ASTM E1687-98

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An American National Standard
Designation: E 1687 – 98
Standard Test Method for
Determining Carcinogenic Potential of Virgin Base Oils in
Metalworking Fluids
This standard is issued under the fixed designation E 1687; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope benzopyrene (5), dibenzopyrene (6), coronene (7). Heterocy-
clic polynuclear compounds are also included in the definition.
1.1 This test method covers a microbiological test procedure
3.1.3 promutagenic compounds, promutagens,
based upon the Salmonella mutagenesis assay of Ames et al
n—compounds that are not directly mutagenic but require
(1) (see also Maron et al (2)). It can be used as a screening
metabolism for expression of mutagenic activity.
technique to detect the presence of potential dermal carcino-
3.1.4 Reference Oil 1, n—straight-run naphthenic vacuum
gens in virgin base oils used in the formulation of metalwork-
distillate (heavy vacuum gas oil) of known MI and PNA
ing oils. Persons who perform this test should be well-versed in
content recommended for use as a reference standard for the
the conduct of the Ames test and conversant with the physical
modified Ames test.
and chemical properties of petroleum products.
3.2 Abbreviations:Abbreviations:
1.2 The test method is not recommended as the sole testing
3.2.1 DMSO (Dimethyl Sulfoxide), n—extraction agent used
procedure for oils which have viscosities less than 18 cSt (90
in the preparation of aromatic-enriched oil fractions for mu-
SUS) at 40°C, or for formulated metalworking fluids.
tagenicity testing.
1.3 The values stated in SI units are to be regarded as the
3.2.2 G-6-P (Glucose-6-Phosphate), n—substrate required
standard. The values given in parentheses are provided for
for the operation of the NADPH generating system involved in
information only.
the biological oxidations described above.
1.4 This standard does not purport to address all of the
3.2.3 MI (Mutagenicity Index), n—the slope of the dose-
safety concerns, if any, associated with its use. It is the
response curve for mutagenicity in the modified Ames test.
responsibility of the user of this standard to establish appro-
3.2.3.1 Discussion—MI is an index of relative mutagenic
priate safety and health practices and determine the applica-
potency.
bility of regulatory limitations prior to use. Section 7 provides
3.2.4 NADP (Nicotinamide Adenine Dinucleotide
general guidelines for safe conduct of this test method.
Phosphate)—required cofactor for the biological oxidations
2. Referenced Documents
involved in activation of PNA to their mutagenic forms.
3.2.5 PNA (Polynuclear Aromatics; also termed CA),
2.1 ASTM Standards:
n—polynuclear aromatic compounds.
29 CFR 1910.1450 Occupational Exposure to Hazardous
3.2.6 S-9, n—fraction prepared from hamster liver which
Chemical in Laboratories
contains the enzymes required for metabolic activation of
3. Terminology
PNAs to their mutagenic forms.
3.1 Definitions of Terms Specific to This Standard:
4. Summary of Test Method
3.1.1 base stock, n—the refined oil component of metal-
4.1 The Ames Salmonella mutagenicity assay is the most
working fluid formulations.
widely used short-term in vitro genotoxicity test. The assay
3.1.2 PCA (Polycyclic Aromatics), n—For the purposes of
employs specific strains of the bacterium Salmonella typhimu-
this test method, PCA refers to fused-ring polycyclic aromatic
rium that have been mutated at a genetic locus precluding the
compounds with three or more rings. For example, the hydro-
biosynthesis of the amino acid histidine which is required for
carbon series is represented by phenanthrene (3), pyrene (4),
growth and reproduction. Additional genetic alterations, some
of which are important markers of strain identity, are also
This test method is under the jurisdiction of ASTM Committee E34 on
present.
Occupational Health and Safety and is the direct responsibility of Subcommittee
4.2 The mutagenicity assay relies upon treating the bacteria
E34.50 on Health and Safety Standards for Metal Working Fluids.
with test material over a range of doses immediately below the
Current edition approved Apr. 10, 1998. Published June 1998. Originally
published as E 1687 – 95. Last previous edition E 1687 – 95. concentration showing significant toxicity to the bacteria.
The boldface numbers refer to the list of references at the end of this standard.
Treated bacteria are then grown on agar plates deficient in
Available from Superintendent of Documents, U.S. Government Printing
Office, Washington, DC 20402.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1687–98
histidine. Bacteria possessing the original mutation in the 6.2 For petroleum refinery streams distilling in the range
histidine locus cannot form colonies under these growth associated with the production of naptha or kerosine or the
conditions, but a certain fraction of treated bacteria which have light end of atmospheric gas oil (that is, median boiling point
undergone a second mutation in the histidine locus revert to <250°C; viscosity< 18 cStat 40°C), the assay is sensitive to
histidine-independence and are able to grow and form visible detecting carcinogenicity related to the presence of polycyclic
colonies. The number of such revertant colonies per agar plate aromatic compounds. However, streams in the range, even
is an indicator of the mutagenic potency of the test material. those with MI less than 1.0, can produce tumors in a standard
4.3 Typically, the test is conducted using a number of mouse dermal carcinogenicity assay through alternative non-
bacterial strains selectively sensitive to various chemical genotoxic mechanisms.
classes of mutagens. Treatment with test compound is carried
out in the presence and absence of a rodent liver extract
7. Hazards
capable of mimicking in vivo metabolic activation of promu-
7.1 The test materials and positive control compounds used
tagenic compounds (see 3.2 for a listing of terms and abbre-
in this assay may present a carcinogenic hazard by ingestion or
viations used.) With this combination of test conditions, the
skin contact. Avoid all contact with test oils and Reference Oil
Ames test becomes a very effective screening tool for chemical
No. 1.
mutagens. Moreover, because many mutagens are also carcino-
7.2 The tester bacteria are attenuated and unlikely to cause
gens, the test is often used as a screen for carcinogenic
illness. However, gloves should be worn during handling of
potential.
bacteria, and care should be taken to avoid injuries with
4.4 Although the ability of the Ames test to assess carcino-
syringes and hypodermic needles contaminated with bacterial
genic potential is good for many classes of compounds, it has
cultures. Waste material generated during testing should be
been shown to be generally unsuited to the testing of water-
regarded as a potential biohazard and disposed of accordingly.
insoluble complex mixtures such as mineral oils. To circum-
Reference 3 provides general guidelines for safe use of this
vent poor solubility and other difficulties, this test method
test method.
employs an extraction of the test oil with DMSO to produce
7.3 Provisions for the safe use of this test method should be
aqueous-compatible solutions which readily interact with the
incorporated into the employer’s compliance with 29 CFR
metabolic activation system (S-9) and with the tester bacteria.
1910.1450.
The concentration of S-9 and of NADP cofactor are increased
relative to the unmodified assay, and hamster rather than rat
8. Materials and Methods
liver S-9 is used. The slope of the dose response curve relating
8.1 Test Organism—Methods for storage, culture, and char-
mutagenicity (TA98 revertants per plate) to the dose of extract
acterization of the test organism are exactly as described by
added is used as an index of mutagenic potency (MI).
Ames et al (1). The test organism used in this assay is
4.5 In this test method, the MI (the slope of the dose
Salmonella typhimurium strain TA98 derived from an original
response curve, and a measure of mutagenic potency) of a
stock produced and supplied by B. N. Ames, University of
DMSO extract of an oil is compared to the mutagenicity
California, Berkeley. Strain TA98 was selected for the test
indices of other oil extracts whose dermal carcinogenicities are
because it is the most sensitive to the class of mutagens present
known. By correlation, the potential dermal carcinogenicity of
in petroleum materials (PNAs) (Hermann et al (4)).
the test oil can be assessed.
8.1.1 Strain TA98 was derived from strain TA1538, and has
5. Significance and Use
the same genetic markers as that strain, including histidine/
biotin requirement, crystal violet sensitivity, and ultraviolet
5.1 The test method is based on a modification of the Ames
sensitivity. In addition, TA98 contains plasmid pKM101,
Salmonella mutagenesis assay. As modified, there is good
which confers ampicillin resistance. Full characterization of
correlation with mouse skin-painting bioassay results for
strain TA98 has been published by Ames et al (1).
samples of raw and refined lubricating oil process streams.
8.1.2 Strain TA98 can be inoculated, either from frozen
5.2 Mutagenic potency in this modified assay and carcino-
stocks maintained at − 80 6 5°C or from master plates
genicity in the skin-painting bioassay also correlate with the
maintained at approximately 4°C, into 25 mL of Oxoid No. 2
content of 3 to 7 ring PNAs, which include polynuclear
nutrient broth in a 125 mL erlenmeyer flask equipped with a
aromatic hydrocarbons and their heterocyclic analogs. The
screw cap. The flask is placed into a shaker-incubator set at
strength of these correlations implies that PNAs are the
approximately 37°C and 100 to 120 rpm. Sixteen hours later, 2
principal mutagenic and carcinogenic species in these oils.
mL of the culture is diluted into 8 mL of fresh Oxoid No. 2, and
Some of the methods that have provided evidence supporting
allowed to regrow for 3 h, or until the turbidity of the regrown
this view are referenced in Appendix X1.
culture, measured spectrophotometrically at 650 nm, is in the
6. Interferences
range from 1.0 to 2.0 absorbance units. A second check on cell
6.1 The test method is designed to detect mutagenicity density may be obtained by serially diluting the culture by a
mediated by PNAs derived from petroleum. The assay is factor of 10 into phosphate-buffered saline (PBS), and plating
disproportionately sensitive to nitroaromatic combustion prod- 1 mL of the resultant dilution onto nutrient agar plates
ucts and as yet unidentified components of catalytically or containing 0.5 % NaCl. After 44 to 48 h incubation at approxi-
thermally cracked stocks such as light or heavy cycle oils. The mately 37°C, the number of colonies can be determined
latter materials are not known to occur in virgin base oils. immediately, or the plates may be refrigerated at 5 6 3°C for
E1687–98
up to five days, and the cell density of the culture calculated Reference Oil No. 1. This oil is tested as part of each assay
from the net dilution factor. Acceptable values range from 1 to according to the procedures outlined in 8.6.
3 3 10 cells/mL. 8.5.2 Assay acceptability is determined using the data
8.2 Sampling and Handling of Oils—Sampling of oils generated for Reference Oil No. 1. An assay is deemed
should be performed with consideration of viscosity and other acceptable only if both of the following criteria are met:
physical properties to ensure that test specimens are represen- 8.5.2.1 Revertant colony counts for the DMSO extract of
tative. Whenever possible, oils should be stored at room Reference Oil No. 1, diluted 1:3 (one volume of oil plus three
temperature in amber bottles under nitrogen to avoid photore- volumes of DMSO), must reach, in a dose-responsive manner,
activity. at least twice the representative mean solvent control value for
8.3 Preparation of DMSO Extract—The mutagenic compo- the method, that is, must exceed 2 3 46 = 92 revertant
nents of oils are extracted into DMSO prior to testing. For oils colonies/plate.
with viscosities low enough to permit accurate volumetric 8.5.2.2 No more than three doses may produce mean rever-
dispensing (< approximately 200 cSt at 40°C), 1 mL of the oil tant counts more than 15 % below the representative mean at
is measured into a 15 mL tube, and 5 mL of reagent grade that dose. The representative data to be used in this analysis are
DMSO added. Volumes of oil rather than 1 mL may be used so provided in Table A2.1.
long as the 1:5 volume ratio of oil to DMSO is preserved. The 8.5.3 For assays done with a single extract and an indepen-
tube is vortexed vigorously either continuously or intermit- dent repeat, three solvent control plates per assay serve as a
tently for a 30-min period to ensure thorough contact between blank (see 8.5.2.1). If a single assay is done on three extracts
the oil and DMSO layers. The sample is then centrifuged for 10 of the test material, two solvent control plates per extract
min in a table-top centrifuge to effect phase separation should be used. The mean revertant count for these plates
(200 3 g). A portion of the lower, DMSO layer, is withdrawn should not fall below 30 colonies/plate or exceed 60 colonies/
with a pipet and reserved for testing. plate. If either of these conditions occur, the effect on the dose
8.4 Preparation of Metabolic Activation Mixture (S-9): response curves of Reference Oil No. 1 and the test materials
8.4.1 Aroclor 1254-induced liver S-9 from Syrian golden should be assessed. If there is a significant change in the slopes
hamsters is prepared according to the following procedure: of those curves, which is directly attributable to the effects of
Adult male hamsters, weighing between 90 and 100 g, are the out-of-range solvent controls, then the assay should be
induced by a single intraperitoneal injection of Aroclor 1254 at repeated.
a dose of 500 mg/kg body weight. Five days after induction,
9. Procedure
the hamsters are sacrificed, the livers are aseptically removed
and rinsed in cold, sterile suspending buffer (isotonic KCl) and
9.1 Perform the following steps in order:
homogenized in a Polytron Tissuemizer at a concentration of
9.1.1 Prepare dosing solutions for the test article and Ref-
1:3 (wet liver wt:volume of suspending buffer).
erence Oil No. 1 by diluting the DMSO extracts
...