ASTM E1687-19

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E1687 − 10 (Reapproved 2014) E1687 − 19 An American National Standard
Standard Test Method for
Determining Carcinogenic Potential of Virgin Base Oils in
Metalworking Fluids
This standard is issued under the fixed designation E1687; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope Scope*
1.1 This test method covers a microbiological test procedure based upon the Salmonella mutagenesis assay of Ames et alal. (1)
(see also Maron et alal. (2)). It can be used as a screening technique to detect the presence of potential dermal carcinogens in virgin
base oils used in the formulation of metalworking oils. Persons who perform this test should be well-versed well versed in the
conduct of the Ames test and conversant with the physical and chemical properties of petroleum products.
1.2 The test method is not recommended as the sole testing procedure for oils which have viscosities less than 18 cSt (90 SUS)
(90 SUS) at 40°C,40 °C, or for formulated metalworking fluids.
1.3 The values stated in SI units are to be regarded as the standard. The values given in parentheses are provided for information
only.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use. Section 7 provides general guidelines for safe conduct of this test method.
1.5 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
E2148 Guide for Using Documents Related to Metalworking or Metal Removal Fluid Health and Safety
E2523 Terminology for Metalworking Fluids and Operations
2.2 Other Standards:Standard:
29 CFR 1910.1450 Occupational Exposure to Hazardous Chemical in Laboratories
3. Terminology
3.1 For definitions of terms used in this test method, see Terminology E2523.
3.2 Definitions of Terms Specific to This Standard: (See also Terminology E2523.)
3.2.1 base stock, n—the refined oil component of metalworking fluid formulations.
3.2.2 PAC (Polycyclic Aromatic Compounds), n—Forfor the purposes of this test method, PAC refers to fused-ring polycyclic
aromatic compounds with three or more rings. For example, the hydrocarbon series is represented by phenanthrene (3), pyrene (4),
benzopyrene (5), dibenzopyrene (6), coronene (7). Heterocyclic polynuclear compounds are also included in the definition.
3.2.3 promutagenic compounds, promutagens, n—compounds that are not directly mutagenic but require metabolism for
expression of mutagenic activity.
This test method is under the jurisdiction of ASTM Committee E34 on Occupational Health and Safety and is the direct responsibility of Subcommittee E34.50 on Health
and Safety Standards for Metal Working Fluids.
Current edition approved Oct. 15, 2014Oct. 1, 2019. Published October 2014October 2019. Originally approved in 1995. Last previous edition approved in 20102014 as
E1687 - 10.E1687 – 10 (2014). DOI: 10.1520/E1687-10R14.10.1520/E1687-19.
The boldface numbers in parentheses refer to thea list of references at the end of this standard.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Available from U.S. Government Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
www.access.gpo.gov.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1687 − 19
3.2.4 Reference Oil 1, n—straight-run naphthenic vacuum distillate (heavy vacuum gas oil) of known MI and PAC content
recommended for use as a reference standard for the modified Ames test.
3.3 Abbreviations:
3.3.1 DMSO (Dimethyl Sulfoxide), n—extraction agent used in the preparation of aromatic-enriched oil fractions for
mutagenicity testing.
3.3.2 G-6-P (Glucose-6-Phosphate), n—substrate required for the operation of the NADPH generating system involved in the
biological oxidations described above.
3.3.3 MI (Mutagenicity Index), n—the slope of the dose-response curve for mutagenicity in the modified Ames test.
3.3.3.1 Discussion—
MI is an index of relative mutagenic potency.
3.3.4 NADP (Nicotinamide Adenine Dinucleotide Phosphate)—required cofactor for the biological oxidations involved in
activation of PAC to their mutagenic forms.
3.3.5 PAC (Polycyclic Aromatic Compounds), n—polycyclic aromatic compounds.
3.3.6 S-9, n—fraction prepared from hamster liver which contains the enzymes required for metabolic activation of PACs to
their mutagenic forms.
4. Summary of Test Method
4.1 The Ames Salmonella mutagenicity assay is the most widely used short-term in vitro genotoxicity test. The assay employs
specific strains of the bacterium Salmonella typhimurium that have been mutated at a genetic locus precluding the biosynthesis of
the amino acid histidine, which is required for growth and reproduction. Additional genetic alterations, some of which are
important markers of strain identity, are also present.
4.2 The mutagenicity assay relies upon treating the bacteria with test material over a range of doses immediately below the
concentration showing significant toxicity to the bacteria. Treated bacteria are then grown on agar plates deficient in histidine.
Bacteria possessing the original mutation in the histidine locus cannot form colonies under these growth conditions, but a certain
fraction of treated bacteria which have undergone a second mutation in the histidine locus revert to histidine-independence and are
able to grow and form visible colonies. The number of such revertant colonies per agar plate is an indicator of the mutagenic
potency of the test material.
4.3 Typically, the test is conducted using a number of bacterial strains selectively sensitive to various chemical classes of
mutagens. Treatment with test compound is carried out in the presence and absence of a rodent liver extract capable of mimicking
in vivo metabolic activation of promutagenic compounds (see 3.23.3 for a listing of terms and abbreviations used). With this
combination of test conditions, the Ames test becomes a very effective screening tool for chemical mutagens. Moreover, because
many mutagens are also carcinogens, the test is often used as a screen for carcinogenic potential.
4.4 Although the ability of the Ames test to assess carcinogenic potential is good for many classes of compounds, it has been
shown to be generally unsuited to the testing of water-insoluble complex mixtures such as mineral oils. To circumvent poor
solubility and other difficulties, this test method employs an extraction of the test oil with DMSO to produce aqueous-compatible
solutions which readily interact with the metabolic activation system (S-9) and with the tester bacteria. The concentration of S-9
and of NADP cofactor are increased relative to the unmodified assay, and hamster rather than rat liver S-9 is used. The slope of
the dose response curve relating mutagenicity (TA98 revertants per plate) to the dose of extract added is used as an index of
mutagenic potency (MI).
4.5 In this test method, the MI (the slope of the dose response curve, and a measure of mutagenic potency) of a DMSO extract
of an oil is compared to the mutagenicity indices of other oil extracts whose dermal carcinogenicities are known. By correlation,
the potential dermal carcinogenicity of the test oil can be assessed.
5. Significance and Use
5.1 The test method is based on a modification of the Ames Salmonella mutagenesis assay. As modified, there is good
correlation with mouse skin-painting bioassay results for samples of raw and refined lubricating oil process streams.
5.2 Mutagenic potency in this modified assay and carcinogenicity in the skin-painting bioassay also correlate with the content
of 3three to 7 ring seven-ring PACs, which include polycyclic aromatic hydrocarbons and their heterocyclic analogs. The strength
of these correlations implies that PACs are the principal mutagenic and carcinogenic species in these oils. Some of the methods
that have provided evidence supporting this view are referenced in Appendix X1.
E1687 − 19
6. Interferences
6.1 The test method is designed to detect mutagenicity mediated by PACs derived from petroleum. The assay is
disproportionately sensitive to nitroaromatic combustion products and as yet as-yet unidentified components of catalytically or
thermally cracked stocks such as light or heavy cycle oils. The latter materials are not known to occur in virgin base oils.
6.2 For petroleum refinery streams distilling in the range associated with the production of naptha or kerosine or the light end
of atmospheric gas oil (that is, median boiling point <250°C;<250 °C; viscosity < 18 <18 cSt at 40°C),40 °C), the assay is sensitive
to detecting carcinogenicity related to the presence of polycyclic aromatic compounds. However, streams in the range, even those
with MI less than 1.0, can produce tumors in a standard mouse dermal carcinogenicity assay through alternative non-genotoxic
mechanisms.
7. Hazards
7.1 The test materials and positive control compounds used in this assay may present a carcinogenic hazard by ingestion or skin
contact. Avoid all contact with test oils and Reference Oil No. 1.
7.2 The tester bacteria are attenuated and unlikely to cause illness. However, gloves should be worn during handling of bacteria,
and care should be taken to avoid injuries with syringes and hypodermic needles contaminated with bacterial cultures. Waste
material generated during testing should be regarded as a potential biohazard and disposed of accordingly. Reference (3) provides
general guidelines for safe use of this test method.
7.3 Provisions for the safe use of this test method should be incorporated into the employer’s compliance with 29 CFR
1910.1450.
8. Materials and Methods
8.1 Test Organism—Methods for storage, culture, and characterization of the test organism are exactly as described by Ames
et alal. (1). The test organism used in this assay is Salmonella typhimurium strain TA98 derived from an original stock produced
and supplied by B. N. Ames, University of California, Berkeley. Strain TA98 was selected for the test because it is the most
sensitive to the class of mutagens present in petroleum materials (PACs) (Hermann et alal. (4)).
8.1.1 Strain TA98 was derived from strain TA1538, and has the same genetic markers as that strain, including histidine/biotin
requirement, crystal violet sensitivity, and ultraviolet sensitivity. In addition, TA98 contains plasmid pKM101, which confers
ampicillin resistance. Full characterization of strain TA98 has been published by Ames et alal. (1).
8.1.2 Strain TA98 can be inoculated, either from frozen stocks maintained at − 80at −80 6 5°C5 °C or from master plates
maintained at approximately 4°C,4 °C, into 25 mL of Oxoid No. 2 nutrient broth in a 125 mL erlenmeyer flask equipped with a
screw cap. The flask is placed into a shaker-incubator set at approximately 37°C37 °C and 100 to 120 rpm. Approximately 16
hoursh later, 3 mL of the culture is diluted into 12 mL of fresh Oxoid No. 2, and allowed to regrow for 3 h, or until the turbidity
of the regrown culture, measured spectrophotometrically at 650 nm, 650 nm, is in the range from 1.0 to 2.0 absorbance units. A
second check on cell density may be obtained by serially diluting the culture by a factor of 10 into phosphate-buffered saline
(PBS), and plating 1 mL of the resultant dilution onto nutrient agar plates containing 0.5 % NaCl. After 44 to 48 h incubation at
approximately 37°C,37 °C, the number of colonies can be determined immediately, or the plates may be refrigerated at 5 6
3°C3 °C for up to five days, and the cell density of the culture calculated from the net dilution factor. Acceptable values range from
1 to 3 × 10 cells/mL.
8.2 Sampling and Handling of Oils—Sampling of oils should be performed with consideration of viscosity and other physical
properties to ensure that test specimens are representative. When possible, oils should be stored at room temperature in amber
bottles under nitrogen to avoid photoreactivity.
8.3 Preparation of DMSO Extract—The mutagenic components of oils are extracted into DMSO prior to testing. For oils with
viscosities low enough to permit accurate volumetric dispensing (< (less than approximately 200 cSt at 40°C),40 °C), 0.2 mL of
the oil is measured into a 13 by 100 mm glass test tube, and 1 mL 1 mL of reagent grade DMSO added. Volumes of oil other than
0.2 mL 0.2 mL may be used so long as the 1:5 volume ratio of oil to DMSO is preserved. The tube is vortexed vigorously either
continuously or intermittently for a 30-min period to ensure thorough contact between the oil and DMSO layers. The sample is
then centrifuged for 10 min in a table-top centrifuge to effect phase separation (200 × g). A portion of the lower,lower DMSO
layer,layer is withdrawn with a pipet and reserved for testing.
8.4 Preparation of Metabolic Activation Mixture (S-9):
8.4.1 Aroclor 1254-induced liver S-9 from Syrian golden hamsters is prepared according to the following procedure: Adultadult
male hamsters, weighing between 90 and 100 g, are induced by a single intraperitoneal injection of Aroclor 1254 at a dose of 500
mg/kg body weight. Five days after induction, the hamsters are sacrificed, the livers are aseptically removed and rinsed in cold,
sterile suspending buffer (isotonic KCl) and homogenized in a Polytron Tissuemizer at a concentration of 1:3 (wet liver wt:volume
of suspending buffer).
E1687 − 19
A
TABLE 1 Dosing Solutions
Dose, μL/Plate
0 12 24 36 48 60
μL Extract 0 36 72 108 144 180
μL DMSO 180 144 108 72 36 0
A
Other dosing regimens over the range 0 to 60 μL may be used.
8.4.2 The supernatant fraction (S-9) is collected following centrifugation at 9000 × g for 10 min in a centrifuge maintained at
approximately 4°C.4 °C. The supernatant is then portioned into aliquots of 5 mL each and stored frozen at − 80at −80 6 5°C5 °C
until used.
8.4.3 S-9 is thawed at approximately 4°C4 °C on the day of the test, and metabolic activation mixture sufficient for one test
article prepared is as follows:
8.4.4 To a sterile container at approximately 4°C4 °C are added in sequence 1.5 mL of 1 M sodium phosphate buffer, pH 7.4;
0.3 mL 0.25 M glucose-6-phosphate; 0.6 mL 0.2 M NADP; 0.6 mL 0.6 mL of a salt solution of 0.2 M MgCl /0.825 M KCl. To
the resulting solution, 12 mL of S-9 are added with gentle swirling.
8.4.5 All steps in the preparation and dispensing of S-9 and S-9 mixture must be performed at approximately 4°C.4 °C. S-9
mixture should not be stored for longer than 2 h prior to use; excess mixture should be discarded when the test is completed.
8.5 Calibration and Standardization:
8.5.1 Reference Standards and Blanks—The reference standard for this test method is a vacuum distillate designated Reference
Oil No. 1. This oil is tested as part of each assay according to the procedures outlined in 8.6.Section 9.
8.5.2 Assay acceptability is determined using the data generated for Reference Oil No. 1. An assay is deemed acceptable if the
revertant colony counts for the DMSO extract of Reference Oil No. 1, diluted 1:3 (one volume of oil plus three volumes of DMSO)
reach, in a dose-responsive manner, at least twice the representative mean solvent control value for that day’s test. (See 8.5.3 for
acceptable solvent control range.)
8.5.3 For assays done with a single extract and an independent repeat, three solvent control plates per assay serve as a blank
(see 8.5.2). If a single assay is done on three extracts of the test material, two solvent control plates per extract should be used.
The mean revertant count for these plates should not fall below 30 colonies/plate or exceed 60 colonies/plate. If either of these
conditions occur,occurs, the effect on the dose response curves of Reference Oil No. 1 and the test materials should be assessed.
If there is a significant change in the slopes of those curves, which is directly attributable to the effects of the out-of-range solvent
controls, then the assay should be repeated.
9. Procedure
9.1 Perform the following steps in order:
9.1.1 Prepare dosing solutions for the test article and Reference Oil No. 1 by diluting the DMSO extracts with DMSO to give
individual doses deliverable in 60 μL. A typical dosing schedule is shown in Table 1, but other dosing protocols are acceptable if
they provide at least four doses on the linear portion of the dose-response curve. For materials which produce a curvilinear dose
response, the original DMSO extract should be diluted with DMSO to yield a linear dose-response dose response over the 0 to 60
μL range. In general, oils with MIs greater than 1.0 will require dilution. A preliminary one plate/dose range-finding assay may
be done to determine the point at which the dose response begins to curve. Based on the results of this assay, the extract is diluted
sufficiently to produce approximately 100 revertants/plate at the 60 μL dose in the full assay.
9.1.2 Either of the following procedures may be used. For single-extract assays with independent repeat, dose three 13 by 100
mm sterile glass test tubes with 60 μL of each dosing solution. Measure doses with a positive displacement micropipet. All tubes
for a day’s test may be dosed together, but the following steps should be performed one test article (30 tubes) at a time.
9.1.3 Add 0.5 mL of S-9 mix to the bottom of each tube.
9.1.4 Add 0.1 mL of a well-mixed suspension of strain TA98 bacteria prepared as described in 8.1.2 to the bottom of each tube.
Bacteria should be maintained at ice temperature until used.
9.1.5 Incubate tubes at approximately 37°C37 °C on a gyratory shaker-incubator at 150 rpm for 20 min.
9.1.6 Add 2.0 mL of top agar to each tube (see Note 1). During dispensing, the top agar is placed on a dry block maintained
at approximately 37°C.37 °C. Vortex the mix, and pour the resulting agar mixture onto a 100 mm petri plate containing 30 mL of
bottom agar consisting of 1.5 % bacteriological grade agar in Vogel-Bonner Minimal E medium supplemented with 2 % dextrose.
NOTE 1—Each 100 mL of top agar contains 0.6 g bacteriological grade agar and 0.5 g NaCl. Top agar is melted, equilibrated to approximately
42°C,42 °C, and supplemented by addition of a volume of 0.5 millimolar histidine -0.5 millimolar biotin equal to 10 % of the original agar volume. The
top agar remains in the water bath until dispensing is complete.
9.1.7 Swirl the plate to obtain a layer of top agar of ev
...