[Code of Federal Regulations]
[Title 40, Volume 14]
[Revised as of July 1, 2003]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR79.68]
[Page 570-574]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 79--REGISTRATION OF FUELS AND FUEL ADDITIVES--Table of Contents
Subpart F--Testing Requirements for Registration
Sec. 79.68 Salmonella typhimurium reverse mutation assay.
(a) Purpose. The Salmonella typhimurium histidine (his) reversion
system is a microbial assay which measures his- [rarr]
his+ reversion induced by chemicals which cause base changes
or frameshift mutations in the genome of the microorganism Salmonella
typhimurium.
(b) Definitions. For the purposes of this section, the following
definitions apply:
Base pair mutagen means an agent which causes a base change in DNA.
In a reversion assay, this change may occur at the site of the original
mutation or at a second site in the chromosome.
Frameshift mutagen is an agent which causes the addition or deletion
of single or multiple base pairs in the DNA molecule.
Salmonella typhimurium reverse mutation assay detects mutation in a
gene of a histidine-requiring strain to produce a histidine independent
strain of this organism.
(c) Reference substances. These may include, but need not be limited
to, sodium azide, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene,
congo red, benzopurpurin 4B, trypan blue or direct blue 1.
(d) Test method.--(1) Principle. Motor vehicle combustion emissions
from fuel or additive/base fuel mixtures are, first, filtered to trap
particulate matter and, then, passed through a sorbent resin to trap
semi-volatile gases. Bacteria are separately exposed to the extract from
both the filtered particulates and the resin-trapped organics. Assays
are conducted using both test mixtures with and without a metabolic
activation system and exposed cells are plated onto minimal medium.
After a suitable period of incubation, revertant colonies are counted in
test cultures and compared to the number of spontaneous revertants in
unexposed control cultures.
(2) Description. Several methods for performing the test have been
described. The procedures described here are for the direct plate
incorporation method and the azo-reduction method. Among those used are:
(i) Direct plate incorporation method;
(ii) Preincubation method;
(iii) Azo-reduction method;
(iv) Microsuspension method; and
(v) Spiral assay.
[[Page 571]]
(3) Strain selection--(i) Designation. Five tester strains shall be
used in the assay. At the present time, TA1535, TA1537, TA98, and TA100
are designated as tester strains. The fifth strain will be chosen from
the pool of Salmonella strains commonly used to determine the degree to
which nitrated organic compounds, i.e., nitroarenes, contribute to the
overall mutagenic activity of a test substance. TA98/1,8-DNP6
or other suitable Rosenkranz nitro-reductase resistant strains will be
considered acceptable. The choice of the particular strain is left to
the discretion of the researcher. However, the researcher shall justify
the use of the selected bacterial tester strains.
(ii) Preparation and storage of bacterial tester strains. Recognized
methods of stock culture preparation and storage shall be used. The
requirement of histidine for growth shall be demonstrated for each
strain. Other phenotypic characteristics shall be checked using such
methods as crystal violet sensitivity and resistance to ampicillin.
Spontaneous reversion frequency shall be in the range expected as
reported in the literature and as established in the laboratory by
historical control values.
(iii) Bacterial growth. Fresh cultures of bacteria shall be grown up
to the late exponential or early stationary phase of growth
(approximately 108-109 cells per ml).
(4) Exogenous metabolic activation. Bacteria shall be exposed to the
test substance both in the presence and absence of an appropriate
exogenous metabolic activation system. For the direct plate
incorporation method, the most commonly used system is a cofactor-
supplemented postmitochondrial fraction prepared from the livers of
rodents treated with enzyme-inducing agents, such as Aroclor 1254. For
the azo-reduction method, a cofactor- supplemented postmitochondrial
fraction (S-9) prepared from the livers of untreated hamsters is
preferred. For this method, the cofactor supplement shall contain flavin
mononucleotide, exogenous glucose 6-phosphate dehydrogenase, NADH and
excess of glucose-6-phosphate.
(5) Control groups--(i) Concurrent controls. Concurrent positive and
negative (untreated) controls shall be included in each experiment.
Positive controls shall ensure both strain responsiveness and efficacy
of the metabolic activation system.
(ii) Strain specific positive controls shall be included in the
assay. Examples of strain specific positive controls are as follows:
(A) Strain TA1535, TA100: sodium azide;
(B) TA98: 2-nitrofluorene (without activation), 2-anthramine (with
activation);
(C) TA1537: 9-aminoacridine; and
(D) TA98/1,8-DNP6: benzo(a)pyrene (with activation).
The papers by Claxton et al., 1991 and 1992 in paragraph (g) in this
section will provide helpful information for the selection of positive
controls.
(iii) Positive controls to ensure the efficacy of the activation
system. The positive control reference substances for tests including a
metabolic activation system shall be selected on the basis of the type
of activation system used in the test. 2-Aminoanthracene is an example
of a positive control compound in plate-incorporation tests using
postmitochondrial fractions from the livers of rodents treated with
enzyme-inducing agents such as Aroclor-1254. Congo red is an example of
a positive control compound in the azo-reduction method. Other positive
control reference substances may be used.
(iv) Class-specific positive controls. The azo-reduction method
shall include positive controls from the same class of compounds as the
test agent wherever possible.
(6) Sampling the test atmosphere.(i) Extracts of test emissions are
collected on Teflon[reg]-coated glass fiber filters using an
exhaust dilution setup. The particulates are extracted with
dichloromethane (DCM) using Soxhlet extraction techniques. Extracts in
DCM can be stored at dry ice temperatures until use.
(ii) Gaseous hydrocarbons passing through the filter are trapped by
a porous, polymer resin, like XAD-2/styrene-divinylbenzene, or an
equivalent product. Methylene chloride is used to extract the resin and
the sample is evaporated to dryness before storage or use.
[[Page 572]]
(iii) Samples taken from this material are then used to expose the
cells in this assay. Final concentration of extracts in solvent/vehicle,
or after solvent exchange, shall not interfere with cell viability or
growth rate. The paper by Stump (1982) in paragraph (g) of this section
is useful for preparing extracts of particulate and semi-volatile
organic compounds from diesel and gasoline exhaust stream.
(iv) Exposure concentrations. (A) The test should initially be
performed over a broad range of concentrations. Among the criteria to be
taken into consideration for determining the upper limits of test
substance concentration are cytotoxicity and solubility. Cytotoxicity of
the test chemical may be altered in the presence of metabolic activation
systems. Toxicity may be evidenced by a reduction in the number of
spontaneous revertants, a clearing of the background lawn or by the
degree of survival of treated cultures. Relatively insoluble samples
shall be tested up to the limits of solubility. The upper test chemical
concentration shall be determined on a case by case basis.
(B) Generally, a maximum of 5 mg/plate for pure substances is
considered acceptable. At least 5 different concentrations of test
substance shall be used with adequate intervals between test points.
(C) When appropriate, a single positive response shall be confirmed
by testing over a narrow range of concentrations.
(e) Test performance. All data developed within this study shall be
in accordance with good laboratory practice provisions under Sec. 79.60.
(1) Direct plate incorporation method. When testing with metabolic
activation, test solution, bacteria, and 0.5 ml of activation mixture
containing an adequate amount of postmitochondrial fraction shall be
added to the liquid overlay agar and mixed. This mixture is poured over
the surface of a selective agar plate. Overlay agar shall be allowed to
solidify before incubation. At the end of the incubation period,
revertant colonies per plate shall be counted. When testing without
metabolic activation, the test sample and 0.1 ml of a fresh bacterial
culture shall be added to 2.0 ml of overlay agar.
(2) Azo-reduction method. When testing with metabolic activation,
0.5 ml of activation mixture containing 150 [mu]l of postmitochondrial
fraction and 0.1 ml of bacterial culture shall be added to a test tube
kept on ice. 0.1 ml of test solution shall be added, and the tubes shall
be incubated with shaking at 30 deg.C for 30 minutes. At the end of the
incubation period, 2.0 ml of agar shall be added to each tube, the
contents mixed and poured over the surface of a selective agar plate.
Overlay agar shall be allowed to solidify before incubation. At the end
of the incubation period, revertant colonies per plate shall be counted.
For tests without metabolic activation, 0.5 ml of buffer shall be used
in place of the 0.5 ml of activation mixture. All other procedures shall
be the same as those used for the test with metabolic activation.
(3) Other methods/modifications may also be appropriate.
(4) Media. An appropriate selective medium with an adequate overlay
agar shall be used.
(5) Incubation conditions. All plates within a given experiment
shall be incubated for the same time period. This incubation period
shall be for 48-72 hours at 37 deg.C.
(6) Number of cultures. All plating shall be done at least in
triplicate.
(f) Data and report--(1) Treatment of results. Data shall be
presented as number of revertant colonies per plate, revertants per
kilogram (or liter) of fuel, and as revertants per kilometer (or mile,
or brake-horsepower/hour, as appropriate) for each replicate and dose.
These same measures shall be recorded on both the negative and positive
control plates. The mean number of revertant colonies per plate,
revertants per kilogram (or liter) of fuel, and revertants per kilometer
(or mile, or brake-horsepower/hour), as well as individual plate counts
and standard deviations shall be presented for the test substance,
positive control, and negative control plates.
(2) Statistical evaluation. Data shall be evaluated by appropriate
statistical methods. Those methods shall include, at a minimum, means
and standard deviations of the reversion data.
[[Page 573]]
(3) Interpretation of results. (i) There are several criteria for
determining a positive result, one of which is a statistically
significant dose-related increase in the number of revertants. Another
criterion may be based upon detection of a reproducible and
statistically significant positive response for at least one of the test
substance concentrations.
(ii) A test substance which does not produce either a statistically
significant dose-related increase in the number of revertants or a
statistically significant and reproducible positive response at any one
of the test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance shall be
considered together in the evaluation.
(4) Test evaluation. (i) Positive results from the Salmonella
typhimurium reverse mutation assay indicate that, under the test
conditions, the test substance induces point mutations by base changes
or frameshifts in the genome of this organism.
(ii) Negative results indicate that under the test conditions the
test substance is not mutagenic in Salmonella typhimurium.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR 79.60, the following specific information shall
be reported:
(i) Sampling method(s) used and manner in which cells are exposed to
sample solution;
(ii) Bacterial strains used;
(iii) Metabolic activation system used (source, amount and
cofactor); details of preparation of postmitochondrial fraction;
(iv) Concentration levels and rationale for selection of
concentration range;
(v) Description of positive and negative controls, and
concentrations used, if appropriate;
(vi) Individual plate counts, mean number of revertant colonies per
plate, number of revertants per kilometer (or mile, or brake-horsepower/
hour), and standard deviation; and
(g) References. For additional background information on this test
guideline, the following references should be consulted.
(1) 40 CFR 798.5265, The Salmonella typhimurium reverse mutation
asay.
(2) Ames, B.N., McCann, J., Yamasaki, E. ``Methods for detecting
carcinogens and mutagens with the Salmonella/mammalian microsome
mutagenicity test,'' Mutation Research 31:347-364 (1975).
(3) Huisingh, J.L., et al.,``Mutagenic and Carcinogenic Potency of
Extracts of Diesel and Related Environmental Emissions: Study Design,
Sample Generation, Collection, and Preparation''. In: Health Effects of
Diesel Engine Emissions, Vol. II, W.E. Pepelko, R., M., Danner and N. A.
Clarke (Eds.), US EPA, Cincinnati, EPA-600/9-80-057b, pp. 788-800
(1980).
(4) [Reserved]
(5) Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., Nestmann,
E., Zeiger, E. ``Guide for the Salmonella typhimurium/mammalian
microsome tests for bacterial mutagenicity'' Mutation Research
189(2):83-91 (1987).
(6) Claxton, L., Houk, V.S., Allison, J.C., Creason, J.,
``Evaluating the relationship of metabolic activation system
concentrations and chemical dose concentrations for the Salmonella
Spiral and Plate Assays'' Mutation Research 253:127-136 (1991).
(7) Claxton, L., Houk, V.S., Monteith, L.G., Myers, L.E., Hughes,
T.J., ``Assessing the use of known mutagens to calibrate the Salmonella
typhimurium mutagenicity assay: I. Without exogenous activation.''
Mutation Research 253:137-147 (1991).
(8) Claxton, L., Houk, V.S., Warner, J.R., Myers, L.E., Hughes,
T.J., ``Assessing the use of known mutagens to calibrate the Salmonella
typhimurium mutagenicity assay: II. With exogenous activation.''
Mutation Research 253:149-159 (1991).
(9) Claxton, L., Creason, J., Lares, B., Augurell, E., Bagley, S.,
Bryant, D.W., Courtois, Y.A., Douglas, G., Clare, C.B., Goto, S.,
Quillardet, P., Jagannath, D.R., Mohn, G., Neilsen, P.A., Ohnishi, Y.,
Ong, T., Pederson, T.C., Shimizu, H., Nylund, L., Tokiwa, H., Vink,
I.G.R., Wang, Y., Warshawsky, D., ``Results of the IPCS Collaborative
Study on Complex Mixtures'' Mutation Research 276:23-32 (1992).
(10) Claxton, L., Douglas, G., Krewski, D., Lewtas, J., Matsushita,
[[Page 574]]
H., Rosenkranz, H., ``Overview, conclusions, and recommendations of the
IPCS Collaborative Study on Complex Mixtures'' Mutation Research 276:61-
80 (1992).
(11) Houk, V.S., Schalkowsky, S., and Claxton, L.D., ``Development
and Validation of the Spiral Salmonella Assay: An Automated Approach to
Bacterial Mutagenicity Testing'' Mutation Research 223:49-64 (1989).
(12) Jones, E., Richold, M., May, J.H., and Saje, A. ``The
Assessment of the Mutagenic Potential of Vehicle Engine Exhaust in the
Ames Salmonella Assay Using a Direct Exposure Method'' Mutation Research
97:35-40 (1985).
(13) Maron, D., and Ames, B. N., Revised methods for the Salmonella
mutagenicity test, Mutation Research, 113:173-212 (1983).
(14) Prival, M.J., and Mitchell, V.D. ``Analysis of a method for
testing azo dyes for mutagenic activity in Salmonella typhimurium in the
presence of flavin mononucleotide and hamster liver S-9,'' Mutation
Research 97:103-116 (1982).
(15) Rosenkranz, H.S., et.al. ``Nitropyrenes: Isolation,
identification, and reduction of mutagenic impurities in carbon black
and toners'' Science 209:1039-43 (1980).
(16) Stump, F., Snow, R., et.al., ``Trapping gaseous hydrocarbons
for mutagenic testing'' SAE Technical Paper Series, No. 820776 (1982).
(17) Vogel, H.J., Bonner, D.M. ``Acetylornithinase of E. coli:
partial purification and some properties,'' Journal of Biological
Chemistry. 218:97-106 (1956).
[59 FR 33093, June 27, 1994, as amended at 61 FR 36513, July 11, 1996]