1、Designation: E1687 10 (Reapproved 2014) An 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 ofo
2、riginal adoption or, 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 procedurebase
3、d upon the Salmonella 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 in
4、the conduct of the 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
5、in SI units are 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 saf
6、ety and health practices 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 Metalworkingor Metal Removal Fluid Health
7、and SafetyE2523 Terminology 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 componen
8、t of metal-working 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. Forexample, the hydrocarbon series is represented by phenan-threne (3), pyrene (4), benzopyrene (5)
9、, dibenzopyrene (6),coronene (7). Heterocyclic polynuclear compounds are alsoincluded in the 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 vacu
10、umdistillate (heavy 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 muta-genicity testing.3.2.2 G-6-P (G
11、lucose-6-Phosphate), 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 m
12、utagenicpotency.3.2.4 NADP (Nicotinamide Adenine DinucleotidePhosphate)required cofactor for the biological oxidationsinvolved in activation of PAC to their mutagenic forms.1This test method is under the jurisdiction of ASTM Committee E34 onOccupational Health and Safety and is the direct responsibi
13、lity of SubcommitteeE34.50 on Health and Safety Standards for Metal Working Fluids.Current edition approved Oct. 15, 2014. Published October 2014. Originallyapproved in 1995. Last previous edition approved in 2010 as E1687 - 10. DOI:10.1520/E1687-10R14.2The boldface numbers refer to the list of refe
14、rences 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 page onthe ASTM website.4Available from U.S. Gover
15、nment Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.2.5 PAC (Polycyclic Aromatic Compounds), npolyc
16、yclicaromatic compounds.3.2.6 S-9, nfraction prepared from hamster liver whichcontains the 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 a
17、ssayemploys specific strains of the bacterium Salmonella typhimu-rium that have been mutated 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,
18、are alsopresent.4.2 The mutagenicity assay relies upon treating the bacteriawith test material 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
19、mutation in thehistidine locus cannot form colonies under these growthconditions, but a certain 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 coloni
20、es 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 to various chemicalclasses of mutagens. Treatment with test compound is carriedout in the presence and absence of a rodent live
21、r extractcapable of mimicking in vivo metabolic activation of promuta-genic compounds (see 3.2 for a listing of terms and abbrevia-tions used). With this combination of test conditions, theAmestest becomes a very effective screening tool for chemicalmutagens. Moreover, because many mutagens are also
22、carcinogens, 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 classes of compounds, it hasbeen shown to be generally unsuited to the testing of water-insoluble complex mixtures such as mineral oi
23、ls. To circum-vent poor solubility and other difficulties, this test methodemploys an 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 cofacto
24、r 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 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
25、 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 carcinogenicities areknown. By correlation, the potential dermal carcinogenicity ofthe test oil can be assessed.5. Significance and Use5.1
26、 The test method is based on a modification of the AmesSalmonella mutagenesis assay. As 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
27、skin-painting bioassay also correlate with thecontent of 3 to 7 ring PACs, which include polycyclic aromatichydrocarbons and their heterocyclic analogs. The strength ofthese correlations implies that PACs are the principal muta-genic and carcinogenic species in these oils. Some of themethods that ha
28、ve provided evidence supporting this view arereferenced in Appendix X1.6. Interferences6.1 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 c
29、atalytically 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 distilling in the rangeassociated with the production of naptha or kerosine or thelight end of atmospheric gas oil (that is, me
30、dian 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 revertant colonies per plate for each doseof the test article and for the solvent control (DMSO) plates.11.1.2 Counts of revertant col
31、onies 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 (MI), mutagenic potency index(MPI) or other quantitative estimate of mutagenicity calculatedby suitable regression analysis of the
32、 dose-response curve formutagenicity (10.1).E1687 10 (2014)411.1.4 Categorization of the probable dermal 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 estimat
33、e 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 potentialfor dermal carcinogenicity, as measured using a standardmouse skin-painting bioassay (10.2.2).12.1.2 Therefore, there are two basic
34、considerations inascertaining the precision of the test method: What are therepeatability and 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 discussio
35、n is based on the results of aninterlaboratory study conducted using five coded oil samplesand 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 partici
36、pated in the study, each reportingdata from two independent assays. Mutagenic potency isrepresented 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
37、 regression was used to fit data that showed 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 TestOi
38、ls 2, 3, and Reference Oil No. 1 were truncated 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 da
39、ta from thesix laboratories participating 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
40、 MIs obtained by linearregression analysis 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 t
41、he calculation.12.1.5.2 Standard deviations 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
42、 or not significantrelative to the solvent 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
43、 of MI determination in six testing laboratories.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 mea
44、n was for Test Oil No. 4 29 %. Theseresults 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 repeatability
45、and reproducibility of assignment to categories of dermalcarcinogenic potential based on MI for six test oils evaluated insix laboratories.TABLE 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 t
46、wo tests. The second row of data is the mean and standard deviationfor the duplicate MI determinationsTest OilMutagenicity IndexLaboratoryABCDEF1 0.1, 0.3 0, 0.3 0.7, 0.6 0.2, 0.1 0.3, 0.5 0.1, 0.10.20 0.14 0.15 0.21 0.65 0.07 0.15 0.07 0.40 0.14 0.1 02 2.1, 2.1 3.2, 2.5 2.4, 2.5 3.3, 3.1 2.8, 3.7 4
47、.4, 3.22.10 2.90.50 2.50.07 3.20.14 3.30.64 3.80.851.7, 1.8 3.2, 3.5 2.3, 2.2 2.8, 1.9 2.2, 2.8 3.7, 3.31.8 0.07 3.4 0.21 2.3 0.07 2.4 0.64 2.5 0.42 3.5 0.283 1.4, 3.0 3.0, 3.4 2.6, 2.6 3.4, 2.3 3.0, 3.3 3.0, 2.32.21.1 3.20.28 2.60 2.90.78 3.20.21 2.70.501.4, 2.0 2.0, 2.0 2.2, 2.2 2.4, 2.0 2.3, 2.5
48、2.2, 1.81.70.42 2.00 2.20 2.20.28 2.40.14 2.00.284 1.0, 1.2 1.0, 1.3 2.4, 2.0 1.3, 1.4 1.3, 1.3 1.7, 1.11.10.14 1.20.21 2.20.28 1.40.07 1.30 1.40.425 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 0.28 0.50 0.14 0.75 0.07 1.0 0.21 0.85 0.07 1.0 0.07Reference Oil 3.9, 3.1 3.3, 3.4 4.1, 4.0
49、 3.4, 5.1 3.8, 4.4 5.7, 4.23.50.57 3.40.07 4.10.07 4.31.2 4.10.42 5.01.12.8, 2.9 3.1, 3.3 3.2, 3.2 3.5, 3.7 2.9, 3.7 4.7, 3.62.90.07 3.20.14 3.20 3.60.14 3.30.57 4.20.78E1687 10 (2014)512.1.7.2 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) were very near the boundary with the consistentgroup. All o
