ASTM E1687-10(2014)
(Test Method)Standard Test Method for Determining Carcinogenic Potential of Virgin Base Oils in Metalworking Fluids
Standard Test Method for Determining Carcinogenic Potential of Virgin Base Oils in Metalworking Fluids
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 3 to 7 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.
SCOPE
1.1 This test method covers a microbiological test procedure based upon the Salmonella mutagenesis assay of Ames et al (1)2 (see also Maron et al (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 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) at 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 and health practices and determine the applicability of regulatory limitations prior to use. Section 7 provides general guidelines for safe conduct of this test method.
General Information
Relations
Buy Standard
Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E1687 − 10 (Reapproved 2014) 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.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2.2 Other Standards:
29 CFR 1910.1450Occupational Exposure to Hazardous
1.1 Thistestmethodcoversamicrobiologicaltestprocedure
Chemical in Laboratories
based upon the Salmonella mutagenesis assay of Ames et al
(1) (see also Maron et al (2)). It can be used as a screening
3. Terminology
technique to detect the presence of potential dermal carcino-
3.1 Definitions of Terms Specific to This Standard:(Seealso
gens in virgin base oils used in the formulation of metalwork-
Terminology E2523.)
ingoils.Personswhoperformthistestshouldbewell-versedin
3.1.1 base stock, n—the refined oil component of metal-
the conduct of theAmes test and conversant with the physical
working fluid formulations.
and chemical properties of petroleum products.
3.1.2 PAC (Polycyclic Aromatic Compounds), n—For the
purposes of this test method, PAC refers to fused-ring polycy-
1.2 The test method is not recommended as the sole testing
clic aromatic compounds with three or more rings. For
procedure for oils which have viscosities less than 18 cSt (90
example, the hydrocarbon series is represented by phenan-
SUS) at 40°C, or for formulated metalworking fluids.
threne (3), pyrene (4), benzopyrene (5), dibenzopyrene (6),
1.3 The values stated in SI units are to be regarded as the
coronene (7). Heterocyclic polynuclear compounds are also
standard. The values given in parentheses are provided for
included in the definition.
information only.
3.1.3 promutagenic compounds, promutagens,
n—compounds that are not directly mutagenic but require
1.4 This standard does not purport to address all of the
metabolism for expression of mutagenic activity.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro- 3.1.4 Reference Oil 1, n—straight-run naphthenic vacuum
distillate (heavy vacuum gas oil) of known MI and PAC
priate safety and health practices and determine the applica-
content recommended for use as a reference standard for the
bility of regulatory limitations prior to use. Section 7 provides
modified Ames test.
general guidelines for safe conduct of this test method.
3.2 Abbreviations:
2. Referenced Documents
3.2.1 DMSO (Dimethyl Sulfoxide), n—extractionagentused
3 in the preparation of aromatic-enriched oil fractions for muta-
2.1 ASTM Standards:
genicity testing.
E2148GuideforUsingDocumentsRelatedtoMetalworking
3.2.2 G-6-P (Glucose-6-Phosphate), n—substrate required
or Metal Removal Fluid Health and Safety
fortheoperationoftheNADPHgeneratingsysteminvolvedin
E2523Terminology for Metalworking Fluids and Opera-
the biological oxidations described above.
tions
3.2.3 MI (Mutagenicity Index), n—the slope of the dose-
response curve for mutagenicity in the modified Ames test.
3.2.3.1 Discussion—MI is an index of relative mutagenic
This test method is under the jurisdiction of ASTM Committee E34 on
potency.
Occupational Health and Safety and is the direct responsibility of Subcommittee
E34.50 on Health and Safety Standards for Metal Working Fluids.
3.2.4 NADP (Nicotinamide Adenine Dinucleotide
Current edition approved Oct. 15, 2014. Published October 2014. Originally
Phosphate)—required cofactor for the biological oxidations
approved in 1995. Last previous edition approved in 2010 as E1687-10. DOI:
involved in activation of PAC to their mutagenic forms.
10.1520/E1687-10R14.
The boldface numbers refer to the 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 AvailablefromU.S.GovernmentPrintingOfficeSuperintendentofDocuments,
Standards volume information, refer to the standard’s Document Summary page on 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
the ASTM website. www.access.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1687 − 10 (2014)
3.2.5 PAC (PolycyclicAromatic Compounds), n—polycyclic 5. Significance and Use
aromatic compounds.
5.1 The test method is based on a modification of theAmes
Salmonella mutagenesis assay. As modified, there is good
3.2.6 S-9, n—fraction prepared from hamster liver which
contains the enzymes required for metabolic activation of correlation with mouse skin-painting bioassay results for
samples of raw and refined lubricating oil process streams.
PACs to their mutagenic forms.
5.2 Mutagenic potency in this modified assay and carcino-
4. Summary of Test Method
genicity in the skin-painting bioassay also correlate with the
contentof3to7ringPACs,whichincludepolycyclicaromatic
4.1 The Ames Salmonella mutagenicity assay is the most
hydrocarbons and their heterocyclic analogs. The strength of
widely used short-term in vitro genotoxicity test. The assay
these correlations implies that PACs are the principal muta-
employs specific strains of the bacterium Salmonella typhimu-
genic and carcinogenic species in these oils. Some of the
rium that have been mutated at a genetic locus precluding the
methods that have provided evidence supporting this view are
biosynthesis of the amino acid histidine which is required for
referenced in Appendix X1.
growth and reproduction. Additional genetic alterations, some
of which are important markers of strain identity, are also
6. Interferences
present.
6.1 The test method is designed to detect mutagenicity
4.2 The mutagenicity assay relies upon treating the bacteria
mediated by PACs derived from petroleum. The assay is
withtestmaterialoverarangeofdosesimmediatelybelowthe
disproportionately sensitive to nitroaromatic combustion prod-
concentration showing significant toxicity to the bacteria.
ucts and as yet unidentified components of catalytically or
Treated bacteria are then grown on agar plates deficient in
thermally cracked stocks such as light or heavy cycle oils.The
histidine. Bacteria possessing the original mutation in the
latter materials are not known to occur in virgin base oils.
histidine locus cannot form colonies under these growth
6.2 For petroleum refinery streams distilling in the range
conditions,butacertainfractionoftreatedbacteriawhichhave
associated with the production of naptha or kerosine or the
undergone a second mutation in the histidine locus revert to
light end of atmospheric gas oil (that is, median boiling point
histidine-independence and are able to grow and form visible
<250°C; viscosity < 18 cSt at 40°C), the assay is sensitive to
colonies.The number of such revertant colonies per agar plate
detecting carcinogenicity related to the presence of polycyclic
is an indicator of the mutagenic potency of the test material.
aromatic compounds. However, streams in the range, even
4.3 Typically, the test is conducted using a number of
those with MI less than 1.0, can produce tumors in a standard
bacterial strains selectively sensitive to various chemical
mouse dermal carcinogenicity assay through alternative non-
classes of mutagens. Treatment with test compound is carried
genotoxic mechanisms.
out in the presence and absence of a rodent liver extract
7. Hazards
capable of mimicking in vivo metabolic activation of promuta-
genic compounds (see 3.2 for a listing of terms and abbrevia-
7.1 The test materials and positive control compounds used
tionsused).Withthiscombinationoftestconditions,theAmes
inthisassaymaypresentacarcinogenichazardbyingestionor
test becomes a very effective screening tool for chemical
skin contact.Avoid all contact with test oils and Reference Oil
mutagens. Moreover, because many mutagens are also
No. 1.
carcinogens, the test is often used as a screen for carcinogenic
7.2 The tester bacteria are attenuated and unlikely to cause
potential.
illness. However, gloves should be worn during handling of
4.4 Although the ability of theAmes test to assess carcino-
bacteria, and care should be taken to avoid injuries with
genic potential is good for many classes of compounds, it has
syringes and hypodermic needles contaminated with bacterial
been shown to be generally unsuited to the testing of water-
cultures. Waste material generated during testing should be
insoluble complex mixtures such as mineral oils. To circum-
regarded as a potential biohazard and disposed of accordingly.
vent poor solubility and other difficulties, this test method
Reference (3) provides general guidelines for safe use of this
employs an extraction of the test oil with DMSO to produce
test method.
aqueous-compatible solutions which readily interact with the
7.3 Provisions for the safe use of this test method should be
metabolic activation system (S-9) and with the tester bacteria.
incorporated into the employer’s compliance with 29 CFR
The concentration of S-9 and of NADP cofactor are increased
1910.1450.
relative to the unmodified assay, and hamster rather than rat
liver S-9 is used.The slope of the dose response curve relating
8. Materials and Methods
mutagenicity (TA98 revertants per plate) to the dose of extract
8.1 Test Organism—Methods for storage, culture, and char-
added is used as an index of mutagenic potency (MI).
acterization of the test organism are exactly as described by
4.5 In this test method, the MI (the slope of the dose Ames et al (1). The test organism used in this assay is
response curve, and a measure of mutagenic potency) of a Salmonella typhimurium strain TA98 derived from an original
DMSO extract of an oil is compared to the mutagenicity stock produced and supplied by B. N. Ames, University of
indicesofotheroilextractswhosedermalcarcinogenicitiesare California, Berkeley. Strain TA98 was selected for the test
known. By correlation, the potential dermal carcinogenicity of becauseitisthemostsensitivetotheclassofmutagenspresent
the test oil can be assessed. in petroleum materials (PACs) (Hermann et al (4)).
E1687 − 10 (2014)
A
TABLE 1 Dosing Solutions
8.1.1 StrainTA98 was derived from strainTA1538, and has
Dose, µL/Plate
the same genetic markers as that strain, including histidine/
0 12 243648 60
biotin requirement, crystal violet sensitivity, and ultraviolet
µL Extract 0 36 72 108 144 180
sensitivity. In addition, TA98 contains plasmid pKM101,
µL DMSO 180 144 108 72 36 0
which confers ampicillin resistance. Full characterization of A
Other dosing regimens over the range 0 to 60 µL may be used.
strain TA98 has been published by Ames et al (1).
8.1.2 Strain TA98 can be inoculated, either from frozen
stocks maintained at−80 6 5°C or from master plates
tainedatapproximately4°C.Thesupernatantisthenportioned
maintained at approximately 4°C, into 25 mL of Oxoid No. 2
into aliquots of 5 mL each and stored frozen at−80 6 5°C
nutrient broth in a 125 mL erlenmeyer flask equipped with a
until used.
screw cap. The flask is placed into a shaker-incubator set at
8.4.3 S-9 is thawed at approximately 4°C on the day of the
approximately 37°C and 100 to 120 rpm. Approximately 16
test, and metabolic activation mixture sufficient for one test
hours later, 3 mL of the culture is diluted into 12 mL of fresh
article prepared is as follows:
Oxoid No. 2, and allowed to regrow for 3 h, or until the
8.4.4 To a sterile container at approximately 4°C are added
turbidity of the regrown culture, measured spectrophotometri-
in sequence 1.5 mL of 1 M sodium phosphate buffer, pH 7.4;
cally at 650 nm, is in the range from 1.0 to 2.0 absorbance
0.3mL0.25Mglucose-6-phosphate;0.6mL0.2MNADP;0.6
units. A second check on cell density may be obtained by
mL of a salt solution of 0.2 M MgCl /0.825 M KCl. To the
serially diluting the culture by a factor of 10 into phosphate-
resultingsolution,12mLofS-9areaddedwithgentleswirling.
buffered saline (PBS), and plating 1 mL of the resultant
8.4.5 All steps in the preparation and dispensing of S-9 and
dilution onto nutrient agar plates containing 0.5% NaCl.After
S-9 mixture must be performed at approximately 4°C. S-9
44 to 48 h incubation at approximately 37°C, the number of
mixture should not be stored for longer than 2 h prior to use;
colonies can be determined immediately, or the plates may be
excessmixtureshouldbediscardedwhenthetestiscompleted.
refrigeratedat5 63°Cforuptofivedays,andthecelldensity
8.5 Calibration and Standardization:
of the culture calculated from the net dilution factor. Accept-
9 8.5.1 Reference Standards and Blanks—The reference stan-
able values range from 1 to 3×10 cells/mL.
dard for this test method is a vacuum distillate designated
8.2 Sampling and Handling of Oils—Sampling of oils
Reference Oil No. 1. This oil is tested as part of each assay
should be performed with consideration of viscosity and other
according to the procedures outlined in 8.6.
physical properties to ensure that test specimens are represen-
8.5.2 Assay acceptability is determined using the data
tative. When possible, oils should be stored at room tempera-
generated for Reference Oil No. 1. An assay is deemed
ture in amber bottles under nitrogen to avoid photoreactivity.
acceptableiftherevertantcolonycountsfortheDMSOextract
8.3 Preparation of DMSO Extract—The mutagenic compo- of Reference Oil No. 1, diluted 1:3 (one volume of oil plus
nents of oils are extracted into DMSO prior to testing. For oils three volumes of DMSO) reach, in a dose-responsive manner,
with viscosities low enough to permit accurate volumetric at least twice the representative mean solvent control value for
dispensing (< approximately 200 cSt at 40°C), 0.2 mL of the
thatday’stest.(See8.5.3foracceptablesolventcontrolrange.)
oil is measured into a 13 by 100 mm glass test tube, and 1 mL 8.5.3 For assays done with a single extract and an indepen-
of reagent grade DMSO added. Volumes of oil other than 0.2
dent repeat, three solvent control plates per assay serve as a
mL may be used so long as the 1:5 volume ratio of oil to blank (see 8.5.2). If a single assay is done on three extracts of
DMSO is preserved. The tube is vortexed vigorously either
the test material, two solvent control plates per extract should
continuously or intermittently for a 30-min period to ensure be used. The mean revertant count for these plates should not
thorough contact between the oil and DMSO layers. The fall below 30 colo
...
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 E1687 − 10 (Reapproved 2014) 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
1.1 This test method covers a microbiological test procedure based upon the Salmonella mutagenesis assay of Ames et al (1)
(see also Maron et al (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 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)
at 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 and health practices and determine the applicability of regulatory
limitations prior to use. Section 7 provides general guidelines for safe conduct of this test method.
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:
29 CFR 1910.1450 Occupational Exposure to Hazardous Chemical in Laboratories
3. Terminology
3.1 Definitions of Terms Specific to This Standard: (See also Terminology E2523.)
3.1.1 base stock, n—the refined oil component of metalworking fluid formulations.
3.1.2 PAC (Polycyclic Aromatic Compounds), n—For 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.
3.1.3 promutagenic compounds, promutagens, n—compounds that are not directly mutagenic but require metabolism for
expression of mutagenic activity.
3.1.4 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.
3.2 Abbreviations:
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 May 1, 2010Oct. 15, 2014. Published June 2010October 2014. Originally approved in 1995. Last previous edition approved in 20042010 as
E1687 - 04.E1687 - 10. DOI: 10.1520/E1687-10.10.1520/E1687-10R14.
The boldface numbers refer to the 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.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1687 − 10 (2014)
3.2.1 DMSO (Dimethyl Sulfoxide), n—extraction agent used in the preparation of aromatic-enriched oil fractions for
mutagenicity testing.
3.2.2 G-6-P (Glucose-6-Phosphate), n—substrate required for the operation of the NADPH generating system involved in the
biological oxidations described above.
3.2.3 MI (Mutagenicity Index), n—the slope of the dose-response curve for mutagenicity in the modified Ames test.
3.2.3.1 Discussion—
MI is an index of relative mutagenic potency.
3.2.4 NADP (Nicotinamide Adenine Dinucleotide Phosphate)—required cofactor for the biological oxidations involved in
activation of PAC to their mutagenic forms.
3.2.5 PAC (Polycyclic Aromatic Compounds), n—polycyclic aromatic compounds.
3.2.6 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 3.2 for a listing of terms and abbreviations used.)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 3 to 7 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.
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 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.
E1687 − 10 (2014)
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; viscosity< viscosity < 18 cSt at 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 al (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 al (3)).
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 al (1).
8.1.2 Strain TA98 can be inoculated, either from frozen stocks maintained at − 80 6 5°C or from master plates maintained at
approximately 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°C and 100 to 120 rpm. Approximately 16 hours 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, 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, the number of colonies can
be determined immediately, or the plates may be refrigerated at 5 6 3°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 (< approximately 200 cSt at 40°C), 0.2 mL of the oil is measured
into a 13 by 100 mm glass test tube, and 1 mL of reagent grade DMSO added. Volumes of oil other than 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, DMSO 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: Adult 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).
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. The supernatant is then portioned into aliquots of 5 mL each and stored frozen at − 80 6 5°C until used.
8.4.3 S-9 is thawed at approximately 4°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°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 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.
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.