ASTM E1619-1995(2003) Standard Test Method for Chronic Oral Toxicity Study in Rats《老鼠口服慢性毒性研究的标准试验方法》.pdf

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1、Designation: E 1619 95 (Reapproved 2003)Standard Test Method forChronic Oral Toxicity Study in Rats1This standard is issued under the fixed designation E 1619; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revisi

2、on. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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

3、 prolonged and repeated 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 relieshe

4、avily on the judgment 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 theresponsibil

5、ity of the user of this 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:2E 609 Terminology Relating to PesticidesE 943 Termin

6、ology Relating to Biological Effects and En-vironmental 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 Adm

7、inistration, Part 58, Good 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. Termin

8、ology3.1 DefinitionsSee Terminology E 609 and TerminologyE 943.3.2 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.

9、2.2 concentration, nthe weight 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 eff

10、ciency, nthis value is a 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 m

11、aterial (ele-ment, chemical 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 ea

12、ch of the four dose levels (control-, low-,medium- and high-dosage groups). If it is determined that aninterim sacrifice is necessary, the number should be increasedby the number of animals scheduled to be sacrificed during thecourse of the study (see CFR, Title 40, Part 798).4.2 The high-dose level

13、 in rats should elicit some signs oftoxicity without causing excessive lethality. The lowest dosagelevel should be one that does not induce any evidence oftoxicity. This level should be higher (if possible) than thatexpected for human exposure. The intermediate-dosage level1This test method is under

14、 the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.26 on Safety to Man.Current edition approved October 1, 2003. Published October 2003. Originallyapproved in 1994. Last previous edition approved in 1999 as E 1619 9

15、5 (1999).2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact 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. Go

16、vernment PrintingOffice, Washington DC 20402.4Benitz, K. F., “Measurement of Chronic 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 19428-2

17、959, United States.should 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 a preliminary 14-day range finding

18、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 treatment and control groups arecom

19、pared 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. Significance and Use5.1 This test m

20、ethod 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, and hematological effects andexposure

21、-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 likely to arise fromrepeated exposure

22、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 substances. Infor-mation concerning acute

23、 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 federal, state, andlocal regulations.

24、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 suggested by the Institute of Laborato

25、ry 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 appropriatewater and food consumption. Maintai

26、n 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 species such asthe rodent; the rat is th

27、e preferred rodent species. If anothermammalian species is used, justification or reasoning for theselection must be recorded.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 nonpregnant.Acclim

28、ate 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 animals may be obt

29、ained 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.9. Diets9.1 The

30、 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 thephysical and ch

31、emical 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 toxic effects i

32、tself 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 acceptable.10. Range-Fi

33、nding Study10.1 Conduct a range-finding study and pilot study to assistin the selection of the appropriate doses 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 appropriate cages for a five-day acclim

34、ation 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 the LD50). One additional group of

35、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 tocontrols or acclimation period values, or both, is reduced bymore than 90 %, c

36、ontinued 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 waterE 1619 95 (2003)2consumption, or both, if necessar

37、y. 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 three dosage levels (low, medium, and high)plus an untreat

38、ed or solvent control. Dose all animals by thesame method during the entire experimental 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 number of animals by thenumber scheduled to be sacrific

39、ed 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 mixtures (dependingon stability of test substance) a

40、t least once on each new batchof test food or water prepared.11.5 Diet PreparationCalculate test substance food mix-ture for the first two weeks using the mean body weights andmean food consumption weights computed during the accli-mation period. Thereafter, prepare the mixtures from the meanbody we

41、ights 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 substance in the diet (grams oftest substance/100 g of g

42、round 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/day.Record food consumption (and water if necessary)

43、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 each animal atleast once per day, with appropriate

44、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, including time of onset, degree, andduration. These

45、 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 least once per week, on the sameday of each week and re

46、cord 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 each group of rats.11.7.1 UrinalysisPerform urinalys

47、is 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, pH, protein,glucose, ketones, bilirubins, urobilin

48、ogen, 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 just prior to the terminal sacrifice at the end of

49、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 period) as follows: electrolytebalance, carbohydrate