OECD GUIDELINE,424

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OECD GUIDELINE,424 FOR THE TESTING OF CHEMICALS:

OECD GUIDELINE,424 FOR THE TESTING OF CHEMICALS Neurotoxicity Study in Rodents Prepared by Nikita modi Roll no. 40 Department of pharmacology. K.B. Institute of pharmaceutical education & research

INTRODUCTION:

INTRODUCTION OECD Guidelines for the Testing of Chemicals are done in order to respond efficiently to scientific progress in hazard identification and related data generation. Test Guidelines can be made by OECD Member countries, the OECD Secretariat, as well as the international scientific community. The final proposal was prepared by an OECD ad hoc Working Group on Neurotoxicity that met in Ottawa, Canada in March 1995.

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This Test Guideline has been designed to obtain the information necessary to confirm or to further characterise the potential neurotoxicity of chemicals in adult animals. It can either be combined with existing Test Guidelines for repeated dose toxicity studies or be carried out as a separate study.

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The potential neurotoxicity of certain classes of chemicals may suggest that they may be more appropriately evaluated using this Guideline without prior indications of potential neurotoxicity from repeated dose systemic toxicity studies. Such considerations include, for example: observation of neurological signs or neuropathological lesions in toxicity studies other than repeated dose systemic toxicity studies, or 2) structural relationship or other information linking them to known neurotoxicants .

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In the past, neurotoxicity was equated with neuropathy involving neuropathological lesions such as seizure, paralysis or tremor. Although neuropathy is an important manifestation of neurotoxicity, it is now clear that there are many other signs of nervous system toxicity (e.g. loss of motor co-ordination, sensory deficits, learning and memory dysfunctions) that may not be reflected in neuropathy or other types of studies. This neurotoxicity Test Guideline is designed to detect major neuropathological effects in adult rodents.

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While behavioural effects, even in the absence of morphological changes, can reflect an adverse impact on the organism, not all behavioural changes are specific to the nervous system. Therefore, any changes observed should be evaluated in conjunction with correlative histopathological , haematological or biochemical data as well as data on other types of systemic toxicity. The testing called for in this Guideline to provide a characterization and quantification of the neurotoxic responses includes specific histopathological and behavioural procedures that may be further supported by electrophysiological and/or biochemical investigations .

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Neurotoxicants may act on a number of targets within the nervous system and by a variety of mechanisms. Since no single array of tests is capable of thoroughly assessing the neurotoxic potential of all substances, it may be necessary to utilise other in vivo or in vitro tests specific to the type of neurotoxicity observed. This neurotoxicity study, when used alone or in combination, provides information that can: 1) identify whether the nervous system is permanently or reversibly affected by the chemical tested; 2) contribute to the characterization of the nervous system alterations associated with exposure to the chemical, and to understanding the underlying mechanism.

PRINCIPLE OF THE TEST:

PRINCIPLE OF THE TEST The test chemical is administered by the oral route across a range of doses to several groups of laboratory rodents. Repeated doses are normally required, and the dosing regimen may be 28 days, subchronic (90 days) or chronic (1 year or longer). The procedures set out in this Guideline may also be used for an acute neurotoxicity study. The animals are tested to allow the detection or the characterization of behavioural and/or neurological abnormalities.

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A range of behaviours that could be affected by neurotoxicants is assessed during each observation period. At the end of the test, a subset of animals of each sex from each group are perfused in situ and sections of the brain, spinal cord, and peripheral nerves are prepared and examined.

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When the study is conducted as a stand-alone study to screen for neurotoxicity ,the animals in each group not used for perfusion and subsequent histopathology, can be used for specific neurobehavioural , neuropathological , neurochemical or electrophysiological procedures that may supplement the data obtained from the standard examinations required by this Guideline . Alternatively, the remaining animals can be used for evaluations such as those called for in Test Guidelines for repeated dose toxicity studies in rodents. When the procedures of this Test Guideline are combined with those of other Test Guidelines, a sufficient number of animals is needed to satisfy the requirements for the observations of both studies.

DESCRIPTION OF THE METHOD:

DESCRIPTION OF THE METHOD Selection of animal species The preferred rodent species is the rat, The females should be nulliparous and non-pregnant. Dosing should normally begin as soon as possible after weaning, between six weeks and nine weeks of age. However, when this study is combined with other studies this age requirement may need adjustment. At the commencement of the study the weight variation of animals used should not exceed ± 20% of the mean weight of each sex.

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Housing and feeding conditions The temperature should be 22°C (± 3°C). The relative humidity between 30-70% . Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. Loud intermittent noise should be kept to a minimum. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. Animals may be housed individually, or be caged in small groups of the same sex.

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Preparation of animals Healthy young animals are randomly assigned to the treatment and control groups. Route of administration and preparation of doses This guideline specifically addresses the oral administration of the test substance. Oral administration may be by gavage , in the diet, in drinking water or by capsules. Other routes of administration (e.g. dermal or inhalation) can be used but may require modification of the procedures recommended. Considerations of the choice of the route of administration depend on the human exposure profile and available toxicological or kinetic information.

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It is recommended that the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/suspension in oil (e.g., corn oil) and then by possible solution/suspension in other vehicles. The toxic characteristics of the vehicle must be known. In addition, consideration should be given to the following characteristics of the vehicle: effects of the vehicle on absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals.

PROCEDURE :

PROCEDURE Number and sex of animals When the study is conducted as a separate study, at least 20 animals (10 females and 10 males) should be used in each dose and control group for the evaluation of detailed clinical and functional observations. When the study is conducted in combination with a repeated dose toxicity study, adequate numbers of animals should be used to meet the objectives of both studies.

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Treatment and control groups At least three dose groups and a control group should generally be used, but if from the assessment of other data, no effects would be expected at a repeated dose of 1000 mg/kg body weight/day, a limit test may be performed. Animals in the control group should be handled in an identical manner to the test group subjects. If a vehicle is used in administering the test substance, the control group should receive the vehicle at the highest volume used.

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Reliability check The laboratory performing the study should present data demonstrating its capability to carry out the study and the sensitivity of the procedures used. Such data should provide evidence of the ability to detect and quantify, as appropriate, changes in the different end points recommended for observation, such as autonomic signs, sensory reactivity, limb grip strength and motor activity. Information on chemicals that cause different types of neurotoxic responses and could be used as positive control substances. Historical data may be used if the essential aspects of the experimental procedures remain the same. Periodic updating of historical data is recommended.

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Dose selection Dose selection is based on previously observed toxicity and kinetic data available for the test compound. The highest dose level should be chosen with the aim of inducing neurotoxic effects or clear systemic toxic effects. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dose-related response and a no-observed-adverse effect (NOAEL) at the lowest dose level. In principle, dose levels should be set so that primary toxic effects on the nervous system can be distinguished from effects related to systemic toxicity. Two to three intervals are frequently optimum and addition of a fourth test group is often preferable to using very large intervals between dosages.

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Limit test For the conduct of an oral acute study, the dose for a limit test should be at least 2000 mg/kg. Administration of doses The animals are dosed with the test substance daily, seven days each week, for a period of at least 28 days; use of a five-day dosing regime or a shorter exposure period needs to be justified. When the test substance is administered by gavage , this should be done in a single dose using a stomach tube or a suitable intubation cannula . The volume should not exceed 1 ml/100 g body weight. However in the case of aqueous solutions, the use of up to 2 ml/100 g body weight can be considered. Except for irritating or corrosive substances.

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For a substance administered by gavage , the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where a repeated dose study is used as a preliminary to a long term study, a similar diet should be used in both studies. For acute studies, if a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours.

OBSERVATIONS:

OBSERVATIONS Frequency of observations and tests In acute studies, 14-day post-treatment period should be observed. Observations should be made with sufficient frequency to maximise the probability of detection of any behavioural and/or neurological abnormalities. Observations should be made preferably at the same times each day with consideration given to the peak period of anticipated effects after dosing.

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Observations of general health condition and mortality/morbidity All animals should be carefully observed at least once daily with respect to their health condition as well as at least twice daily for morbidity and mortality. Detailed clinical observations clinical observations should be made outside the home cage in a standard arena. They should be carefully recorded using scoring systems that include criteria or scoring scales for each measurement in the observations.

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It is recommended that the observations be carried out in a "normal" range to each animal at each observation time. The “normal” range should be adequately documented. All observed signs should be recorded. Clinical observations should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Any unusual responses with respect to body position, activity and co-ordination of movement should also be noted. Changes in gait (e.g., waddling, ataxia), posture (e.g., hunched-back) and reactivity to handling, placing or other environmental stimuli, as well as the presence of clonic or tonic movements, convulsions or tremors, stereotypies or aggression should be recorded.

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Functional tests Functional tests should also be conducted once prior to exposure and frequently thereafter in all animals selected for this purpose . The frequency of functional testing is also dependant on the study duration . Functional tests should include sensory reactivity to stimuli of different modalities [e.g., auditory, visual and proprioceptive stimuli, assessment of limb grip strength and assessment of motor activity. Motor activity should be measured with an automated device capable of detecting both decreases and increases in activity. Each device should be tested to ensure reliability across time and consistency between devices.

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Body weight and food/water consumption For studies up to 90 days duration, all animals should be weighed at least once a week and measurements should be made of food consumption (water consumption, when the test substance is administered by that medium) at least weekly. For long term studies, all animals should be weighed at least once a week for the first 13 weeks and at least once every 4 weeks thereafter.

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Ophthalmology For studies longer that 28 days duration, ophthalmological examination, using an ophthalmoscope or an equivalent suitable instrument, should be made prior to the administration of the test substance and at the termination of the study, preferably on all animals, but at least on animals in the high dose and control groups. If changes in the eyes are detected or, if clinical signs indicate the need, all animals should be examined. For long term studies, an ophthalmological examination should also be carried out at 13 weeks.

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Haematology and clinical biochemistry Haematological examinations and clinical biochemistry determinations should be carried out as set out in the respective Guideline of the systemic toxicity study. Collection of samples should be carried out in such a way that any potential effects on neurobehaviour are minimized. Histopathology When the study is conducted as a stand-alone study to screen for neurotoxicity , animals may be used either for specific neurobehavioura , neuropathological , neurochemical or electrophysiological procedures that may supplement the procedures and examinations described here, or to increase the number of subjects examined for histopathology.

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A general staining procedure, such as haematoxylin and eosin (H&E), should be performed on all tissue specimens embedded in paraffin and microscopic examination should be carried out. Representative sections of the central and peripheral nervous system should be examined histologically . The spinal cord and peripheral nerve sections should include both cross or transverse and longitudinal sections. Attention should be given to the vasculature of the nervous system. Special attention should be paid to sites with cellular and fibre structure and pattern in the CNS and PNS known to be particularly affected by neurotoxicants .

DATA AND REPORTING:

DATA AND REPORTING Data Individual data should be provided. All data should be summarised in tabular form showing for each test or control 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, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, type and severity of any toxic effects, the number of animals showing lesions, including the type and severity of the lesion(s). When possible, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods should be selected during the design of the study.

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Test report The test report must include the following information: 1) Test substance: physical nature (including isomerism, purity and physicochemical properties); identification data. 2) Vehicle (if appropriate): justification for choice of vehicle. 3) Test animals: species/strain used; number, age and sex of animals; source, housing conditions, acclimatization, diet, etc.; individual weights of animals at the start of the test.

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4) Test conditions: details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation; specification of the doses administered, including details of the vehicle, volume and physical form of the material administered; details of the administration of the test substances; rationale for dose levels selected; rationale for the route and duration of the exposure; conversion from diet/drinking water test substance concentration ( ppm ) to the actual dose (mg/kg body weight/day), if applicable; details of food and water quality.

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5) Observation and Test Procedures: Details of the assignment of animals in each group to the perfusion subgroups; details of scoring systems, including criteria and scoring scales for each measurement in the detailed clinical observations; details of ophthalmological examinations and, if appropriate, haematological examinations and clinical biochemistry tests with relevant base-line values; details for specific neurobehavioural , neuropathological , neurochemical or electrophysiological procedures.

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6) Results: body weight/body weight changes including body weight at kill; food consumption and water consumption, as appropriate; toxic response data by sex and dose level, including signs of toxicity or mortality; a detailed description of all functional test results; necropsy findings; a detailed description of all neurobehavioural , neuropathological , and neurochemical or electrophysiological findings, if available; absorption and metabolism data, if available; statistical treatment of results, where appropriate.

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7) Discussion of results: dose response information; relationship of any other toxic effects to a conclusion about the neurotoxic potential of the test chemical; no-observed-adverse effect level. 8) Conclusions: a specific statement of the overall neurotoxicity of the test chemical is encouraged.

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