Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

Housing and feeding conditions

• 17.Animals should generally be housed individually during the testing period. Group housing might be justified in special circumstances. Lighting should be artificial, the sequence being 12 h light/12 h dark. The temperature of the experimental animal room should be 22 °C (± 3 °C) and the relative humidity 30-70 %. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.

Test Chemical

• 18.A radiolabelled test chemical using ¹⁴C should be used for all mass balance and metabolite identification aspects of the study; however, if it can be demonstrated that:

• — mass balance and metabolite identification can be adequately evaluated using the unlabelled test chemical,

• — the analytical specificity and sensitivity of the method used with non-radioactive test chemical is equal to or greater than that which could be obtained with the radiolabelled test chemical,

• then a radiolabelled test chemical does not need to be used. Furthermore, other radioactive and stable isotopes may be used, particularly if the element is responsible for or is a part of the toxic portion of the test chemical. If possible, the radiolabel should be located in a core portion of the molecule which is metabolically stable (it is not exchangeable, is not removed metabolically as CO2, and does not become part of the one-carbon pool of the organism). Labelling of multiple sites or specific regions of the molecule may be necessary to follow the metabolic fate of the test chemical.

• 19.The radiolabelled and non-radiolabelled test chemicals should be analysed using appropriate methods to establish purity and identity. The radio-purity of the radioactive test chemical should be the highest attainable for a particular test chemical (ideally it should be greater than 95 %) and reasonable effort should be made to identify impurities present at or above 2 %. The purity, along with the identity and proportion of any impurities which have been identified, should be reported. Individual regulatory programmes may choose to provide additional guidance to assist in the definition and specifications of test chemicals composed of mixtures and methods for determination of purity.

Dose Selection

Pilot Study

• 20.Usually a single oral dose is sufficient for the pilot study. The dose should be non-toxic, but high enough to allow for metabolite identification in excreta (and plasma, if appropriate) as well as to meet the stated purpose of the pilot study as noted in paragraph 12 of this Test Method.

Main Studies

• 21.For the main studies, a minimum of two doses is preferred since information gathered from at least two dose groups may aid in dose setting in other toxicity studies, and help in the dose-response assessment of already available toxicity tests.

• 22.Where two doses are administered, both doses should be high enough to allow for metabolite identification in excreta (and plasma, if appropriate). Information from available toxicity data should be considered for dose selection. If information is not available (e.g. from acute oral toxicity studies recording clinical signs of toxicity, or from repeated dose toxicity studies) a value for the higher dose that is below the LD50 (oral and dermal routes) or LC50 (inhalation route) estimate or below the lower value of the acute toxicity range estimate may be considered. The lower dose should be some fraction of the higher dose.

• 23.If only one dose level is investigated, ideally the dose should be high enough to allow for metabolite identification in excreta (and plasma, if appropriate), while not producing apparent toxicity. A rationale should be provided as to why no second dose level has been included.

• 24.If the effect of dose on kinetic processes needs to be established, two doses may not be sufficient and at least one dose should be high enough so as to saturate these processes. If the area under the plasma concentration-time curve (AUC) is not linear between two dose levels used in the main study, this is a strong indication that saturation of one or more of the kinetic processes is occurring somewhere between the two dose levels.

• 25.For test chemicals of low toxicity, a maximum dose of 1 000 mg/kg body weight (oral and dermal routes) should be used (if administration is by the inhalation route, refer to Chapter B.2 of this Annex for guidance; typically this dose would not exceed 2 mg/l). Chemical-specific considerations may necessitate a higher dose depending on regulatory needs. Dose selection should always be justified.

• 26.Single dose toxicokinetic and tissue distribution data may be adequate to determine the potential for accumulation and/or persistence. However in some circumstances repeated dose administration may be needed (i) to address more fully the potential for accumulation and/or persistence or changes in TK (i.e. for instance, enzyme induction and inhibition), or (ii) as required by the applicable regulatory system. In studies involving repeated dosing, while repeated low dose administration is usually sufficient, under certain circumstances repeated high dose administration may also be necessary (see also paragraph 57).

Administration of Test Chemical

• 27.The test chemical should be dissolved or suspended homogeneously in the same vehicle employed for the other oral gavage toxicity studies performed with the test chemical, if such vehicle information is available. Rationale for the choice of vehicle should be provided. The choice of the vehicle and the volume of dosing should be considered in the design of the study. The customary method of administration is by gavage; however, administration by gelatine capsule or as a dietary mixture may be advantageous in specific situations (in both cases, justification should be given). Verification of the actual dose administered to each animal should be provided.

• 28.The maximum volume of liquid to be administered by oral gavage at one time depends on the size of the test animals, the type of dose vehicle, and whether or not feed is withheld prior to administration of the test chemical. The rationale for administering or restricting food prior to dosing should be provided. Normally the volume should be kept as low as practical for either aqueous or non-aqueous vehicles. Dose volumes should not normally exceed 10 ml/kg body weight for rodents. Volumes of vehicles used for more lipophilic test chemicals might start at 4 ml/kg body weight. For repeated dosing, when daily fasting would be contraindicated, lower dose volumes (e.g. 2-4 ml/kg body weight) should be considered. Where possible, consideration may be given to the use of a dose volume consistent with that administered in other oral gavage studies for a test chemical.

• 29.Intravenous (IV) administration of the test chemical and measurement of the test chemical in blood and/or excreta may be used to establish bioavailability or relative oral absorption. For the IV study, a single dose (usually equivalent to but not to exceed the lower oral dose – see dose selection) of test chemical is administered using an appropriate vehicle. This material should be administered in a suitable volume (e.g. 1 ml/kg bw) at the chosen site of administration to at least four animals of the appropriate sex (both sexes might be used, if warranted, see paragraph 16). A fully dissolved or suspended dose preparation is necessary for IV administration of the test chemical. The vehicle for IV administration should not interfere with blood integrity or blood flow. If the test chemical is infused, the infusion rate should be reported and standardised between animals, provided an infusion pump is used. Anaesthesia should be used if one cannulates the jugular vein (for administration of test chemical and/or collection of blood) or if one uses the femoral artery for administration. Due consideration should be given to the type of anaesthesia as it may have effects on toxicokinetics. Animals should be allowed to recover adequately before administration of the test chemical plus the vehicle.

• 30.Other routes of administration, such as dermal and inhalation, (see paragraphs 74-78) may be applicable for certain test chemicals, considering their physico-chemical properties and the expected human use or exposure.

Measurements

Mass Balance

• 31.Mass balance is determined by summation of the percent of the administered (radioactive) dose excreted in urine, faeces, and expired air, and the percent present in tissues, residual carcass, and cage wash (see paragraph 46). Generally, total recoveries of administered test chemical (radioactivity) in the order of > 90 % are considered to be adequate.

Absorption

• 32.An initial estimation of absorption can be achieved by excluding the percentage of dose in the gastro-intestinal (GI) tract and/or faeces from the mass balance determination. For the calculation of percent absorption, see paragraph 33. For investigation of excreta, see paragraphs 44-49. If the exact extent of absorption following oral dosing cannot be established from mass balance studies (e.g. where greater than 20 % of the administered dose is present in faeces), further investigations might be necessary. These studies could comprise either 1) oral administration of test chemical and measurement of test chemical in bile or 2) oral and IV administration of test chemical and measurement of net test chemical present in urine plus expired air plus carcass by each of the two routes. In either study design, measurement of radioactivity is conducted as a surrogate method for chemical-specific analysis of test chemical plus metabolites.

• 33.If a biliary excretion study is undertaken, the oral route of administration is typically used. In this study, the bile ducts of at least four animals of the appropriate sex (or of both sexes, if warranted) should be cannulated and a single dose of the test chemical should be administered. Following administration of the test chemical, excretion of radioactivity/test chemical in bile should be monitored as long as necessary to estimate the percentage of the administered dose that is excreted via this route, which can be used to directly calculate the extent of oral absorption, as follows:

Image omitted; see official source

• 34.With some classes of test chemical, direct secretion of the absorbed dose can occur across intestinal membranes. In such cases the measurement of % dose in faeces following an oral dose in the bile duct cannulated rat is not considered to be representative of the unabsorbed dose. It is recommended that where intestinal secretion is thought to occur then the % dose absorbed be based on the absorption calculated from a comparison of the excretion following the oral versus IV route (intact or bile duct cannulated rat) (see paragraph 35). It is also recommended that where quantification of the intestinal secretion is considered necessary, excretion in the bile duct cannulated rat following IV dose administration be measured.

Bioavailability

• 35.Bioavailability can be determined from plasma/blood kinetics of the oral and IV groups, as described in paragraphs 50-52, by specific chemical analysis of the test chemical and/or relevant metabolite(s), therefore not requiring radiolabelled test chemical. The calculation of bioavailability (F) of the test chemical or relevant metabolite(s) can then be made as follows:

Image omitted; see official source

where AUC is the area under the plasma concentration-time curve, and exp is the experimental route (oral, dermal or via inhalation).

• 36.For use in risk assessment of systemic effects, bioavailability of the toxic component is in general preferred over the percent absorption when comparing systemic concentrations from animal studies with analogous biomonitoring data from worker exposure studies. The situation may become more complex if doses are in the non-linear range so it is important that toxicokinetic screening determines doses in the linear range.

Tissue Distribution

• 37.Knowledge of tissue distribution of a test chemical and/or its metabolites is important for the identification of target tissues, and understanding of the underlying mechanisms of toxicity, and in order to get information on the potential for test chemical and metabolite accumulation and persistence. The percent of the total (radioactive) dose in tissues as well as residual carcass should at a minimum be measured at the termination of the excretion experiment (e.g. typically up to 7 days post dose or less depending on test chemical specific behaviour). When no test chemical is detected in tissues at study termination (e.g. because the test chemical might have been eliminated before study termination due to a short half-life), care should be taken in order to prevent misinterpretation of the data. In this type of situation, tissue distribution should be investigated at the time of test chemical (and/or metabolite) peak plasma/blood concentration (Tmax) or peak rate of urinary excretion, as appropriate (see paragraph 38). Furthermore, tissue collection at additional time points may be needed to determine tissue distribution of the test chemical and/or its metabolites, to evaluate time dependency (if appropriate), to aid in establishing mass balance, and/or as required by a competent authority. Tissues that should be collected include liver, fat, GI tract, kidney, spleen, whole blood, residual carcass, target organ tissues and any other tissues (e.g. thyroid, erythrocytes, reproductive organs, skin, eye (particularly in pigmented animals) of potential significance in the toxicological evaluation of the test chemical. Analysis of additional tissues at the same time points should be considered to maximise utilisation of animals and in the event that target organ toxicity is observed in sub-chronic or chronic toxicity studies. The (radioactive) residue concentration and tissue-to-plasma (blood) ratios should also be reported.

• 38.The evaluation of tissue distribution at additional time points such as the time of peak plasma/blood concentration (e.g. Tmax) or the peak rate of urinary excretion, obtained from the respective plasma/blood kinetic or excretion experiments, may also be needed or required by a competent authority. This information can be useful for understanding toxicity and the potential for test chemical and metabolite accumulation and persistence. Justification for sample selection should be provided; samples for analysis generally should be the same as those above (see paragraph 37).

• 39.Quantification of radioactivity for tissue distribution studies can be performed using organ dissection, homogenisation, combustion and/or solubilisation, followed by liquid scintillation counting (LSC) of trapped residues. Certain techniques, currently at various stages of development, e.g. Quantitative whole-body autoradiography and receptor microscopic autoradiography, may prove useful in determining the distribution of a test chemical in organs and/or tissues (3) (4).

• 40.For routes of exposure other than oral, specific tissues should be collected and analysed, such as lungs in inhalation studies and skin in dermal studies. See paragraphs 74-78.

Metabolism

• 41.Excreta (and plasma, if appropriate) should be collected for identification and quantitation of unchanged test chemical and metabolites as described under paragraphs 44-49. Pooling of excreta to facilitate metabolite identification within a given dose group is acceptable. Profiling of metabolites from each time period is recommended. However, if lack of sample and/or radioactivity precludes this, pooling of urine and faeces across several time points is acceptable but pooling across sexes or doses is not acceptable. Appropriate qualitative and quantitative methods should be used to assay urine, faeces, expired radioactivity from treated animals, and bile if appropriate.

• 42.Reasonable efforts should be made to identify all metabolites present at 5 % or greater of the administered dose and to provide a metabolic scheme for the test chemical. Test chemicals which have been characterised in excreta as comprising 5 % or greater of the administered dose should be identified. Identification refers to the exact structural determination of components. Typically, identification is accomplished either by co-chromatography of the metabolite with known standards using two dissimilar systems or by techniques capable of positive structural identification such as mass spectrometry, nuclear magnetic resonance (NMR), etc. In the case of co-chromatography, chromatographic techniques utilising the same stationary phase with two different solvent systems are not considered to be an adequate two-method verification of metabolite identity, since the methods are not independent. Identification by co-chromatography should be obtained using two dissimilar, analytically independent systems such as reverse and normal phase thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Provided that the chromatographic separation is of suitable quality, then additional confirmation by spectroscopic means is not necessary. Alternatively, unambiguous identification can also be obtained using methods providing structural information such as: liquid chromatography/mass spectrometry (LC-MS), or liquid chromatography/tandem mass spectrometry (LC-MS/MS), gas chromatography/mass spectrometry (GC-MS), and NMR spectrometry.

• 43.If identification of metabolites at 5 % or greater of the administered dose is not possible, a justification/explanation should be provided in the final report. It might be appropriate to identify metabolites representing less than 5 % of the administered dose to gain a better understanding of the metabolic pathway for hazard and/or risk assessment of the test chemical. Structural confirmation should be provided whenever possible. This may include profiling in plasma or blood or other tissues.

Excretion

• 44.The rate and extent of excretion of the administered dose should be determined by measuring the percent recovered (radioactive) dose from urine, faeces and expired air. These data will also assist in establishing mass balance. The quantities of test chemical (radioactivity) eliminated in the urine, faeces, and expired air should be determined at appropriate time intervals (see paragraphs 47-49). Repeated dose experiments should be properly designed to allow for collection of excretion data to meet the objectives described in the paragraph 26. This will allow for comparison to single dose experiments.

• 45.If a pilot study has shown that no significant amount of test chemical (radioactivity) (according to paragraph 49) is excreted in expired air, then expired air does not need to be collected in the definitive study.

• 46.Each animal is to be placed in a separate metabolic unit for collection of excreta (urine, faeces and expired air). At the end of each collection period (see paragraphs 47-49), the metabolic units should be rinsed with appropriate solvent (this is known as the ‘cage wash’) to ensure maximum recovery of the test chemical (radioactivity). Collection of excreta should be terminated at 7 days, or after at least 90 % of the administered dose has been recovered, whichever occurs first.

• 47.The total quantities of test chemical (radioactivity) in urine are to be determined for at least two time points on day 1 of collection, one of which should be at 24 h post dosing, and daily thereafter until study termination. The selection of more than two sampling points on day one (e.g. at 6, 12 and 24 h) is encouraged. The results of pilot studies should be analysed for information on alternate or additional time points for collection. A rationale should be provided for the collection schedules.

• 48.The total quantities of test chemical (radioactivity) in faeces should be determined on a daily basis beginning at 24 h post-dosing until study termination, unless pilot studies suggest alternate or additional time points for collection. A rationale should be provided for alternative collection schedules.

• 49.The collection of expired CO2 and other volatile materials may be discontinued in a given study experiment when less than 1 % of the administered dose is found in the exhaled air during a 24-h collection period.

Time Course Studies

Plasma/Blood Kinetics

• 50.The purpose of these studies is to obtain estimates of basic TK parameters [e.g. Cmax, Tmax, half-life (t1/2), AUC] for the test chemical. These studies may be conducted at one dose or, more likely, at two or more doses. Dose setting should be determined by the nature of the experiment and/or the issue being addressed. Kinetic data may be needed to resolve issues such as test chemical bioavailability and/or to clarify the effect of dose on clearance (e.g. to clarify whether clearance is saturated in a dose-dependent fashion).

• 51.For these studies a minimum of four animals of one sex per dose group should be used. Justification should be provided for the sex of the animals used. The use of both sexes (four males and four females) should be considered if there is evidence to support significant sex-related differences in toxicity.

• 52.Following administration of the test chemical (radiolabelled), blood samples should be obtained from each animal at suitable time points using appropriate sampling methodology. The volume and number of blood samples which can be obtained per animal might be limited by potential effects of repeated sampling on animal health/physiology and/or the sensitivity of the analytical method. Samples should be analysed for each individual animal. In some circumstances (e.g. metabolite characterisation), it might be necessary to pool samples from more than one animal. Pooled samples should be clearly identified and an explanation for pooling provided. If a radiolabelled test chemical is used, analysis of total radioactivity present might be adequate. If so, total radioactivity should be analyzed in whole blood and plasma or plasma and red blood cells to allow calculation of the blood/plasma ratio. In other circumstances, more specific investigations requiring the identification of parent compound and/or metabolites, or to assess protein binding might be necessary.

Other Tissue Kinetics

• 53.The purpose of these studies is to obtain time course information to address questions related to issues such as toxic mode of action, bioaccumulation and bio-persistence via determination of levels of test chemical in various tissues. The selection of tissues and the number of time points evaluated will depend on the issue to be addressed and the toxicological database for the test chemical. The design of these additional tissue kinetics studies should take into account information gathered as described in paragraphs 37-40. These studies might involve single or repeated dosing. A detailed rationale for the approach used should be provided.

• 54.Reasons for performing other tissue kinetic studies might include:

• — Evidence of extended blood half-life, suggesting possible accumulation of test chemical in various tissues, or

• — interest in seeing if a steady state level has been achieved in specific tissues (e.g. in repeated dosing studies, even though an apparent blood steady state level of test chemical may have been achieved, there may be interest in ascertaining that a steady state level has also been attained in target tissues).

• 55.For these types of time-course studies, an appropriate oral dose of test chemical should be administered to a minimum of four animals per dose per time point and the time course of distribution monitored in selected tissues. Only one sex may be used, unless gender specific toxicity is observed. Whether total radioactivity or parent chemical and/or metabolites are analysed will also depend on the issue being addressed. Assessment of tissue distribution should be made using appropriate techniques.

Enzyme Induction/Inhibition

• 56.Studies addressing the possible effects of enzyme induction/inhibition or biotransformation of test chemical under study may be needed under one or more of the following cases:

• (1) Available evidence indicates a relationship between biotransformation of test chemical and enhanced toxicity;

• (2) The available toxicity data indicate a non-linear relationship between dose and metabolism;

• (3) The results of metabolite identification studies show identification of a potentially toxic metabolite that might have been produced by an enzyme pathway induced by the test chemical;

• (4) In explaining effects which are postulated to be linked to enzyme induction phenomena;

• (5) If toxicologically significant alterations in the metabolic profile of the test chemical are observed through either in vitro or in vivo experiments with different species or conditions, characterisation of the enzyme(s) involved may be needed (e.g. Phase I enzymes such as isoenzymes of the Cytochrome P450-dependent mono-oxygenase system, Phase II enzymes such as isoenzymes of sulfotransferase or uridine diphosphate glucuronosyl transferase, or any other relevant enzymes). This information might be used to evaluate the pertinence of species to species extrapolations.

• 57.Appropriate study protocols to evaluate test chemical related changes in TK, suitably validated and justified should be used. Example study designs consist of repeated dosing with unlabelled test chemical, followed by a single radiolabelled dose on day 14, or repeated dosing with radiolabelled test chemical and sampling at days 1, 7 and 14 for determination of metabolite profiles. Repeated dosing with radiolabelled test chemical may also provide information on bioaccumulation (see paragraph 26).

SUPPLEMENTAL APPROACHES

• 58.Supplemental approaches beyond the in vivo experiments described in this Test Method may provide useful information on the absorption, distribution, metabolism or elimination of a test chemical in certain species.

Use of in vitro information

• 59.Several questions concerning the metabolism of the test chemical may be addressed in in vitro studies using appropriate test systems. Freshly isolated or cultured hepatocytes and subcellular fractions (e.g. microsomes and cytosol or S9 fraction) from liver may be used to study possible metabolites. Local metabolism in the target organ, e.g. lung, may be of interest for risk assessment. For these purposes, microsomal fractions of target tissues may be useful. Studies with microsomes may be useful to address potential gender and life-stage differences and characterise enzyme parameters (Km and Vmax) which can aid in the assessment of dose dependency of metabolism in relation to exposure levels. In addition microsomes may be useful to identify the specific microsomal enzymes involved in the metabolism of the test chemical which can be relevant in species extrapolation (see also paragraph 56). The potential for induction of biotransformation can also be examined by using liver subcellular fractions (e.g. microsomes and cytosol) of animals pre-treated with the test chemical of interest, in vitro via hepatocyte induction studies or from specific cell lines expressing relevant enzymes. In certain circumstances and under appropriate conditions, subcellular fractions coming from human tissues might be considered for use in determining potential species differences in biotransformation. The results from in vitro investigations may also have utility in the development of PBTK models (5).

• 1. In vitro dermal absorption studies may provide supplemental information to characterise absorption (6).

• 61.Primary cell cultures from liver cells and fresh tissue slices may be used to address similar questions as with liver microsomes. In certain cases, it may be possible to answer specific questions using cell lines with defined expression of the relevant enzyme or engineered cell lines. In certain cases, it may be useful to study the inhibition and induction of specific cytochrome P450 isozymes (e.g. CYP1A1, 2E1, 1A2, and others) and/or phase II enzymes by the parent compound using in vitro studies. Information obtained may have utility for similarly structured compounds.

Use of Toxicokinetic Data from Toxicity Studies as Complementary Information

• 62.Analysis of blood, tissue and/or excreta samples obtained during the conduct of any other toxicity studies can provide data on bioavailability, changes in plasma concentration in time (AUC, Cmax), bioaccumulation potential, clearance rates, and gender or life-stage changes in metabolism and kinetics.

• 63.Consideration of the study design can be used to answer questions relating to: saturation of absorption, biotransformation or excretion pathways at higher dose levels; the operation of new metabolic pathways at higher doses and the limitation of toxic metabolites to higher doses.

• 64.Other hazard assessment considerations could include issues such as:

• — Age-related sensitivity due to differences in the status of the blood-brain barrier, the kidney and/or detoxification capacities;

• — Sub-population sensitivity due to differences in biotransformation capacities or other TK differences;

• — Extent of exposure of the foetus by transplacental transfer of chemicals or of the newborn through lactation.

Use of Toxicokinetic Modelling

• 65.Toxicokinetic models may have utility for various aspects of hazard and risk assessment as for example in the prediction of systemic exposure and internal tissue dose. Furthermore specific questions on mode of action may be addressed, and these models can provide a basis for extrapolation across species, routes of exposure, dosing patterns, and for human risk assessment. Data useful for developing PBTK models for a test chemical in any given species include (1) partition coefficients, (2) biochemical constants and physiological parameters, (3) route-specific absorption parameters and 4) in vivo kinetic data for model evaluation [e.g. clearance parameters for relevant (> 10 %) excretion pathways, Km and Vmax for metabolism]. The experimental data used in model development should be generated with scientifically sound methods and the model results validated. Test chemical- and species-specific parameters such as absorption rates, blood-tissue partitioning and metabolic rate constants are often determined to facilitate development of non-compartmental or physiologically-based models (7).

DATA AND REPORTING

• 66.It is recommended that the study report include a table of contents.

Body of the Report

• 67.The body of the report should include information covered by this Test Method organised into sections and paragraphs as follows:

Summary

• 68.This section of the study report should include a summary of the study design and a description of methods used. It should also highlight the key findings regarding mass balance, the nature and magnitude of metabolites, tissue residue, rate of clearance, bioaccumulation potential, sex differences, etc. The summary should be presented in sufficient detail to permit evaluation of the findings.

Introduction

• 69.This section of the report should include the study objectives, rationale and design, as well as, appropriate references and any background history.

Materials and Methods

• 70.This section of the report should include detailed descriptions of all pertinent information including:

• (a) Test Chemical This subsection should include identification of the test chemical: chemical name, molecular structure, qualitative and quantitative determination of its chemical composition, chemical purity and whenever possible, type and quantities of any impurities. It should also include information on physical/chemical properties including physical state, colour, gross solubility and/or partition coefficient, stability, and if appropriate, corrosivity. If applicable, information on isomers should be provided. If the test chemical is radiolabelled, information on the following should be included in this subsection: the type of radionuclide, position of label, specific activity, and radiochemical purity. The type or description of any vehicle, diluents, suspending agents, and emulsifiers or other materials used in administering the test chemical should be stated.

• (b) Test Animals This subsection should include information on the test animals, including selection and justification for species, strain, and age at study initiation, sex as well as body weight, health status, and animal husbandry.

• (c) Methods This subsection should include details of the study design and methodology used. It should include a description of:
• (1) Justification for any modification of route of exposure and exposure conditions, if applicable;
• (2) Justification for selection of dose levels;
• (3) Description of pilot studies used in the experimental design of the follow-up studies, if applicable. Pilot study supporting data should be submitted;
• (4) How the dosing solution was prepared and the type of solvent or vehicle, if any, used;
• (5) Number of treatment groups and number of animals per group;
• (6) Dosage levels and volume (and specific activity of the dose when radioactivity is used);
• (7) Route(s) and methods of administration;
• (8) Frequency of dosing;
• (9) Fasting period (if used);
• (10) Total radioactivity per animal;
• (11) Animal handling;
• (12) Sample collection and handling;
• (13) Analytical methods used for separation, quantitation and identification of metabolites;
• (14) Limit of detection for the employed methods;
• (15) Other experimental measurements and procedures employed (including validation of methods for metabolite analysis).

• (d) Statistical Analysis If statistical analysis is used to analyse the study findings, then sufficient information on the method of analysis and the computer program employed should be included, so that an independent reviewer/statistician can re-evaluate and reconstruct the analysis. In the case of systems modelling studies such as PBTK, presentation of models should include a full description of the model to allow independent reconstruction and validation of the model (see paragraph 65 and Appendix: Definitions).

Results

• 71.All data should be summarised and tabulated with appropriate statistical evaluation and described in the text of this section. Radioactivity counting data should be summarised and presented as appropriate for the study, typically as microgram or milligram equivalents per mass of sample, although other units may be used. This section should include graphic illustrations of the findings, reproduction of representative chromatographic and spectrometric data, metabolite identification/quantification and proposed metabolic pathways including molecular structure of metabolites. In addition the following information is to be included in this section, if applicable:

• (1) Quantity and percent recovery of radioactivity in urine, faeces, expired air, and urine and faeces cage wash.
• — For dermal studies, also include data on test chemical recovery from treated skin, skin washes, and residual radioactivity in the skin covering apparatus and metabolic unit as well as results of the dermal washing study. For further discussion, see paragraphs 74-77.
• — For inhalation studies, also include data on recovery of test chemical from lungs and nasal tissues (8). For further discussion, see paragraph 78.

• (2) Tissue distribution reported as percent of administered dose and concentration (microgram equivalents per gram of tissue), and tissue-to-blood or tissue-to-plasma ratios;

• (3) Material balance developed from each study involving the assay of body tissues and excreta;

• (4) Plasma concentrations and toxicokinetic parameters (bioavailability, AUC, Cmax, Tmax, clearance, half-life) after administration by the relevant route(s) of exposure;

• (5) Rate and extent of absorption of the test chemical after administration by the relevant route(s) of exposure;

• (6) Quantities of the test chemical and metabolites (reported as percent of the administered dose) collected in excreta;

• (7) Reference to appendix data which contain individual animal data for all measurement endpoints (e.g. dose administration, percent recovery, concentrations, TK parameters, etc.);

• (8) A figure with the proposed metabolic pathways and the molecular structures of the metabolites.

Discussion and Conclusions

• 72.In this section the author(s) should:

• (1) Provide a proposed metabolic pathway based on the results of the metabolism and disposition of the test chemical;

• (2) Discuss any potential species and sex differences regarding the disposition and/or biotransformation of the test chemical;

• (3) Tabulate and discuss the identification and magnitude of metabolites, rates of clearance, bioaccumulation potential, and level of tissue residues of parent, and/or metabolite(s), as well as possible dose-dependent changes in TK parameters, as appropriate;

• (4) Integrate into this section any relevant TK data obtained in the course of conducting toxicity studies;

• (5) Provide a concise conclusion that can be supported by the findings of the study;

• (6) Add Sections (as needed or appropriate).

• 73.Additional sections should be used to include supporting bibliographic information, tables, figures, appendices, etc.

ALTERNATIVE ROUTES OF EXPOSURE

Dermal

Dermal Treatment

• 74.This section provides specific information on the investigation of the toxicokinetics of the test chemical by the dermal route. For dermal absorption, chapter B.44 of this Annex [Skin absorption: in vivo method (9)] should be consulted. For other endpoints such as distribution and metabolism, this Test Method B.36 can be used. One or more dose levels for the test chemical should be used in the dermal treatment. The test chemical (e.g. neat, diluted or formulated material containing the test chemical which is applied to the skin) should be the same (or a realistic surrogate) as that to which humans or other potential target species might be exposed. The dose level(s) should be selected in accordance with paragraphs 20-26 of this Test Method. Factors that could be taken into consideration in dermal dose selection include expected human exposure and/or doses at which toxicity was observed in other dermal toxicity studies. The dermal dose(s) should be dissolved, if necessary, in a suitable vehicle and applied in a volume adequate to deliver the doses. Shortly before testing, fur should be clipped from the dorsal area of the trunk of the test animals. Shaving may be employed, but it should be carried out approximately 24 h before the test. When clipping or shaving the fur, care should be taken to avoid abrading the skin, which could alter its permeability. Approximately 10 % of the body surface should be cleared for application of the test chemical. With highly toxic chemicals, the surface area covered may be less than approximately 10 %, but as much of the area as possible is to be covered with a thin and uniform film. The same treatment surface area should be used for all dermal test groups. The dosed areas are to be protected with a suitable covering which is secured in place. The animals should be housed separately.

• 75.A dermal washing study should be conducted to assess the amount of the applied dose of the test chemical that may be removed from the skin by washing the treated skin area with a mild soap and water. This study can also aid in establishing mass balance when the test chemical is administered by the dermal route. For this dermal washing study, a single dose of the test chemical should be applied to two animals. Dose level selection is in accordance with paragraph 23 of this Test Method (also see paragraph 76 for discussion of skin contact time). The amounts of test chemical recovered in the washes should be determined to assess the effectiveness of removal of the test chemical by the washing procedure.

• 76.Unless precluded by corrosiveness, the test chemical should be applied and kept on the skin for a minimum of 6 h. At the time of removal of the covering, the treated area should be washed following the procedure as outlined in the dermal washing study (see paragraph 75). Both covering and the washes should be analysed for residual test chemical. At the termination of the studies, each animal should be humanely killed in accordance with (2), and the treated skin removed. An appropriate section of treated skin should be analysed to determine residual test chemical (radioactivity).

• 77.For the toxicokinetic assessment of pharmaceuticals, different procedures, in accordance with the appropriate regulatory system, may be needed.

Inhalation

• 78.A single concentration (or more if needed) of test chemical should be used. The concentration(s) should be selected in accordance with paragraphs 20-26 of this Test Method. Inhalation treatments are to be conducted using a ‘nose-cone’ or ‘head-only’ apparatus to prevent absorption by alternate routes of exposure (8). If other inhalation exposure conditions are used, justification for the modification should be documented. The duration of exposure by inhalation should be defined; a typical exposure is 4-6 h.

LITERATURE:

• (1) OECD (2009). Preliminary Review of OECD Test Guidelines for their Applicability to Manufactured Nanomaterials, Series on the Safety of Manufactured Nanomaterials No 15, ENV/JM/MONO(2009)21, OECD, Paris.

• (2) OECD (2000). Guidance Document on Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation; Environmental Health and Safety Publications, Series on Testing and Assessment No 19, ENV/JM/MONO(2000), OECD, Paris.

• (3) Solon E G, Kraus L (2002). Quantitative whole-body autoradiography in the pharmaceutical industry; Survey results on study design, methods, and regulatory compliance, J Pharm and Tox Methods 46: 73-81.

• (4) Stumpf WE (2005). Drug localization and targeting with receptor microscopic autoradiography. J. Pharmacological and Toxicological Methods 51: 25-40.

• (5) Loizou G, Spendiff M, Barton HA, Bessems J, Bois FY, d’Yvoire MB, Buist H, Clewell HJ 3rd, Meek B, Gundert-Remy U, Goerlitz G, Schmitt W. (2008). Development of good modelling practice for physiologically based pharmacokinetic models for use in risk assessment: The first steps. Regulatory Toxicology and Pharmacology 50: 400 – 411.

• (6) Chapter B.45 of this Annex, Skin Absorption: In Vitro Method.

• (7) IPCS (2010). Characterization and application of Physiologically-Based Pharmacokinetic Models in Risk Assessment. IPCS Harmonization Project Document No 9. Geneva, World Health Organization, International Programme on Chemical Safety.

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

• (9) Chapter B.44 of this Annex, Skin Absorption: In Vivo Method.

• (10) Barton HA, et al. (2006). The Acquisition and Application of Absorption, Distribution, Metabolism, and Excretion (ADME) Data in Agricultural Chemical Safety Assessments, Critical Reviews in Toxicology 36: 9-35.

• (11) Gibaldi M and Perrier D, (1982), Pharmacokinetics, 2nd edition, Marcel Dekker, Inc., New York.

Appendix

DEFINITIONS

Absorption : Process(es) of uptake of chemicals into or across tissues. Absorption refers to parent compound and all its metabolites. Not to be confused with ‘bioavailability’.

Accumulation (Bioaccumulation) : Increase of the amount of a test chemical over time within tissues (usually fatty tissues, following repeated exposure); if the input of a test chemical into the body is greater than the rate at which it is eliminated, the organism accumulates the test chemical and toxic concentrations of a test chemical might be achieved.

ADME : Acronym for ‘Absorption, Distribution, Metabolism, and Excretion’.

AUC : (Area under the plasma concentration-time curve): Area under the curve in a plot of concentration of test chemical in plasma over time. It represents the total amount of test chemical absorbed by the body within a predetermined period of time. Under linear conditions, the AUC (from time zero to infinity) is proportional to the total amount of a test chemical absorbed by the body, irrespective of the rate of absorption.

Autoradiography : (Whole-body autoradiography): Used to determine qualitatively and/or quantitatively the tissue localisation of a radioactive test chemical, this technique uses X-ray film or more recently digital phosphorimaging to visualize radioactively labelled molecules or fragments of molecules by recording the radiation emitted within the object under study. Quantitative whole-body autoradiography, compared to organ dissection, may have some advantages for the evaluation of test chemical distribution and the assessment of overall recovery and resolution of radioactive material in tissues. One significant advantage, for example, is it can be used in a pigmented animal model to assess possible association of the test chemical with melanin, which can bind certain molecules. However, while it may provide convenient whole body overviews of the high-capacity-low-affinity binding sites, this technique might be limited in recognising specific target sites such as receptor-binding sites where relatively high-resolution and high-sensitivity are needed for detection. When autoradiography is used, experiments intended to determine mass balance of administered compound should be conducted as a separate group or in a separate study from the tissue distribution experiment, where all excreta (which may also include expired air) and whole carcasses are homogenised and assayed by liquid scintillation counting.

Biliary excretion : Excretion via the bile ducts.

Bioaccumulation : See ‘Accumulation’.

Bioavailability : Fraction of an administered dose that reaches the systemic circulation or is made available at the site of physiological activity. Usually, bioavailability of a test chemical refers to the parent compound, but it could refer to its metabolite. It considers only one chemical form. Nota Bene: bioavailability and absorption are not the same. The difference between e.g. oral absorption (i.e. presence in gut wall and portal circulation) and bioavailability (i.e. presence in systemic blood and in tissues) can arise from chemical degradation due to gut wall metabolism or efflux transport back to the intestinal lumen or presystemic metabolism in the liver, among other factors (10). Bioavailability of the toxic component (parent compound or a metabolite) is a critical parameter in human risk assessment (high-to-low dose extrapolation, route-to-route extrapolation) for derivation of an internal value from the external NOAEL or BMD (applied dose). For liver effects upon oral administration, it is the oral absorption that suffices. However, for every effect other than at the portal of entry, it is the bioavailability that is in general a more reliable parameter for further use in risk assessment, not the absorption.

Biopersistence : See ‘Persistence’.

Biotransformation : (Usually enzymatic) chemical conversion of a test chemical of interest into a different chemical within the body. Synonymous with ‘metabolism’.

Cmax : Either maximal (peak) concentration in blood (plasma/serum) after administration or maximal (peak) excretion (in urine or faeces) after administration.

Clearance rate : Quantitative measure of the rate at which a test chemical is removed from the blood, plasma or a certain tissue per unit time.

Compartment : Structural or biochemical portion (or unit) of a body, tissue or cell, that is separate from the rest.

Detoxification pathways : Series of steps leading to the elimination of toxic chemicals from the body, either by metabolic change or excretion.

Distribution : Dispersal of a test chemical and its derivatives throughout an organism.

Enzymes/Isozymes : Proteins that catalyse chemical reactions. Isozymes are enzymes that catalyse similar chemical reactions but differ in their amino acid sequence.

Enzymatic Parameters : Km: Michaelis constant and Vmax: maximum velocity.

Excretion : Process(es) by which an administered test chemical and/or its metabolites are removed from the body.

Exogenously : Introduced from or produced outside the organism or system.

Extrapolation : Inference of one or more unknown values on the basis of that which is known or has been observed.

Half-life (t1/2) : The time taken for the concentration of the test chemical to decrease by one-half in a compartment. It typically refers to plasma concentration or the amount of the test chemical in the whole body.

Induction/Enzyme induction : Enzyme synthesis in response to an environmental stimulus or inducer molecule.

Linearity/linear kinetics : A process is linear in terms of kinetics when all transfer rates between compartments are proportional to the amounts or concentrations present, i.e. first order. Consequently, clearance and distribution volumes are constant, as well as half-lives. The concentrations achieved are proportional to the dosing rate (exposure), and accumulation is more easily predictable. Linearity/Non-linearity can be assessed by comparing the relevant parameters, e.g. AUC, after different doses or after single and repeated exposure. Lack of dose dependency may be indicative of saturation of enzymes involved in the metabolism of the compound, an increase of AUC after repeated exposure as compared to single exposure may be an indication for inhibition of metabolism and a decrease in AUC may be an indication for induction of metabolism [see also (11)].

Mass balance : Accounting of test chemical entering and leaving the system.

Material balance : See ‘mass balance’.

Mechanism (Mode) of toxicity/Mechanism (Mode) of action : Mechanism of action refers to specific biochemical interactions through which a test chemical produces its effect. Mode of action refers to more general pathways leading to the toxicity of a test chemical.

Metabolism : Synonymous with ‘biotransformation’.

Metabolites : Products of metabolism or metabolic processes.

Oral Absorption : The percentage of the dose of test chemical absorbed from the site of administration (i.e. GI tract). This critical parameter can be used to understand the fraction of the administered test chemical that reaches the portal vein, and subsequently the liver.

Partition coefficient : Also known as the distribution coefficient, it is a measure of the differential solubility of a chemical in two solvents.

Peak blood (plasma/serum) levels : Maximal (peak) blood (plasma/serum) concentration after administration (see also ‘Cmax’).

Persistence (biopersistence) : Long-term presence of a chemical (in a biological system) due to resistance to degradation/elimination.

Read-across : The endpoint information for one or more chemicals is used to make a prediction of the endpoint for the target chemical.

Receptor Microscopic Autoradiography (or Receptor Microautoradiography) : This technique may be used to probe xenobiotic interaction with specific tissue sites or cell populations as for instance in receptor binding or specific mode of action studies that may require high-resolution and high sensitivity which may not be feasible with other techniques such as whole-body autoradiography.

Route of administration (oral, IV, dermal, inhalation, etc.) : Refers to the means by which chemicals are administered to the body (e.g. orally by gavage, orally by diet, dermal, by inhalation, intravenously, etc.).

Saturation : State whereby one or more of the kinetic (e.g. absorption, metabolism or clearance) process(es) are at a maximum (read ‘saturated’).

Sensitivity : Capability of a method or instrument to discriminate between measurement responses representing different levels of a variable of interest.

Steady-state blood (plasma) levels : Non-equilibrium state of an open system in which all forces acting on the system are exactly counter-balanced by opposing forces, in such a manner that all its components are stationary in concentration although matter is flowing through the system.

Systems Modelling (Physiologically-based Toxicokinetic, Pharmacokinetic-based, Physiologically-based Pharmacokinetic, Biologically-based, etc.) : Abstract model that uses mathematical language to describe the behaviour of a system.

Target tissue : Tissue in which a principal adverse effect of a toxicant is manifested.

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

Tissue distribution : Reversible movement of a test chemical from one location in the body to another. Tissue distribution can be studied by organ dissection, homogenisation, combustion and liquid scintillation counting or by qualitative and/or quantitative whole body autoradiography. The former is useful to obtain concentration and percent of recovery from tissues and remaining carcass of the same animals, but may lack resolution for all tissues and may have less than ideal overall recovery (< 90 %). See definition for the latter above.

Tmax : Time to reach Cmax.

Toxicokinetics (Pharmacokinetics) : Study of the absorption, distribution, metabolism, and excretion of chemicals over time.

Validation of models : Process of assessing the adequacy of a model to consistently describe the available toxicokinetic data. Models may be evaluated via statistical and visual comparison of model predictions with experimental values against a common independent variable (e.g. time). The extent of evaluation should be justified in relation to the intended use of the model.

B.37.   DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS SUBSTANCES FOLLOWING ACUTE EXPOSURE

1. METHOD

1.1.   INTRODUCTION

In the assessment and evaluation of the toxic effects of substances, it is important to consider the potential of certain classes of substances to cause specific types of neurotoxicity that might not be detected in other toxicity studies. Certain organophosphorus substances have been observed to cause delayed neurotoxicity and should be considered as candidates for evaluation.

In vitro screening tests could be employed to identify those substances which may cause delayed polyneuropathy; however, negative findings from in vitro studies do not provide evidence that the test substance is not a neurotoxicant.

See General introduction Part B.

1.2.   DEFINITIONS

Organophopsphorus substances include uncharged organophosphorus esters, thioesters or anhydrides of organophosphoric, organophosphonic or organophosphoramidic acids or of related phosphorothioic, phosphonothioic or phosphorothioamidic acids, or other substances that may cause the delayed neurotoxicity sometimes seen in this class of substances.

Delayed neurotoxicity is a syndrome associated with prolonged delayed onset of ataxia, distal axonopathies in spinal cord and peripheral nerve, and inhibition and aging of neuropathy target esterase (NTE) in neural tissue.

1.3.   REFERENCE SUBSTANCES

A reference substance may be tested with a positive control group as a means of demonstrating that under the laboratory test conditions, the response of the tested species has not changed significantly.

An example of a widely used neurotoxicant is tri-o-tolyl phosphate (CAS 78-30-8, Einecs 201-103-5, CAS nomenclature: phosphoric acid, tris(2-methylphenyl)ester), also known as tris-o-cresylphosphate.

1.4.   PRINCIPLE OF THE TEST METHOD

The test substance is administered orally in a single dose to domestic hens which have been protected from acute cholinergic effects, when appropriate. The animals are observed for 21 days for behavioural abnormalities, ataxia, and paralysis. Biochemical measurements, in particular neuropathy target esterase inhibition (NTE), are undertaken on hens randomly selected from each group, normally 24 and 48 hours after dosing. Twenty-one days after exposure, the remainder of the hens are killed and histopathological examination of selected neural tissues is undertaken.

1.5.   DESCRIPTION OF THE TEST METHOD

1.5.1.   Preparations

Healthy young adult hens free from interfering viral diseases and medication and without abnormalities of gait should be randomised and assigned to treatment and control groups and acclimatised to the laboratory conditions for at least five days prior to the start of the study.

Cages or enclosures which are large enough to permit free mobility of the hens, and easy observation of gait should be used.

Dosing with the test substance should normally be by the oral route using gavage, gelatine capsules, or a comparable method. Liquids may be given undiluted or dissolved in an appropriate vehicle such as corn oil; solids should be dissolved if possible since large doses of solids in gelatine capsules may not be absorbed efficiently. For non-aqueous vehicles the toxic characteristics of the vehicle should be known, and if not known should be determined before the test.

1.5.2.   Test conditions

1.5.2.1.   Test animals

The young adult domestic laying hen (Gallus gallus domestícus), aged eight to 12 months, is recommended. Standard size breeds and strains should be employed and the hens normally should have been reared under conditions which permitted free mobility.

1.5.2.2.   Number and sex

In addition to the treatment group, both a vehicle control group and a positive control group should be used. The vehicle control group should be treated in a manner identical to the treatment group, except that administration of the test substance is omitted.

Sufficient number of hens should be utilised in each group of birds so that at least six birds can be killed for biochemical determination (three at each of two time points) and six can survive the 21-day observation period for pathology.

The positive control group may be run concurrently or be a recent historical control group. It should contain at least six hens, treated with a known delayed neurotoxicant, three hens for biochemistry and three hens for pathology. Periodic updating of historical data is recommended. New positive control data should be developed when some essential element (e.g. strain, feed, housing conditions) of the conduct of the test has been changed by the performing laboratory.

1.5.2.3.   Dose levels

A preliminary study using an appropriate number of hens and dose levels groups should be performed to establish the level to be used in the main study. Some lethality is typically necessary in this preliminary study to define an adequate main study dose. However, to prevent death due to acute cholinergic effects, atropine or another protective agent, known to not interfere with delayed neurotoxic responses, may be used. A variety of test methods may be used to estimate the maximum non-lethal dose of test substances (See method B.1bis). Historical data in the hen or other toxicological information may also be helpful in dose selection.

The dose level of the test substance in the main study should be as high as possible taking into account the results of the preliminary dose selection study and the upper limit dose of 2 000 mg/kg body weight. Any mortality which might occur should not interfere with the survival of sufficient animals for biochemistry (six) and histology (six) at 21 days. Atropine or another protective agent, known to not interfere with delayed neurotoxic responses, should be used to prevent death due to acute cholinergic effects.

1.5.2.4.   Limit test

If a test at a dose level of at least 2 000 mg/kg body weight/day, using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data from structurally related substances, then a study using a higher dose may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.

1.5.3.   Observation period

Observation period should be 21 days.

1.5.4.   Procedure

After administration of a protective agent to prevent death due to acute cholinergic effect, the test substance is administered in a single dose.

General observation

Observations should start immediately after exposure. All hens should be carefully observed several times during the first two days and thereafter at least once daily for a period of 21 days or until scheduled kill. All signs of toxicity should be recorded, including the time of onset, type, severity and duration of behavioural abnormalities. Ataxia should be measured on an ordinal grading scale consisting of at least four levels, and paralysis should be noted. At least twice a week the hens selected for pathology should be taken outside the cages and subjected to a period of forced motor activity, such as ladder climbing, in order to facilitate the observation of minimal toxic effects. Moribund animals and animals in severe distress or pain should be removed when noticed, humanely killed and necropsied.

Body weight

All hens should be weighed just prior to administration of the test substance and at least once a week thereafter.

Biochemistry

Six hens randomly selected from each of the treatment and vehicle control groups, and three hens from the positive control group (when this group is run concurrently), should be killed within a few days after dosing, and the brain and lumbar spinal cord prepared and assayed for neuropathy target esterase inhibition activity. In addition, it may also be useful to prepare and assay sciatic nerve tissue for neuropathy target esterase inhibition activity. Normally, three birds of the control and each treatment group are killed after 24 hours and three at 48 hours, whereas the three hens of the positive controls should be killed at 24 hours. If observation of clinical signs of intoxication (this can often be assessed by observation of the time of onset of cholinergic signs) indicates that the toxic agent may be disposed of very slowly then it may be preferable to sample tissue from three birds at each of two times between 24 and as late as 72 hours after dosing.

Analyses of acetylcholinesterase (AChE) may also be performed on these samples, if deemed appropriate. However, spontaneous reactivation of AChE may occur in vivo, and so lead to underestimation of the potency of the substance as an AChE inhibitor.

Gross necropsy

Gross necropsy of all animals (scheduled killed and killed when moribund) should include observation of the appearance of the brain and spinal cord.

Histopathological examination

Neural tissue from animals surviving the observation period and not used for biochemical studies should be subjected to microscopic examination. Tissues should be fixed in situ, using perfusion techniques. Sections should include cerebellum (mid-longitudinal level), medulla oblongata, spinal cord, and peripheral nerves. The spinal cord sections should be taken from the upper cervical segment, the mid-thoracic and the lumbo-sacral regions. Sections of the distal region of the tibial nerve and its branches to the gastrocnemial muscle and of the sciatic nerve should be taken. Sections should be stained with appropriate myelin and axon-specific stains.

2. DATA

Negative results on the endpoints selected in this method (biochemistry, histopathology and behavioural observation) would not normally require further testing for delayed neurotoxicity. Equivocal or inconclusive results for these endpoints may require further evaluation.

Individual data should be provided. Additionally, all data should be summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, behavioural or biochemical effects, the types and severity of these lesions or effects, and the percentage of animals displaying each type and severity of lesion or effect.

The findings of this study should be evaluated in terms of the incidence, severity, and correlation of behavioural, biochemical and histopathological effects and any other observed effects in the treated and control groups.

Numerical results should be evaluated by appropriate and generally acceptable statistical methods. The statistical methods used should be selected during the design of the study.

3. REPORTING

TEST REPORT

The test report shall, if possible, include the following information:

• 3.1. Test animals:
• — strain used,
• — number and age of animals,
• — source, housing conditions, etc.,
• — individual weights of animals at the start of the test.

• 3.2. Test conditions:
• — details of test substance preparation, stability and homogeneity, where appropriate,
• — justification for choice of vehicle,
• — details of the administration of the test substance,
• — details of food and water quality,
• — rationale for dose selection,
• — specification of doses administered, including details of the vehicle, volume and physical form of the material administered,
• — identity and details of the administration of any protective agent.

• 3.3. Results:
• — body weight data,
• — toxic response data by group, including mortality,
• — nature, severity and duration of clinic observations (whether reversible or not),
• — a detailed description of biochemical methods and findings,
• — necropsy findings,
• — a detailed description of all histopathological findings,
• — statistical treatment of results, where appropriate.

Discussion of results.

Conclusions.

4. REFERENCES

This method is analogius to OECD TG 418.

B.38.   DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS SUBSTANCES 28-DAY REPEATED DOSE STUDY

1. METHOD

1.1.   INTRODUCTION

In the assessment and evaluation of the toxic effects of substances, it is important to consider the potential of certain classes of substances to cause specific types of neurotoxicity that might not be detected in other toxicity studies. Certain organophosphorus substances have been observed to cause delayed neurotoxicity and should be considered as candidates for evaluation.

In vitro screening tests could be employed to identify those substances which may cause delayed polyneuropathy; however, negative findings from in vitro studies do not provide evidence that the test substance is not a neurotoxicant.

This 28-day delayed neurotoxicity test provides information on possible health hazards likely to arise from repeated exposures over a limited period of time. It will provide information on dose response and can provide an estimate of a no-observed-adverse effect level, which can be of use for establishing safety criteria for exposure.

See also General introduction Part B.

1.2.   DEFINITIONS

Organophosphorus substances include uncharged organophosphorus esters, thioesters or anhydrides of organophosphoric, organophosphonic or organophosphoramidic acids or of related phosphorothioic, phosphonothioic or phosphorothioamidic acids or other substances that may cause the delayed neurotoxicity sometimes seen in this class of substances.

Delayed neurotoxicity is a syndrome associated with prolonged delayed onset of ataxia, distal axonopathies in spinal cord and peripheral nerve, and inhibition and ageing of neuropathy target esterase (NTE) in neural tissue.

1.3.   PRINCIPLE OF THE TEST METHOD

Daily doses of the test substance are administered orally to domestic hens for 28 days. The animals are observed at least daily for behavioural abnormalities, ataxia and paralysis until 14 days after the last dose. Biochemical measurements, in particular neuropathy target esterase inhibition (NTE), are undertaken, on hens randomly selected from each group, normally 24 and 48 hours after the last dose. Two weeks after the last dose, the remainder of the hens are killed and histopathological examination of selected neural tissues is undertaken.

1.4.   DESCRIPTION OF THE TEST METHOD

1.4.1.   Preparations

Healthy young adult hens free from interfering viral diseases and medication, and without abnormalities of gait should be randomised and assigned to treatment and control groups and acclimatised to the laboratory conditions for at least five days prior to the start of the study.

Cages or enclosures which are large enough to permit free mobility of the hens and easy observation of gait should be used.

Oral dosing each day, seven days per week, should be carried out, preferably by gavage or administration of gelatine capsules. Liquids may be given undiluted or dissolved in an appropriate vehicle such as corn oil; solids should be dissolved if possible since large doses of solids in gelatine capsules may not be absorbed efficiently. For non-aqueous vehicles the toxic characteristics of the vehicle should be known, and if not known should be determined before the test.

1.4.2.   Test conditions

1.4.2.1.   Test animals

The young adult domestic laying hen (Gallus gallus domesticus), aged eight to 12 months, is recommended. Standard size, breeds and strains should be employed and the hens normally should have been reared under conditions which permitted free mobility.

1.4.2.2.   Number and sex