ASTM E1619-2011 Standard Test Method for Chronic Oral Toxicity Study in Rats《用老鼠进行口服慢性毒性研究的标准试验方法》.pdf

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1、Designation: E1619 11Standard Test Method forChronic Oral Toxicity Study in Rats1This standard is issued under the fixed designation E1619; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in pare

2、ntheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers a long-term study to determinethe effects of a substance in a mammalian species such as therat following prolonged and repeat

3、ed oral exposure. Under theconditions of the chronic toxicity test, effects that require along latency period or that are cumulative should becomemanifest.1.2 This test method assumes that the user is knowledgeablein mammalian toxicology and related pertinent areas, and reliesheavily on the judgment

4、 of the evaluator.1.3 The values stated in SI units are to be regarded as thestandard. The inch-pound units given in parentheses are forinformation only.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of th

5、is standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. For specific hazardstatements, see Section 6.2. Referenced Documents2.1 ASTM Standards:2E609 Terminology Relating to PesticidesE943 Terminology Relating to Biolo

6、gical Effects and Envi-ronmental Fate2.2 Federal Standards:3Title 40, Code of Federal Regulations (CFR), Environmen-tal Protection Agency, Subchapter E, Pesticide Programs:Part 160, Good Laboratory Practice StandardsTitle 21, Code of Federal Regulations (CFR). Food andDrug Administration, Part 58, G

7、ood Laboratory Practicefor Nonclinical StudiesTitle 40, CFR, Toxic Substance ControlAct, Part 792, GoodLaboratory Practice StandardsTitle 40, CFR, Environmental Protection Agency, Part 798,Health Effects Testing Guidelines, Subpart D, ChronicExposure, Chronic Toxicity3. Terminology3.1 DefinitionsSee

8、 Terminology E609 and TerminologyE943.3.2 Definitions of Terms Specific to This Standard:3.2.1 chronic toxicity, nthe adverse effects occurring as aresult of the daily exposure of mammalian species to a testsubstance by diet, water, capsule, or gavage for a one-yearperiod.3.2.2 concentration, nthe w

9、eight of test substance per unitweight of the diet (expressed as milligrams per kilogram ofdiet). The weight of test substance per volume of H2O(expressed as milligrams per millilitre of water), or at aconstant rate in the diet (expressed as parts per million).3.2.3 feed effciency, nthis value is a

10、measure of theefficiency with which the animals convert food to body weight.The calculation is the total body weight gain per total foodconsumed.3.2.4 gavage, nforced feeding, as by tube that is passeddown the throat to the stomach.3.2.5 test substance, npesticide or other material (ele-ment, chemic

11、al compound, formulation, known mixture) ad-ministered orally for the purpose of determining chronictoxicity.4. Summary of Test Method44.1 One mammalian species, a rodent, is required; the rat isthe preferred rodent. Forty rats (twenty females and twentymales) are used at each of the five dose level

12、s (control-, low-,two intermediate levels-, and high-dosage groups). If it isdetermined that an interim sacrifice is necessary, the numbershould be increased by the number of animals scheduled to besacrificed during the course of the study (see CFR, Title 40,Part 798).4.2 The high-dose level in rats

13、 should elicit some signs oftoxicity without causing excessive lethality. The lowest dosagelevel should be one that does not induce any evidence of1This test method is under the jurisdiction of ASTM Committee E47 onBiological Effects and Environmental Fate and is the direct responsibility ofSubcommi

14、ttee E47.02 on Terrestrial Assessment and Toxicology.Current edition approved Oct. 1, 2011. Published November 2011. Originallyapproved in 1994. Last previous edition approved in 2003 as E1619 95 (2003).DOI: 10.1520/E1619-11.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcon

15、tact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from Superintendent of Documents, U.S. Government PrintingOffice, Washington DC 20402.4Benitz, K. F., “Measurement of Chronic

16、 Toxicity,” Methods of Toxicology, ed.G. E. Paget, Blackwell Scientific Publications, Oxford, England, 1970, pp. 32-131.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.toxicity. This level should be higher (if possible) than thatexpe

17、cted for human exposure. The intermediate-dosage levelshould produce a minimal observable effect. Where appropri-ate, a vehicle control (the volume of which corresponds to thevolume of vehicle at the highest exposure level) should beused. The selection of test substance dosages may be estimatedfrom

18、a preliminary 14-day range finding study.4.3 Daily observations of all individual animals for signs oftoxicity and mortality are recorded.4.4 After one year, prior to necropsy, urine, hematology, andblood samples are collected for analysis and then test animalsare sacrificed.4.5 Data collected from

19、treatment and control groups arecompared statistically to detect changes in food or waterconsumption, or both, body weights, organ-to-body weight,and organ-to-brain weight ratios, hematology, and clinicalblood and urine values. Histopathological examinations arealso performed on selected tissues.5.

20、Significance and Use5.1 This test method should generate data to identify themajority of chronic effects and shall serve to define long-termdose response relationships. In addition the test should allowfor the detection of general toxic effects including neurologi-cal, physiological, biochemical, an

21、d hematological effects andexposure-related morphological (pathology) effects.5.2 This test method should provide information on targetorgans, the possibilities of accumulation, and may be used forestablishing safety criteria for human exposure. It providesinformation on potential health hazards lik

22、ely to arise fromrepeated exposure over a long period of time.6. Hazards6.1 Minimize contact with all test substances and solutionswith appropriate protective clothing, gloves, eye protection,etc. The use of fume hoods and increased ventilation in testrooms is necessary when handling volatile substa

23、nces. Infor-mation concerning acute mammalian toxicity and specialhandling procedures should be known before this test methodis used.6.2 Dispose excess test substance, solutions, diets, excreta,and treated animals with consideration for health and environ-mental safety, and in accordance with all fe

24、deral, state, andlocal regulations.7. Facilities7.1 No precise physical requirements concerning facilitiesare set forth. However, the animal facility shall meet theestablished standard(s) that may be required by law or regula-tions. It is desirable that the animal facilities meet the guide-lines sug

25、gested by the Institute of Laboratory Resources orfacilities that have been approved by such organizations as theAmerican Association for Accreditation of Laboratory AnimalCare (AAALAC).7.2 EnvironmentHouse test and control animals in cagesdesigned to hold laboratory animals. Provide for appropriate

26、water and food consumption. Maintain all animals in atemperature-, humidity-, and light-controlled room. The con-ditions should be 18 to 26C (64.4 to 78.8F) for temperature,40 to 70 % for humidity, and a 12-h light, 12-h dark lightingcycle.8. Test Animals8.1 Perform the test with one mammalian speci

27、es; the rat isthe preferred rodent species. If another mammalian species isused, justification or reasoning for the selection must berecorded.8.2 Obtain rats three weeks post-weaning. The Sprague-Dawley (COBS/CD) rat is an example of a strain frequentlyused. The females should be nulliparious and no

28、npregnant.Acclimate the animals for a period of no less than seven days.Dosing of rats should begin ideally before six weeks old, butno later than eight weeks of age.8.3 All animals for a given test must come from one sourceand strain and be approximately the same age to minimizevariability. Test an

29、imals may be obtained from commercialsources or reared in laboratory colonies, but they must not havebeen used in a previous test. Animals should be healthy anddisease free and those that are deformed, injured, emaciated orphenotypically different from normal animals must not be usedas test subjects

30、.9. Diets9.1 The preferred administration of test substance is incor-porated into a diet. However, the test substance may beadministered dissolved in drinking water or a solvent, or givenby gavage or capsule for a period of at least twelve months.The choice of route of administration depends upon th

31、ephysical and chemical characteristics of the test substance.9.1.1 If the test substance is administered by gavage, a five-day/week dosing regimen is acceptable.9.1.2 When necessary, dissolve or suspend the test sub-stance in a suitable solvent. If a vehicle or diluent is needed, itshould not elicit

32、 toxic effects itself nor substantially alter thechemical or toxicological properties of the test substance.9.2 Formulate diets in accordance with the nutrient require-ments of the test species. Any unmedicated commercial dietthat meets the minimum nutritional standards of the test speciesis accepta

33、ble.10. Range-Finding Study10.1 Previous data or a range-finding study should beused/conducted to assist in the selection of the appropriatedoses for the chronic study.10.2 Use groups of six male and six female rats between sixand eight weeks of age. Randomize, number, and place allanimals in approp

34、riate cages for a five-day acclimation period.During this period, all rats will receive rodent diet minus thetest substance. Dietary levels of the test substance to beadministered may approximate the acute oral LD50dosagesand fractions thereof (such as 1X, 0.5X, 0.25X, 0.125X,0.0625X, 0.03125X of th

35、e LD50). One additional group of eachsex will serve as a solvent or untreated control.10.3 It is strongly recommended that a dietary group beremoved from testing for humane purposes when food con-sumption is markedly reduced. If consumption as compared toE1619 112controls or acclimation period value

36、s, or both, is reduced bymore than 90 %, continued exposure will result in mortality oftest animals in that group.10.4 Base the no-effect and effect levels on the followingparameters: body weight, organ-to-body weight ratios, hema-tology, clinical chemistry, gross necropsy, and food or waterconsumpt

37、ion, or both, if necessary. Histology on a limitedselection of organs may be necessary in some instances.10.5 If a lethal dose is not found, set the highest dietarydosage at 1000 mg/kg, since dosage below this value isassumed to be nontoxic.11. Procedure511.1 Select four dosage levels (low, two inte

38、rmediate con-centrations, and high) plus an untreated or solvent control.Dose all animals by the same method during the entireexperimental period.11.2 Randomize, number, and assign at least 40 rats (20females and 20 males) to each dosage group. If additionalsacrifices are planned, increase the numbe

39、r of animals by thenumber scheduled to be sacrificed during the course of thestudy.11.3 Administer the test substance (if in the diet or drinkingwater) ad libitum throughout the study and depending on thestability of the test material replace at least weekly.11.4 Perform chemical analysis of test mi

40、xtures (dependingon stability of test substance) at least once on each new batchof test food or water prepared.11.5 Diet PreparationCalculate test substance food mix-tures for the first two weeks using the mean body weights andmean food consumption weights computed during the accli-mation period. Th

41、ereafter, prepare the mixtures from the meanbody weights and mean food consumption weights computedfrom the first week of the previous two-week period.11.5.1 Compute test substance food-mixture concentrationsfor each dosage using the following formula:X 5 100K/Gwhere:X = percent of active test subst

42、ance in the diet (grams oftest substance/100 g of ground food),K = dosage of substance that is desired and is expressed asgrams of test substance/kilogram of body weight/day,G = amount of food consumed/day over a one-week periodand is expressed as grams of food consumed perkilogram of body weight/da

43、y.Record food consumption (and water if necessary) through-out the study.11.5.2 An alternative to this test method would be todetermine the concentration of test substance in the feed priorto study initiation and then have it remain constant throughoutthe study.11.6 ObservationsMake observations of

44、each animal atleast once per day, with appropriate actions taken to minimizeloss of animals to the study (for example, necropsy orrefrigeration of animals found dead and isolation or sacrifice ofweak or moribund animals).11.6.1 Record signs of toxicity (by dosage group and sex) asthey are observed,

45、including time of onset, degree, andduration. These signs include, but are not limited to, changes inskin and fur, eyes and mucous membranes, and also respira-tory, circulatory, autonomic and central nervous systems,somatomotor activity, and unusual behavior patterns.11.6.2 Weigh all animals at leas

46、t once per week, on the sameday of each week and record weights.11.6.3 Record temperature and humidity continuallythroughout the study.11.6.4 At the end of the one year, sacrifice all survivinganimals.11.7 Clinical ExaminationsMake the following clinicalexaminations on at least five of each sex in e

47、ach group of rats.11.7.1 UrinalysisPerform urinalysis at the termination ofthe testing period. Place randomly selected animals in fromeach group and from each sex in metabolism cages for urinecollection. Evaluate each urine sample individually and includethe following measurements: specific gravity,

48、 pH, protein,glucose, ketones, bilirubins, urobilinogen, as well as micro-scopic examination of formed elements.11.7.2 HematologyMake the following hematology deter-minations at least twice during the test period on all groups ofanimals including concurrent controls (at six months into thestudy and

49、just prior to the terminal sacrifice at the end of thestudy) as follows: hematocrit, hemoglobin concentration,erythrocyte count, total and differential leukocyte counts, and ameasure of clotting potential such as prothrombin time orplatelet count, and reticulocyte count, if signs of anemia arepresent.11.7.3 Blood ChemistryMake clinical biochemical teststhat are considered appropriate to all studies, at least twiceduring the test period on all groups of animals includingconcurrent controls (at six months and just prior to the terminalsacrifice at the end of the test p