historical control data may be valuable in the interpretation of the results of the study, e.g. in the case when there are indications that the data provided by the concurrent controls are substantially out of line when compared to recent data from control animals from the same test facility/colony. Historical control data, if evaluated, should be submitted from the same laboratory and relate to animals of the same age and strain generated during the five years preceding the study in question.
|
51.
|
When applicable, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods and the data to be analysed should be selected during the design of the study (paragraph 8). Selection should make provision for survival adjustments, if needed.
|
Test Report
52.
|
The test report should include the following information:
|
Test chemical:
—
|
physical nature, purity, and physicochemical properties;
|
—
|
certificate of chemical analysis
|
|
|
Vehicle (if appropriate):
—
|
justification for choice of vehicle (if other than water).
|
|
|
Test animals:
—
|
species/strain used and justification for choice made;
|
—
|
number, age, and sex of animals at start of test;
|
—
|
source, housing conditions, diet, etc.;
|
—
|
individual weights of animals at the start of the test.
|
|
|
Test conditions:
—
|
rationale for route of administration and dose selection;
|
—
|
when applicable, the statistical methods used to analyse the data;
|
—
|
details of test chemical formulation/diet preparation;
|
—
|
analytical data on achieved concentration, stability and homogeneity of the preparation;
|
—
|
route of administration and details of the administration of the test chemical;
|
—
|
for inhalation studies, whether nose only or whole body;
|
—
|
actual doses (mg/kg body weight/day), and conversion factor from diet/drinking water test chemical concentration (mg/kg or ppm) to the actual dose, if applicable;
|
—
|
details of food and water quality.
|
|
|
Results (summary tabulated data and individual animal data should be presented):
—
|
body weight/body weight changes;
|
—
|
food consumption, calculations of food efficiency, if made, and water consumption if applicable;
|
—
|
toxic response data by sex and dose level, including signs of toxicity;
|
—
|
nature, incidence (and, if scored, severity), and duration of clinical observations ((whether transitory or permanent);
|
—
|
ophthalmological examination;
|
—
|
clinical biochemistry tests;
|
—
|
outcome of any investigations of neurotoxicity or immunotoxicity;
|
—
|
organ weights (and their ratios, if applicable);
|
—
|
a detailed description of all treatment-related histopathological findings;
|
—
|
absorption data if available;
|
|
|
Statistical treatment of results, as appropriate
|
|
Discussion of results including:
—
|
Dose: response relationships
|
—
|
Consideration of any mode of action information
|
—
|
Discussion of any modelling approaches
|
—
|
BMD, NOAEL or LOAEL determination
|
—
|
Historical control data
|
|
|
LITERATURE:
(1)
|
OECD (1995). Report of the Consultation Meeting on Sub-chronic and Chronic Toxicity/Carcinogenicity Testing (Rome, 1995), internal working document, Environment Directorate, OECD, Paris.
|
(2)
|
Combes RD, Gaunt I, Balls M (2004). A Scientific and Animal Welfare Assessment of the OECD Health Effects Test Guidelines for the Safety Testing of Chemicals under the European Union REACH System. ATLA 32: 163-208.
|
(3)
|
Barlow SM, Greig JB, Bridges JW et al. (2002). Hazard identification by methods of animal-based toxicology. Food. Chem. Toxicol. 40, 145-191.
|
(4)
|
Chhabra RS, Bucher JR, Wolfe M, Portier C (2003). Toxicity characterization of environmental chemicals by the US National Toxicology Programme: an overview. Int. J. Hyg. Environ. Health 206: 437-445.
|
(5)
|
Chapter B.27 of this Annex, Sub-chronic Oral Toxicity Test Repeated Dose 90-day Oral Toxicity Study in Non-Rodents.
|
|