1、Designation: E 1687 04An American National StandardStandard Test Method forDetermining Carcinogenic Potential of Virgin Base Oils inMetalworking Fluids1This standard is issued under the fixed designation E 1687; the number immediately following the designation indicates the year oforiginal adoption
2、or, in the case of revision, the year of last revision. 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 microbiological test procedurebased upon the Salmo
3、nella mutagenesis assay of Ames et al(1)2(see also Maron et al (2). It can be used as a screeningtechnique to detect the presence of potential dermal carcino-gens in virgin base oils used in the formulation of metalwork-ing oils. Persons who perform this test should be well-versed inthe conduct of t
4、he Ames test and conversant with the physicaland chemical properties of petroleum products.1.2 The test method is not recommended as the sole testingprocedure for oils which have viscosities less than 18 cSt (90SUS) at 40C, or for formulated metalworking fluids.1.3 The values stated in SI units are
5、to be regarded as thestandard. The values given in parentheses are provided 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 this standard to establish appro-priate safety and health p
6、ractices and determine the applica-bility of regulatory limitations prior to use. Section 7 providesgeneral guidelines for safe conduct of this test method.2. Referenced Documents2.1 ASTM Standards:3E 2148 Guide for Using Documents Related to Metalwork-ing or Metal Removal Fluid Health and Safety2.2
7、 Other Standards:29 CFR 1910.1450 Occupational Exposure to HazardousChemical in Laboratories43. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 base stock, nthe refined oil component of metal-working fluid formulations.3.1.2 PCA (Polycyclic Aromatics), nFor the purposes ofthis te
8、st method, PCA refers to fused-ring polycyclic aromaticcompounds with three or more rings. For example, the hydro-carbon series is represented by phenanthrene (3), pyrene (4),benzopyrene (5), dibenzopyrene (6), coronene (7). Heterocy-clic polynuclear compounds are also included in the definition.3.1
9、.3 promutagenic compounds, promutagens,ncompounds that are not directly mutagenic but requiremetabolism for expression of mutagenic activity.3.1.4 Reference Oil 1, nstraight-run naphthenic vacuumdistillate (heavy vacuum gas oil) of known MI and PNAcontent recommended for use as a reference standard
10、for themodified Ames test.3.2 Abbreviations:Abbreviations:3.2.1 DMSO (Dimethyl Sulfoxide), nextraction agent usedin the preparation of aromatic-enriched oil fractions for mu-tagenicity testing.3.2.2 G-6-P (Glucose-6-Phosphate), nsubstrate requiredfor the operation of the NADPH generating system invo
11、lved inthe biological oxidations described above.3.2.3 MI (Mutagenicity Index), nthe slope of the dose-response curve for mutagenicity in the modified Ames test.3.2.3.1 DiscussionMI is an index of relative mutagenicpotency.3.2.4 NADP (Nicotinamide Adenine DinucleotidePhosphate)required cofactor for
12、the biological oxidationsinvolved in activation of PNA to their mutagenic forms.3.2.5 PNA (Polynuclear Aromatics; also termed CA),npolynuclear aromatic compounds.3.2.6 S-9, nfraction prepared from hamster liver whichcontains the enzymes required for metabolic activation ofPNAs to their mutagenic for
13、ms.4. Summary of Test Method4.1 The Ames Salmonella mutagenicity assay is the mostwidely used short-term in vitro genotoxicity test. The assayemploys specific strains of the bacterium Salmonella typhimu-rium that have been mutated at a genetic locus precluding thebiosynthesis of the amino acid histi
14、dine which is required for1This test method is under the jurisdiction of ASTM Committee E34 onOccupational Health and Safety and is the direct responsibility of SubcommitteeE34.50 on Health and Safety Standards for Metal Working Fluids.Current edition approved April 1, 2004. Published April 2004. Or
15、iginallyapproved in 1995. Last previous edition approved in 1998 as E 1687 - 98.2The boldface numbers refer to the list of references at the end of this standard.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book
16、of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.4Available from Superintendent of Documents, U.S. Government PrintingOffice, Washington, DC 20402.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,
17、United States.growth and reproduction. Additional genetic alterations, someof which are important markers of strain identity, are alsopresent.4.2 The mutagenicity assay relies upon treating the bacteriawith test material over a range of doses immediately below theconcentration showing significant to
18、xicity to the bacteria.Treated bacteria are then grown on agar plates deficient inhistidine. Bacteria possessing the original mutation in thehistidine locus cannot form colonies under these growthconditions, but a certain fraction of treated bacteria which haveundergone a second mutation in the hist
19、idine locus revert tohistidine-independence and are able to grow and form visiblecolonies. The number of such revertant colonies per agar plateis an indicator of the mutagenic potency of the test material.4.3 Typically, the test is conducted using a number ofbacterial strains selectively sensitive t
20、o various chemicalclasses of mutagens. Treatment with test compound is carriedout in the presence and absence of a rodent liver extractcapable of mimicking in vivo metabolic activation of promu-tagenic compounds (see 3.2 for a listing of terms and abbre-viations used.) With this combination of test
21、conditions, theAmes test becomes a very effective screening tool for chemicalmutagens. Moreover, because many mutagens are also carcino-gens, the test is often used as a screen for carcinogenicpotential.4.4 Although the ability of the Ames test to assess carcino-genic potential is good for many clas
22、ses of compounds, it hasbeen shown to be generally unsuited to the testing of water-insoluble complex mixtures such as mineral oils. To circum-vent poor solubility and other difficulties, this test methodemploys an extraction of the test oil with DMSO to produceaqueous-compatible solutions which rea
23、dily interact with themetabolic activation system (S-9) and with the tester bacteria.The concentration of S-9 and of NADP cofactor are increasedrelative to the unmodified assay, and hamster rather than ratliver S-9 is used. The slope of the dose response curve relatingmutagenicity (TA98 revertants p
24、er plate) to the dose of extractadded is used as an index of mutagenic potency (MI).4.5 In this test method, the MI (the slope of the doseresponse curve, and a measure of mutagenic potency) of aDMSO extract of an oil is compared to the mutagenicityindices of other oil extracts whose dermal carcinoge
25、nicities areknown. By correlation, the potential dermal carcinogenicity ofthe test oil can be assessed.5. Significance and Use5.1 The test method is based on a modification of the AmesSalmonella mutagenesis assay. As modified, there is goodcorrelation with mouse skin-painting bioassay results forsam
26、ples of raw and refined lubricating oil process streams.5.2 Mutagenic potency in this modified assay and carcino-genicity in the skin-painting bioassay also correlate with thecontent of 3 to 7 ring PNAs, which include polynucleararomatic hydrocarbons and their heterocyclic analogs. Thestrength of th
27、ese correlations implies that PNAs are theprincipal mutagenic and carcinogenic species in these oils.Some of the methods that have provided evidence supportingthis view are referenced in Appendix X1.6. Interferences6.1 The test method is designed to detect mutagenicitymediated by PNAs derived from p
28、etroleum. The assay isdisproportionately sensitive to nitroaromatic combustion prod-ucts and as yet unidentified components of catalytically orthermally cracked stocks such as light or heavy cycle oils. Thelatter materials are not known to occur in virgin base oils.6.2 For petroleum refinery streams
29、 distilling in the rangeassociated with the production of naptha or kerosine or thelight end of atmospheric gas oil (that is, median boiling point2.0 have a high probability of beingcarcinogenic in a mouse skin-painting bioassay.11. Report11.1 Report the following information:11.1.1 Counts of revert
30、ant colonies per plate for each doseof the test article and for the solvent control (DMSO) plates.11.1.2 Counts of revertant colonies per plate for each doseof Reference Oil No. 1. One test of the positive control oil willserve for all test articles concurrently assayed.11.1.3 Amutagenicity index (M
31、I), mutagenic potency index(MPI) or other quantitative estimate of mutagenicity calculatedby suitable regression analysis of the dose-response curve formutagenicity (10.1).11.1.4 Categorization of the probable dermal carcinogenicpotential of the test article, using the criteria cited in 10.2.12. Pre
32、cision and Bias12.1 Precision:12.1.1 The fundamental data produced from the use of thistest method is an estimate of the mutagenic potency of test oils(MI). This value, which is calculated by the procedure detailedin 10.1.1, is used to categorize oils according to their potentialE1687044for dermal c
33、arcinogenicity, as measured using a standardmouse skin-painting bioassay (10.2.2).12.1.2 Therefore, there are two basic considerations inascertaining the precision of the test method: What are therepeatability and reproducibility of the assay in terms of MIdetermination, and what are the repeatabili
34、ty and reproducibil-ity of the categorization of dermal carcinogenic potential of theoils.12.1.3 The following discussion is based on the results of aninterlaboratory study conducted using five coded oil samplesand Reference Oil No. 1. Six laboratories participated in thestudy, each reporting data f
35、rom two independent assays.Mutagenic potency is represented by MI, the slope of thedose-response curve as determined by regression analysis. Forthe purposes of determining precision of the test method, MIwas determined using the steps in 10.1.1.12.1.4 Linear regression was used to fit data that show
36、ed alinear increase in revertants over the entire dose-range. Qua-dratic regression was used to fit data that exhibited a decline inthe rate of increase in revertants with dose at the high end ofthe dose range (a plateau). In addition, dose ranges for TestOils 2, 3, and Reference Oil No. 1 were trun
37、cated to the 20 Ldose and fit by linear regression analysis. The same regressionprocedure was used to fit the data from all laboratories for agiven oil.12.1.5 Repeatability of Mutagenicity Index Determination:12.1.5.1 Based on analysis of the repeat assay data from thesix laboratories participating
38、in the interlaboratory study, Table2 illustrates intralaboratory repeatability. Note that the methodused for the interlaboratory study was different from that nowrecommended in that extracts were not diluted to achievelinearity of dose response. However, the MIs obtained by linearregression analysis
39、 of the initial linear regions (up to 20L/plate) should be similar to those obtained for dilutedextracts. Repeatability and reproducibility of MI determinationon diluted extracts would be expected to be somewhat bettersince the entire dose range is used in the calculation.12.1.5.2 Standard deviation
40、s ranged from a low of zero to ahigh of 50 % of the mean of the two replicates for those oilswith MI greater than 0.5. (Percent standard deviations for OilNo. 1 were higher in tests where MIs were less than 0.5, andrevertant increases were barely significant or not significantrelative to the solvent
41、 control (Laboratories A, B, and D).These deviations were not considered an accurate reflection ofthe repeatability of the assay.)12.1.6 Reproducibility of Mutagenicity Index Determina-tion:12.1.6.1 The data in Table 3 show the interlaboratoryreproducibility of MI determination in six testing labora
42、tories.12.1.6.2 Standard deviations of the mean MIs from sixdeterminations for each oil range from a low of 14 % of meanto a high of 67 % of mean for the weakly active Test Oil No.1. For oils with MIs 0.5, the highest standard deviation as apercentage of mean was for Test Oil No. 4 29 %. Theseresult
43、s indicate that interlaboratory reproducibility is similar tointralaboratory repeatability.12.1.7 Repeatability and Reproducibility of Assignment ofOils to Categories of Dermal Carcinogenic Potential:12.1.7.1 Table 4 provides an analysis of the repeatabilityand reproducibility of assignment to categ
44、ories of dermalcarcinogenic potential based on MI for six test oils evaluated insix laboratories.12.1.7.2 The data in Table 4 indicate that the originalmethod produced MIs leading to consistent classificationaccording to dermal carcinogenic potential in thirty-two out ofthe thirty-six tests. Two of
45、the four inconsistently classified oils(5D and 5F) were very near the boundary with the consistentgroup. All of the tests that led to inconsistent classificationwere paired with tests that indicated a need for corroborativedata for correct classification. The revised method changed theTABLE 2 Repeat
46、ability of Duplicate MI Determinations of Six Oils in Six LaboratoriesNOTE 1The first row of data for each oil provides the replicate MIs for the two tests. The second row of data is the mean and standard deviationfor the duplicate MI determinationsTest OilMutagenicity IndexLaboratoryABCDEF1 0.1, 0.
47、3 0, 0.3 0.7, 0.6 0.2, 0.1 0.3, 0.5 0.1, 0.10.20 6 0.14 0.15 6 0.21 0.65 6 0.07 0.15 6 0.07 0.40 6 0.14 0.1 6 02 2.1, 2.1 3.2, 2.5 2.4, 2.5 3.3, 3.1 2.8, 3.7 4.4, 3.22.1 6 02.96 0.50 2.5 6 0.07 3.26 0.14 3.3 6 0.64 3.8 6 0.851.7, 1.8 3.2, 3.5 2.3, 2.2 2.8, 1.9 2.2, 2.8 3.7, 3.31.86 0.07 3.4 6 0.21 2
48、.3 6 0.07 2.4 6 0.64 2.5 6 0.42 3.56 0.283 1.4, 3.0 3.0, 3.4 2.6, 2.6 3.4, 2.3 3.0, 3.3 3.0, 2.32.2 6 1.1 3.2 6 0.28 2.6 6 02.96 0.78 3.2 6 0.21 2.7 6 0.501.4, 2.0 2.0, 2.0 2.2, 2.2 2.4, 2.0 2.3, 2.5 2.2, 1.81.7 6 0.42 2.0 6 02.26 02.6 0.28 2.4 6 0.14 2.0 6 0.284 1.0, 1.2 1.0, 1.3 2.4, 2.0 1.3, 1.4
49、1.3, 1.3 1.7, 1.11.1 6 0.14 1.2 6 0.21 2.2 6 0.28 1.4 6 0.07 1.36 01.46 0.425 0.4, 0.8 0.6, 0.4 0.8, 0.7 1.1, 0.8 0.9, 0.8 0.9, 1.00.60 6 0.28 0.50 6 0.14 0.75 6 0.07 1.0 6 0.21 0.85 6 0.07 1.0 6 0.07Reference Oil 3.9, 3.1 3.3, 3.4 4.1, 4.0 3.4, 5.1 3.8, 4.4 5.7, 4.23.5 6 0.57 3.4 6 0.07 4.16 0.07 4.3 6 1.2 4.1 6 0.42 5.0 6 1.12.8, 2.9 3.1, 3.3 3.2, 3.2 3.5, 3.7 2.9, 3.7 4.7, 3.62.9 6 0.07 3.2 6 0.14 3.2 6 03.66 0.14 3.36 0.57 4.2 6 0.78E1687045classification of results for six tests as shown in Table 4. Theassay designations in bold type are new categories for theass
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