1、Designation: E1687 10An American National StandardStandard Test Method forDetermining Carcinogenic Potential of Virgin Base Oils inMetalworking Fluids1This standard is issued under the fixed designation E1687; the number immediately following the designation indicates the year oforiginal adoption or
2、, in the case of revision, the year of last revision. A number in parentheses 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 microbiological test procedurebased upon the Salmonel
3、la 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 the
4、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 to
5、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 prac
6、tices 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:3E2148 Guide for Using Documents Related to Metalwork-ing or Metal Removal Fluid Health and SafetyE2523 T
7、erminology for Metalworking Fluids and Opera-tions2.2 Other Standards:29 CFR 1910.1450 Occupational Exposure to HazardousChemical in Laboratories43. Terminology3.1 Definitions of Terms Specific to This Standard: (See alsoTerminology E2523.)3.1.1 base stock, nthe refined oil component of metal-workin
8、g fluid formulations.3.1.2 PAC (Polycyclic Aromatic Compounds), nFor thepurposes of this test method, PAC refers to fused-ring polycy-clic aromatic compounds with three or more rings. For ex-ample, the hydrocarbon series is represented by phenanthrene(3), pyrene (4), benzopyrene (5), dibenzopyrene (
9、6), coronene(7). Heterocyclic polynuclear compounds are also included inthe definition.3.1.3 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 (hea
10、vy vacuum gas oil) of known MI and PACcontent recommended for use as a reference standard for themodified Ames test.3.2 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-Phosphat
11、e), nsubstrate requiredfor the operation of the NADPH generating system involved 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
12、.2.4 NADP (Nicotinamide Adenine DinucleotidePhosphate)required cofactor for the biological oxidationsinvolved in activation of PAC to their mutagenic forms.3.2.5 PAC (Polycyclic Aromatic Compounds),npolycyclic aromatic compounds.1This test method is under the jurisdiction of ASTM Committee E34 onOcc
13、upational Health and Safety and is the direct responsibility of SubcommitteeE34.50 on Health and Safety Standards for Metal Working Fluids.Current edition approved May 1, 2010. Published June 2010. Originallyapproved in 1995. Last previous edition approved in 2004 as E1687 - 04. DOI:10.1520/E1687-10
14、.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 of ASTMStandards volume information, refer to the standards Document Summary pa
15、ge 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, United States.3.2.6 S-9, nfraction prepared from hamster liver whichcontains th
16、e enzymes required for metabolic activation ofPACs to their mutagenic forms.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 muta
17、ted at a genetic locus precluding thebiosynthesis of the amino acid histidine which is required forgrowth 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 mat
18、erial over a range of doses immediately below theconcentration showing significant toxicity 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 c
19、ertain fraction of treated bacteria which haveundergone a second mutation in the histidine 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 Typica
20、lly, the test is conducted using a number ofbacterial strains selectively sensitive to 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 (se
21、e 3.2 for a listing of terms and abbre-viations used.) With this combination of test 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
22、 the ability of the Ames test to assess carcino-genic potential is good for many classes 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
23、extraction of the test oil with DMSO to produceaqueous-compatible solutions which readily 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
24、is used. The slope of the dose response curve relatingmutagenicity (TA98 revertants per 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 oi
25、l is compared to the mutagenicityindices of other oil extracts whose dermal carcinogenicities 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. A
26、s modified, there is goodcorrelation with mouse skin-painting bioassay results forsamples 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 PACs, which inclu
27、de polycyclic aromatichydrocarbons and their heterocyclic analogs. The strength ofthese correlations implies that PACs are the principal mu-tagenic and carcinogenic species in these oils. Some of themethods that have provided evidence supporting this view arereferenced in Appendix X1.6. Interference
28、s6.1 The test method is designed to detect mutagenicitymediated by PACs derived from petroleum. 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 m
29、aterials are not known to occur in virgin base oils.6.2 For petroleum refinery streams 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-pai
30、nting bioassay.11. Report11.1 Report the following information:11.1.1 Counts of revertant 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
31、oil willserve for all test articles concurrently assayed.11.1.3 Amutagenicity index (MI), 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 derm
32、al carcinogenicpotential of the test article, using the criteria cited in 10.2.12. Precision 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 detailed
33、in 10.1.1, is used to categorize oils according to their potentialfor dermal carcinogenicity, as measured using a standardmouse skin-painting bioassay (10.2.2).E1687 10412.1.2 Therefore, there are two basic considerations inascertaining the precision of the test method: What are therepeatability and
34、 reproducibility of the assay in terms of MIdetermination, and what are the repeatability 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 samplesan
35、d Reference Oil No. 1. This study was done prior to arevision in the method that advised dilution of DMSO extractsto produce linear responses over the 0 to 60 Ldose range (See9.1.1). Six laboratories participated in the study, each reportingdata from two independent assays. Mutagenic potency isrepre
36、sented by MI, the slope of the dose-response curve asdetermined by regression analysis. For the purposes of deter-mining precision of the test method, MI was determined usingthe steps in 10.1.1.12.1.4 Linear regression was used to fit data that showed alinear increase in revertants over the entire d
37、ose-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 truncated to the 20 Ldose and fit by linear regression
38、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 in the interlaboratory study, Table2 illustrates in
39、tralaboratory 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 of the initial linear regions (up to 20L/plate) sh
40、ould 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 deviations ranged from a low of zero to ahigh of 50 % of the
41、 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 control (Laboratories A, B, and D).These deviation
42、s 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 laboratories.12.1.6.2 Standard deviations of the mean MIs
43、 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 %. Theseresults indicate that interlaboratory reproducibility is
44、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 categories of dermalcarcinogenic potential based on MI f
45、or 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 the four inconsistently classified oils(5D and 5F)
46、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 theclassification of results for six tests as shown in Table 4. TheT
47、ABLE 2 Repeatability 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 IndexLaboratoryAB
48、CDEF1 0.1, 0.3 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
49、 3.4 6 0.21 2.3 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 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
