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OAR Box 1846 

Prepped by Ollie Stewart 

Document Number: 

76) 111 -A-2 

Docket Number: 

A-90-07 

Printed 11/18/2008 7:30:17 AM Sheet 76 of 93 
_E.£, 

ENVIRONMENrAL PROTECTION AGENCY 

40 CFR Part 79 

Fuels and Fuel Additives Program 

Notice of Proposed Rulemaking: 

Proposed Regulatory Text 

i>. 
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For the reasons described in the preamble, the Code of Federal 

Regulations is proposed to be amended as follows: 

1. The authority citation for Part 79 is proposed to be amended to 

read as follows: 

Authority: Sees. 205, 211 and 301(a) of the Clean Air Act, 

42 U.S.C. 7524, 7545 and 7601. 
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2. Section 79.3 is proposed to be amended to read as follows: 

"§79.3 Availability of information. 

"The availability to the public of information provided to, or 

otherwise obtained by, the Administrator under this Part shall be 

governed by Part 2 of this chapter except as expressly noted in 

Subpart E of this Part." 
F 

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3. Section 79.6 is proposed to be deleted and reserved. 
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4. A new Subpart E is proposed to be added to Part 79 to read as 

follows: 

SUBPART S — Testing Requirements for Registration 

79.40 Definitions. 

79.41 General requirements and provisions. 

79.42 Tier 1. 

79.43 Tier 2. 

79.44 Tier 3. 

79.45 Base fuel specifications. 

79.46 Fuel and fuel additive grouping system. 

79.47 Emission generation. 

79.48 Special provisions. 

79.49 Reporting requirements. 

79.50 Vehicle emissions inhalation exposure guideline. 

79.51 Salmonella typhimurium reverse mutation (Ames) assay. 

79.52 In vivo micronucleus assay. 

79.53 In vivo sister chromatid exchange assay. 

79.54 Inhalation developmental toxicity study. 

79.55 Reproduction and fertility screen. 

79.56 Sub-acute pulmonary toxicity study. 

79.57 Functional observational neurotoxicity battery. 

§ 79.40 Definitions. 

The definitions of §79.2 remain effective unless expressly 

modified in this §79.40. The definitions listed in this section 

apply only to Subpart E of Part 79. 

(a) "Additive/base fuel mixture" means the mixture resulting 

when a fuel additive is added in specified proportion to the base 

fuel of the fuel family to which the additive belongs. 

(b) "Aftermarket additive" means a product marketed for 

introduction directly into the fuel system of a motor vehicle. 

(c) "Alternative fuels and fuel additives" means fuels and 

fuels additives other than those classified in the unleaded 

gasoline, leaded gasoline and diesel fuel families. 

(d) "Atmospheric transformation products" means compounds 

formed in the atmosphere from the reactions of primary emissions 

such as volatile organic compounds (VOCs) in the presence of 

atmospheric gases such as nitrogen oxides (NOx) . 

(e) "Atypical element" means any chemical element or 

characteristic other than those typical of the base fuel 

formulation for that fuel family. 

(f) "Base fuel" means a generic fuel formulated from a set of 

specifications to be representative of a particular family of 
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fuels. It is used in conjunction with the testing of related 

additives. 

(g) "Bulk additive" means a product which is added at the 

refinery as part of the original blending stream or after the fuel 

is transported from the refinery and before being purchased for 

introduction into the fuel tank of a motor vehicle. 

(h) "Combustion emissions" means the exhaust products of the 

combustion of fuel and/or additives in a motor vehicle engine. For 

the purposes of subpart E, this designation refers to controlled 

exhaust products (i.e., post-aftertreatment tailpipe emissions) 

unless otherwise specified. 

(i) "Compressed natural gas" or "CNG" means a gaseous motor 

vehicle fuel marketed commercially as compressed natural gas, whose 

primary constituent is methane. 

(j) "Conventional fuels and fuel additives" means fuels and 

fuel additives classified in the unleaded gasoline, leaded gasoline 

and diesel fuel families. 

(k) "Designated Evaporative Fuel" means a fuel designated 

pursuant to subpart D which has a Reid Vapor Pressure (RVP) of 2.0 

pounds per square inch or greater and is not supplied to motor 

vehicle engines by way of sealed containment and delivery systems. 

(1) "Designated Evaporative Fuel Additive" means a fuel 

additive pursuant to subpart D which, when mixed with its specified 

base fuel as specified in Section 79.41(g)(1), causes an increase 

in the RVP of 0.1 psi or more relative to the RVP of the base fuel 

alone, or has a partial pressure greater than 0.1 psi in the vapor 

phase of the additive/base fuel mixture at atmospheric pressure and 

100° Fahrenheit. Excluded from this definition are fuel additives 

used with fuels which are supplied to motor vehicle engines by way 

of sealed containment and delivery systems. 

(m) "Emission characterization" means the determination of 

the chemical composition of emissions. 

(n) "Emission collection" means the isolation of generated 

emissions from the ambient air so that, to the greatest extent 

possible, the emission characteristics are kept unchanged. 

(o) "Emission generation" means the operation of a vehicle 

and/or the vaporization of a fuel or an additive/fuel mixture under 

controlled conditions for the purpose of creating emissions to be 

used for testing purposes. 

(p) "Emission sampling" means the removal of a fraction of 

collected emissions for testing purposes. 

(q) "Emission speciation" means the performance of analysis 

to determine the chemical compounds which comprise the emissions. 
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(r) "Evaporation chamber" means an apparatus which simulates 

the fuel tank of an emission generation vehicle and is used to 

generate evaporative emissions for testing purposes. 

(s) "Evaporative emissions" means hydrocarbons and other 

chemical compounds emitted into the atmosphere by vaporization of 

contents of a fuel or additive/fuel mixture from the fuel system of 

a motor vehicle. 

(t) "Fuel Family" means a set of fuels and fuel additives 

which share basic chemical and physical formulation characteristics 

and can be used in the same engine or vehicle. 

(u) "Halogens" (when used in quotation marks) means any one 

of the following four elements found on a standard Periodic Table 

of the Elements: fluorine, chlorine, bromine, iodine. 

(v) "Hydrocarbon compounds" means all compounds whose 

molecular structure includes only hydrogen and carbon atoms. 

(w) "Liquified petroleum gas" or "LPG" means a motor fuel 

marketed commercially as liquified petroleum gas, whose primary 

constituent is propane. 

(x) "MlOO" means a motor vehicle fuel marketed commercially 

as MlOO and composed of at least 96 percent methanol. 

(y) "M85" means a motor vehicle fuel marketed commercially as 

M85 and composed of 85 +/- 5 percent methanol and 15 +\- 5 percent 

unleaded gasoline and additives. 

(z) "Metals" (when used in quotation marks) means any one of 

the following elements found on a standard Periodic Table of the 

Elements: aluminum, bismuth, cadmium, cerium, chromium, cobalt, 

copper, iron, manganese, mercury, molybdenum, nickel, lead, 

platinum, selenium, titanium, vanadium, zinc, and zirconium. 

(aa) "NPAH" or "nitrated polycyclic aromatic hydrocarbon" 

means the class of compounds whose molecular structure is the same 

as that of PAH except for the addition of one or more nitrogen 

compounds on the molecule. 

(bb) "Oxygenate compound" means an oxygen-containing, 

ashless, organic compound, such as an alcohol or ether, which may 

be used as a fuel or fuel supplement. 

(cc) "PAH" or "polycyclic aromatic hydrocarbons" means the 

class of hydrocarbons whose molecular structure includes more than 

one aromatic ring. 

(dd) "Particulate phase" or "particulates" means any material 

collected on a specified filter medium after diluting exhaust to 

less than or equal to 325 K with clean filtered air. 
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(ee) "Relabeled additive" means a fuel additive which was 

manufactured and registered by its original manufacturer and is 

also registered and sold, unchanged in composition but under a 

different label, by a different manufacturer. 

(ff) "Regulated emissions" means the emissions that are 

controlled for new motor vehicles under the vehicle certification 

program found in 40 CFR Part 86. The regulated emissions vary 

between different vehicle types and classes. 

(gg) "Semi-volatile organic compounds" means compounds with 

ten or more carbon atoms which have a partition coefficient of 2.0 

or greater and which can be trapped on sorbent polymer resins. 

(hh) "Steady state of atypical emissions" means the condition 

reached when the mass of atypical elements that appears in a 

vehicle's combustion emissions during the performance of one or 

more urban Dynamometer Driving Schedules/Engine Dynamometer 

Schedules is within a specified percentage of the mass of atypical 

elements that enters the combustion chamber. 

(ii) "T90" means the temperature at which 90 percent by 

volume of a liquid mixture is evaporated. 

(jj) "Vapor phase" means the gaseous matter fraction of 

exhaust emissions. 

(kk) "Vehicle class" means the primary divisions of vehicle 

groups within a vehicle type, including light-duty vehicles, lightduty trucks, and heavy-duty vehicles. 

(11) "Vehicle subclass" means the divisions of vehicle weight 

within a vehicle class, including light, medium, and heavy 

subclasses. Generally, this applies only to heavy-duty vehicles. 

(mm) "Vehicle type" means the divisions of on-road motor 

vehicles according to combustion cycle and intended fuel class, 

including Otto cycle gasoline-fueled vehicles, Otto cycle methanolfueled vehicles, diesel cycle diesel-fueled vehicles, and diesel 

cycle methanol-fueled vehicles. 

(nn) "Whole emissions" means unfiltered combustion emissions. 

§79.41 General requirements and provisions. 

(a) Registration: All manufacturers of highway fuels and 

fuel additives are required to register such fuels or fuel 

additives pursuant to the requirements of this subpart. Each 

manufacturer (subject to the provisions of §79.41(b) and 79.48(a)) 

shall submit all information required by §§79.42, 79.43 and 79.49 

for each fuel or additive, provided that the Tier 1 emission 

characterization requirements (§79.42(a)) and/or the Tier 2 testing 

requirements (§79.43) may be satisfied by adequate information 

pursuant to the Tier 1 literature search requirements (§79.42(c)). 
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In any case where the Administrator determines further testing is 

required, the provisions of Tier 3 (§79.44) shall apply. 

(b) Group Applications: EPA will consider any testing 

requirements of this subpart to have been met for any fuel or fuel 

additive when a fuel or fuel additive which meets the criteria for 

inclusion in the same group as such fuel or fuel additive has met 

that testing requirement, provided that all fuels and additives 

must be registered as described in §79.49. For purposes of this 

subpart, a determination of which group contains a particular fuel 

or additive will be made pursuant to the provisions of §79.46. 

Nothing in this subsection (b) shall be deemed to require a 

manufacturer to rely on another manufacturer's testing. 

(c) Date of Application; existing fuels and fuel additives: 

The registration procedures described in §§ 79.42, 79.43 and 79.49 

must be completed within three years of the effective date of this 

subpart for any designated fuel or fuel additive which is 

registered under Subpart B of this Part 79 as of such effective 

date. Each application shall be signed by the manufacturer of the 

designated fuel or additive to be registered or his agent and shall 

be submitted in the format prescribed in §79.49. Manufacturers 

which choose to comply as part of a group must submit their 

applications jointly. 

(d) Date of Application; new fuels and fuel additives: For 

any fuel or additive which is not registered pursuant to Subpart B 

of this Part 79 as of the effective date of this subpart, a 

manufacturer must complete the registration procedures described in 

§§ 79.42, 79.43 and 79.49, and receive a notification pursuant to 

§79.41(e) that such procedures have been completed properly, prior 

to the date on which such fuel or additive manufacturer may begin 

to sell, offer for sale, introduce into commerce or import such 

fuel or fuel additive. Each application shall be signed by the 

fuel or additive manufacturer or his agent and shall be submitted 

in the format prescribed in §79.49. Manufacturers which choose to 

comply as part of a group must submit their applications jointly. 

Manufacturers of fuels and additives which meet the criteria for 

inclusion in the same group as previously registered fuels and fuel 

additives pursuant to §79.46 may rely on the information in the 

previous registration provided that they notify the manufacturers 

of the previously registered fuels or additives that they have used 

the data and that the previous registrant (s) have a right to be 

reimbursed. Reimbursement arrangements will be the responsibility 

of the parties involved. If suitable arrangements cannot be agreed 

upon among the affected manufacturers, then manufacturers may 

initiate arbitration proceedings similar to those described in 40 

CFR Part 791. The previous registrant (s) shall be entitled to such 

reimbursement for a period of five years following the date on 

which the registration information was submitted. 

(e) Notification of Manufacturers: Upon receipt of a 

manufacturer's application, EPA will notify such manufacturer that 

(a) the application has been received and (b) what, if any, 

information, testing, or retesting is necessary to bring the 

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application into compliance with the reporting requirements of this 

subpart. Reliance upon inadequate existing test data pursuant to 

§79.43, or use of inadequate test protocols or substandard 

laboratory techniques in compliance with the provisions of this 

subpart, will invalidate a registration under this subpart. EPA 

shall use its best efforts to provide such notification within 

eighteen (18) months of receipt of each such application. The 

manufacturer of a new fuel or additive (pursuant to §79.41[d]), 

upon receiving EPA's notification that the application for that 

product contains adequate information pursuant to the requirements 

of §§79.42, 79.43, and 79.49(a) through (c) , may attempt to sell or 

import the fuel or additive. Similarly, upon receiving such EPA 

notification, the manufacturer of an existing fuel or additive 

(pursuant to §79.41[(c]) may continue to sell or import the fuel or 

additive. For manufacturers of both new and existing fuels and 

fuel additives, these permissions are conditional upon satisfactory 

completion of any Tier 3 requirements which EPA may imposed 

pursuant to §79.41(f) and §79.44. 

(f) Notification of Tier 3 Requirements: 

(1) Within five (5) years of receipt of a manufacturer's 

application pursuant to this subpart, EPA shall, according to 

subpart §79.44 (b), notify manufacturers of any Tier 3 testing which 

is required based upon the information contained in the application 

or any other information the Agency then has. Nothing shall 

preclude EPA from making this determination prior to five years 

after receipt of the Tier 1 and 2 data. A 30-day comment period 

shall commence upon receipt of such notice as described under 

§79.44(b). 

(2) Three years after promulgation of these regulations, and 

updated at least every two years thereafter while program activity 

remains high, EPA shall publish a notice in the Federal Register 

containing the following three lists: 1) products (or groups) for 

which EPA has prescribed Tier 3 testing according to §79.44, 2) 

products (or groups) for which EPA intends not to prescribe Tier 3 

tests based on current information, and 3) products (or groups) for 

which an adequate Tier 1 and 2 submittal has been received by EPA 

but for which a decision on the necessity of Tier 3 is still 

pending. Only those products for which EPA has received the 

respective Tier 1 and 2 submittal and has reviewed it for adequate 

compliance with the Tier 1 and 2 requirements shall be included in 

this notice. The public shall have six months following each 

Federal Register notice in which to petition EPA to prescribe Tier 

3 testing for those products for which EPA does not currently 

intend to require such testing or for which a decision is pending. 

Such petitions shall include substantive reasons for the specific 

testing required and shall not be binding on EPA. Each petition 

and EPA's formal response shall be placed in the public docket. 

(g) Penalties and Injunctive Relief: 

(1) Any person who violates these regulations shall forfeit 

and pay to the United States a civil penalty of up to the sum of 
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$25,000.00 for each and every day of the continuance of the 

violation and the economic benefit or savings resulting from the 

violation. Such civil penalties shall be commenced in accordance 

with subsection (b) of §205 of the Clean Air Act or assessed in 

accordance with subsection (c) of §205 of the Clean Air Act, 42 

U.S.C. §7524(b) and (c). 

(2) Under §205(b), the Administrator may commence a civil 

action for violation of this rule in the district court of the 

United States for the district in which the violation is alleged to 

have occurred or in which the defendant resides or has a principal 

place of business. 

(3) Under §205(c), the Administrator may assess a civil 

penalty of $25,000 for each and every day of the continuance of the 

violation and the economic benefit or savings resulting from the 

violation, except that the maximum penalty assessment shall not 

exceed $200,000, unless the Administrator and the Attorney General 

jointly determine that a matter involving a larger penalty amount 

is appropriate for administrative penalty assessment. Any such 

determination by the Administrator and the Attorney General shall 

be subject to judicial review. 

(4) The Administrator may, upon application by the person 

against whom any such penalty has been assessed, remit or mitigate 

any such penalty. 

(5) The district courts of the United States shall have 

jurisdiction to compel the furnishing of information and the 

conduct of tests required by the Administrator under these 

regulations and to award other appropriate relief. Actions to 

compel such actions shall be brought by and in the name of the 

United States. In any such action, subpoenas for witnesses who are 

required to attend a district court in any district may run into 

any other district. 

(6) Cancellation. 

(i) The Administrator of EPA may issue a notice of intent to 

cancel a fuel or fuel additive registration if the Administrator 

determines that a manufacturer has failed to submit data required 

by the Administrator under Section 211 (b) and (e) of the Clean Air 

Act. 

(ii) EPA will notify the registrant by certified mail at the 

address of record of the Agency's intent to cancel the 

registration, and will state the reasons for the proposed 

cancellation. 

(iii) The registrant may, within 30 days after receipt of the 

notice of intent to cancel, demonstrate compliance with the 

requirement to submit data or information as required by the 

Administrator. 

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(iv) The registrant may request a hearing within 30 days 

after receipt of this notice of intent to cancel. Such a hearing 

request shall be sent to the following address: 

Director, Field Operations and Support Division 

EN-397F 

U.S. Environmental Protection Agency 

3d Floor, Fairchild Building 

499 S. Capitol Street, S.W. 

Washington, DC 204 60 

If no hearing is requested within 30 days from receipt of the 

notice of intent to cancel, the cancellation shall become effective 

as of the thirty-first day from receipt of the notice. 

(v) Any hearing will be conducted according to Part 22 of 

this chapter. 

(h) Modification of Regulation. 

(1) Any person stabject to this rule may request the 

Administrator to modify the mandatory testing requirements in the 

test standard for any test required by this rule by application to 

Director, Field Operations and Support Division, at the above 

address. 

(2) Such request shall be made as soon as the test sponsor is 

aware that the modification is necessary, but in no event shall the 

request be made after 15 days following the event which 

precipitated the request. Except in the event of force majeure 

(e.g., act of God, or circumstances beyond control), in no event 

shall a request be made less than 8 months before the date on which 

the submission is due. 

(3) Upon such request, the Administrator may, in 

circumstances which are outside the control of the manufacturer(s) 

or his/their agent and which could not have been reasonably 

foreseen or avoided, modify the mandatory testing requirements in 

the rule if such requirements are infeasible. The Administrator 

may not extend the deadlines for submission contained in §79.41(c) 

and (d). 

(4) If the Administrator determines that such modifications 

would not significantly alter the scope of the test, EPA will not 

ask for public comment before approving the modification. The 

Administrator will notify the test sponsor by certified mail of the 

response to the request. EPA will place copies of each application 

and EPA response in the public docket for this rulemaking. EPA 

will publish a notice in the Federal Register annually describing 

such changes which have occurred during the previous year. Until 

such a Federal Register notice is published, any modification 

approved by EPA shall apply only to the person or group who 

requested the modification; EPA shall state the applicability of 

each modification in such notice. 

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(5) Where, in EPA's judgment, the requested modification of 

a test standard would significantly change the scope of the test, 

EPA will publish a notice in the Federal Register requesting 

comment on the request and proposed modification. However, EPA may 

approve a requested modification of a test standard without first 

seeking public comment if necessary to preserve the validity of an 

ongoing test undertaken in good faith. 

(i) Special Requirements for Additive Manufacturers: The 

following requirements shall apply when the product undergoing 

health effects testing or emission characterization is a fuel 

additive: 

(1) All required procedures shall be performed on the mixture 

which results when the additive is added to the base fuel for the 

appropriate fuel family at the maximum concentration recommended by 

the manufacturer for use in such fuels pursuant to §79.21 (d) . The 

appropriate fuel family is the one for which the additive is 

recommended by the manufacturer; if the additive is recommended for 

more than one fuel family, then the applicable fuel family of which 

the most gallons of fuel are sold annually is the appropriate fuel 

family; and 

(2) The results of the emission characterization or health 

effects testing performed on the mixture described in paragraph (1) 

of this section shall be compared, for purposes of a control, with 

the results of the same procedures when performed on the base fuel 

alone, provided that the same equipment (including vehicle model) 

and test protocols are used for both tests; and 

(3) The control data obtained for the comparison described in 

paragraph (2) of this section may have been generated for the 

purpose of registering a group for which the appropriate base fuel 

was the designated representative, or for registering a different 

additive (or group of additives), provided appropriate 

reimbursement arrangements are made with the original testers. 

§79.42 Tier 1. 

Tier 1 requires manufacturers of designated fuels or fuel 

additives to supply to the Administrator (1) the identity and 

concentration of certain emissions products of such fuels or 

additives, (2) any available information regarding the health and 

welfare effects of those emissions, and (3) the results of certain 

modeling analyses. In addition to any information required under 

§79.49 and in conformance with the requirements thereof, 

manufacturers shall provide, pursuant to the timing provisions of 

§79.41 (c) or (d) as appropriate, the following information: 

(a) Emissions Characterization: Manufacturers must provide 

a characterization of the emission products which are generated by 

evaporation or combustion of the fuel or additive mixture in a 

motor vehicle. Manufacturers may perform the characterization or 

rely on earlier characterizations contained in the data compiled 

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pursuant to the requirements of §79.42 (c). The emissions must be 

obtained by the process described in §7 9.47. The whole emissions 

must be characterized into individual products by the following 

process: 

(1) General Provisions 

(i) Characterization shall be performed separately on a 

sample of the emissions collected during each applicable segment of 

the emission generation procedure, i.e., on a sample of each bag of 

emissions collected. 

(ii) The emissions shall be generated three times, and each 

time shall be analyzed according to the provisions of paragraphs 

(2) and (3) of this subsection. 

(iii) Measurement of background emissions is not required; 

however, if such background emissions are not measured, the results 

of emission characterization procedures shall be attributed 

entirely to the fuel or the additive/base fuel mixture being 

tested. 

(iv) Concentrations of emission products shall be determined 

in units of grams per mile. 

(v) Laboratory practice must be of high quality and must be 

consistent with state of the art methods as presented in current 

environmental and analytical chemistry literature and related 

literature. 

(2) Characterization of the combustion emissions of fuels and 

fuel additives shall include: 

(i) Determination of the concentration of the regulated 

emissions specified for the applicable vehicle type and class in 40 

CFR 86, using the analytic procedures specified therein, except 

that individual regulated emissions which are required to be 

measured in other emission characterization procedures in this 

section need not be measured as regulated emissions. 

(ii) With respect to the vapor phase of combustion emissions, 

determination of the identity and concentration of individual 

species of hydrocarbon compounds containing 12 or fewer carbon 

atoms. Such characterization shall begin within 20 minutes after 

emission collection is completed. 

(iii) With respect, to the vapor phase of combustion 

emissions, determination of the identity and concentration of 

individual species of aldehyde and ketone compounds containing 7 or 

fewer carbon atoms. Characterization of these emissions captured 

in cartridges shall be performed within two weeks if the cartridge 

is stored at room temperature, and one month if the cartridge is 

stored at 0°C or less. If the emissions are sampled using the 

impinger method, the sample must be stored in a capped sample vial 

at 0°C or less and characterized within one week. 

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(iv) With respect to the vapor phase of combustion emissions 

generated from those fuels and additive/base fuel mixtures which 

contain alcohol and/or ether compounds in the uncombusted state, 

determination of the identity and concentration of individual 

species of alcohol and ether compounds containing six or fewer 

carbon atoms. Such characterization shall begin within four hours 

after emission collection is completed. 

(v) With respect to both the semi-volatile and particulate 

phases of combustion emissions, determination of the identity and 

concentration of individual species of polycyclic aromatic 

hydrocarbons (PAH) containing two or more rings. Particulate 

matter may be stored on the filter in a sealed container. The 

soluble organic fraction may be extracted and stored in a separate 

sealed container. Both the particulate and the extract shall be 

shielded from ultraviolet light and stored at -20°C or less. 

Particulate emissions shall be tested no later than six months from 

the date they were generated. 

(vi) With respect to both the semi-volatile and particulate 

phases of combustion emissions, determination of the identity and 

concentration of individual species of nitrated polycyclic aromatic 

hydrocarbons (NPAH) containing two or more rings. Particulate 

matter may be stored on the filter at -20°C in a sealed container 

shielded from ultraviolet light. Particulate emissions shall be 

tested no later than six months from the date they were generated. 

The soluble organic fraction of semi-volatile emissions shall be 

extracted immediately and tested within six months of being 

generated. The extract shall be stored in a sealed container which 

is shielded from ultraviolet light and stored at -20'C or less. 

The particulate and semi-volatile phase emission collection, 

handling and extraction methods chosen shall not alter the 

composition of the collected material, to the extent possible. 

(vii) With respect to both the vapor phase and the 

particulate phase of combustion emissions generated from those 

fuels and additive/base fuel mixtures which contain atypical 

elements, determination of the identity and concentration of 

individual emission products containing such atypical elements. 

Such characterization shall be performed after the requisite amount 

of mileage has been accumulated as specified in §79.47. 

(3) For designated evaporative fuels and designated 

evaporative fuel additives, characterization of the evaporative 

emissions shall include: 

(i) Determination of the concentration of the regulated 

emissions specified for the applicable vehicle type and class in 40 

CFR 86, using the analytic procedures specified therein, except 

that individual regulated emissions which are required to be 

measured in other emission characterization procedures in this 

section need not be measured as regulated emissions. 

(ii) Determination of the identity and concentration of 

individual species of hydrocarbon compounds containing 12 or fewer 

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carbon atoms. Such characterization shall begin within three hours 

after emission collection is completed. 

(iii) In the case of those fuels and additive/base fuel 

mixtures which contain alcohol and/or ether compounds, 

determination of the identity and concentration of individual 

species of alcohol and ether compounds containing six or fewer 

carbon atoms. Such characterization shall begin within four hours 

after emission collection is completed. 

(iv) In the case of those fuels and additive/base fuel 

mixtures which contain atypical elements, determination of the 

identity and concentration of individual emission products 

containing such atypical elements. 

(4) Laboratory quality control 

(i) Laboratories performing the procedures specified in this 

section shall be required to conduct verification testing every six 

months or before testing using known samples representative of the 

types of compounds being characterized if possible. Verification 

testing shall be consistent with the verification procedures 

designated for vehicle certification in 40 CFR 86.119-91 for 

light-duty vehicles and 40 CFR 86.1319-90 for heavy-duty vehicles 

and engines. 

(ii) Laboratories performing the procedures specified in this 

section shall agree to permit quality control inspections by EPA, 

and for this purpose shall admit any EPA Enforcement Officer, upon 

proper presentation of credentials, to any facility where vehicles 

are conditioned or where emissions are generated, collected, 

stored, sampled, or characterized in meeting the requirements of 

this section. Such laboratory audits may include EPA distribution 

of "blind" samples for analysis by participating laboratories. 

(b) Data Modeling and Analvsis: Using up-to-date modeling 

and/or analytic approaches, manufacturers are required to provide 

(1) estimates of ambient exposures to the emission products of 

fuels and fuel additives, (2) the expected atmospheric 

transformation products of such emissions, and (3) the predicted 

environmental partitioning of such emissions tg the air, soil, 

water, and biota. 

(c) Literature Search: A literature search and compilation 

of information on the potential toxicologic, environmental, and 

other public welfare effects of the emissions of such fuels and 

additives. The literature search shall include all available 

relevant information from in-house, industry, government, and 

public sources pertaining to the emissions of the subject fuel or 

fuel additive or the emissions of similar fuels or additives, with 

such similarity determined according to the provisions of §79.46. 

(1) The literature search shall address the potential adverse 

effects of whole combustion emissions, evaporative emissions of 

designated evaporative fuels and designated evaporative fuel 

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additives, relevant emission fractions, and individual emission 

products of the subject fuel or fuel additive. The individual 

emission products to be included are those identified pursuant to 

the emission characterization procedures specified in section 

79.42(b). However, the emission products addressed in the 

literature search need not include the regulated emissions. 

(2) Data bases: The literature search must include the 

results of searching appropriate commercially available toxicologic 

and environmental data bases. The data bases shall be searched by, 

at a minimum, CAS numbers (when applicable), chemical names, and 

common synonyms. 

(3) Search period: The literature search shall cover a time 

period beginning at least fifteen years prior to the date of 

submission of the reports specified in subsections 79.49(b)-(c) and 

ending no earlier than six months prior to the date on which 

testing is commenced or reports are submitted in compliance with 

this Subpart. 

§79.43 Tier 2. 

(a) Generally: Subject to the provisions of §79.43(b) 

through (d), the combustion emissions of each fuel or fuel additive 

subject to testing under this subpart must be tested according to 

each of the testing guidelines in §§79.50-79.57. In addition, 

subject to the provisions of §79.43(b) through (d) the evaporative 

emissions of each "designated evaporative fuel" and each 

"designated evaporative fuel additive" subject to testing under 

this subpart must be tested according to each of the testing 

guidelines in §§79.50 and 79.52-79.57. 

(b) Manufacturer Determination: Manufacturers shall 

determine whether the information gathered pursuant to §79.42(c) 

contains the results of adequately performed and adequately 

documented previous testing which provides information reasonably 

comparable to that supplied by the tests described in §§79.51 

through 79.57 regarding the carcinogenicity, mutagenicity, 

neurotoxicity, teratogenicity, reproductive toxicity and/or 

pulmonary effects of the emissions of the fuel or additive. When 

manufacturers make an affirmative determination they shall submit 

only the information gathered pursuant to §79.42 (c) for such tests. 

Manufacturer's shall submit such information at their own risk. 

EPA maintains final authority in judging whether the information is 

an adequate substitution for actual testing. When manufacturers 

make a negative determination on any one of the health or welfare 

effects listed, then they shall perform testing as described in 

subsection (c) of this section in order to supply the missing 

information. 

(c) Testing: All testing required pursuant to this §79.43 

must be done in accordance with the procedures described in §7 9.47 

regarding emissions generation and in §79.50 regarding exposure as 

well as any other requirements described in this subpart. 

16 
P.19 

Carcinogenic or mutagenic effects shall be determined pursuant to 

§79.51, Salmonella typhimurium Reverse Mutation (Ames) Assav, 

§79.52, In vivo Micronucleus Assav, §79.53, In vivo Sister 

Chromatid Exchange Assav. Teratogenic effects shall be determined 

pursuant to §79.54, Inhalation Developmental Toxicity Study. 

Reproductive toxicity effects shall be determined pursuant to 

§7 9.55, Reproduction and Fertility Screen. Pulmonary toxicity 

shall be determined pursuant to §79.56, Sub-Acute Pulmonary 

Toxicity Study. Neurotoxic effects shall be determined pursuant to 

§79.57, Functional Observational Neurotoxicity Battery. 

(d) EPA Determination: 

(1) After submission of all information and testing, EPA in 

its judgment shall determine whether previously conducted tests 

relied upon in the registration submission are adequately performed 

and documented and provide information reasonably comparable to 

that which would be provided by the tests described herein. 

(2) EPA shall give appropriate weight when making this 

determination to the following factors: (i) the age of the data, 

(ii) the adequacy of documentation of procedures, findings, and 

conclusions, (iii) the extent to which the testing conforms to 

generally accepted scientific principles and practices, (iv) the 

type and number of test subjects, (v) the number and adequacy of 

dosages (i.e. emission dilutions) and (vii) the degree to which the 

test procedures conform to the testing guidelines set forth in 

§§7 9.50-79.57 and/or provide information comparable to that 

provided by such testing. 

(3) In general, EPA will not make a finding of adequacy 

unless the duration of the exposure is at least as long as 

specified in the related guidelines, except that shorter durations 

may be acceptable if the test results are positive, including a 

demonstrable dose-response relationship. EPA shall not make a 

finding of adequacy unless the testing directly addresses the 

effects of inhalation exposure to the whole combustion of 

evaporative emissions of the respective fuel or additive or to the 

whole combustion or evaporative emissions of other fuels or 

additives which satisfy the criteria in §79.46 for classification 

into the same group as the subject fuel or fuel additive. However, 

if emission characterization studies for a fuel additive show that 

it has only one emission product that differentiates its emissions 

from that of the base fuel, then health studies on this emission 

product may be acceptable in lieu of whole emission studies. 

(4) If EPA finds that a previously conducted test is 

inadequate, the corresponding test(s) in §§79.51 through 79.57 must 

be performed. For new manufacturers, the registration application 

shall be considered incomplete until the results of such tests have 

been submitted. 

§79.44 Tier 3. 

17 
P.20 

(a) General Criteria for Reguiring Tier 3 Testing. Tier 3 

testing shall be required on the basis of Tier 1 and Tier 2 

information if non-definitive results or remaining uncertainties 

about the significance of observed effects and/or exposures 

interferes with EPA's ability to make reasonable estimates of the 

attendant public health risks posed by the fuel or fuel additive. 

In addition to the criteria specific to particular tests as 

summarized and detailed in the testing guidelines (§79.51-79.57), 

EPA may consider any of the following factors: (1) the number of 

positive and negative outcomes related to each endpoint, (2) the 

identification of concentration-effect relationships, (3) the 

statistical sensitivity and significance of such studies, (4) the 

severity of the observed effects (e.g., whether the effects would 

be likely to lead to incapacitating or irreversible conditions), 

(5) the number of species involved in the reported tests, (6) the 

consistency and clarity of apparent mechanisms, target organs, and 

outcomes. Also, as described in subparagraph (c), EPA will also 

consider exposure in deciding whether to require Tier 3 testing. 

(b) Notice. EPA will determine whether Tier 3 testing is 

necessary within five years of receipt of a Tier 1 and 2 submittal 

as prescribed under §79.41(f). If, based on the Tier 1 and 2 data, 

along with any other available information, EPA determines that 

further testing is necessary, EPA will require the manufacturer (s) 

of the fuels (s) or fuel additive (s) to conduct certain tests as 

described elsewhere in this section. EPA will notify the 

manufacturer(s) by certified letter of the testing to be conducted 

and the deadlines for completing the testing. EPA will place a 

copy of such notification in the public docket for this rulemaking. 

EPA will provide the manufacturer a 30-day comment period after the 

manufacturer's receipt of such notice. EPA may extend the comment 

period if it appears from the nature of the issues raised that 

further comment is warranted. In response to the comments, EPA may 

thereafter modify the schedule and/or testing requirements and 

notify the manufacturer of such change. The comments and EPA's 

response to the comments will be placed in the public docket for 

this rulemaking. In the event that no comment is received by EPA 

within the comment period, the manufacturer shall be deemed to have 

consented in full to the Tier 3 requirements. Prior to beginning 

any required Tier 3 testing, the manufacutrer shall submit detailed 

test protocols to EPA for approval. Once EPA finally determines 

the Tier 3 requirements and approves the test protocols, any 

modification to the requirements shall be governed by §79.41(h). 

(c) Consideration of exposure. It is within EPA's discretion 

to consider, in addition to available toxicity data, any Tier 1 

data on exposure to a fuel or fuel additive (or group of fuels and 

fuel additives) in determining whether to require Tier 3 testing. 

EPA may choose to consider the following: (1) historical and/or 

projected production volumes, (2) types and emission rates of 

speciated emission components, (3) estimated population and/or 

environmental exposures obtained through exposure modeling 

approaches, (4) evidence for bioconcentration from environmental 

modeling results, and (5) possible literature search findings on 

ambient, occupational, or epidemiological inhalation exposures. 

18 
P.21 

(d) Carcinogenicity and Mutagenicity Testing. (1) A 

potential need for upper tier carcinogenicity and/or mutagenicity 

testing will be indicated if the results of the Salmonella 

typhimurium Reverse Mutation (Ames) Assay, required under §79.51, 

the In Vivo Micronucleus Assay, required under §79.52, or the In 

Vivo Sister Chromatid Exchange Assay, required under §79.53, 

demonstrate a statistically significant dose-related positive 

response as compared with appropriate controls. Alternatively, 

upper tier carcinogenicity testing and/or mutagenicity testing may 

be required if there are positive outcomes for at least one 

concentration in two or more of the tests required under §§ 79.51, 

79.52, or 79.53. 

(2) The testing for carcinogenicity required under this 

paragraph shall be conducted in accordance with 40 CFR 798.3300 or 

798.3320. The testing for mutagenicity required under this 

paragraph shall be conducted in accordance with 40 CFR Parts 

798.5195, 798.5500, 798.5955, and/or 798.7100. These guidelines 

may be modified or supplemented by EPA as required to ensure that 

the prescribed testing addresses the identified areas of concern. 

(e) Reproductive and Developmental Effects Testing. (1) A 

potential need for upper tier teratogenicity testing will be 

indicated if the results of the Inhalation Developmental Toxicity 

Study required under §79.54 or the Reproduction and Fertility 

Screen required under §79.55 demonstrate, in comparison with 

appropriate controls, a decrease in neonatal viability, a 

significant change in the proportion of viable male v. female 

fetuses or offspring, the presence of soft tissue or skeletal 

abnormalities, or an increased rate of embryonic or fetal 

resorption. In addition, a potential need for upper tier testing 

of reproductive and fertility effects will be indicated if the 

results of the Reproduction and Fertility Screen required under 

§7 9.55 demonstrate, in comparison with appropriate controls, 

changes in the number and type of cells in vaginal smears, 

decreases in sperm number or motility or abnormalities of sperm 

structure, or pathologic changes found during gross or microscopic 

examination of male or female reproductive organs. 

(2) The testing for reproductive and developmental effects 

required under this paragraph shall be conducted in accordance with 

40 CFR Part 798.4700 and/or by performance of a reproductive assay 

by continuous breeding. These guidelines may be modified or 

supplemented by EPA as required to ensure that the prescribed 

testing addresses the identified areas of concern. 

(f) Neurotoxicity Testing. (1) A potential need for upper 

tier neurotoxicity testing will be indicated if the results of the 

Functional Observational Neurotoxicity Battery required under 

§79.57 demonstrate, in comparison with appropriate controls, any of 

the following responses: unusual body position, activity level, 

coordination, gait, or behavior, tremors or convulsions, increased 

lacrimation or salivation, piloerection, pupillary changes, unusual 

respiration, diarrhea, increased or decreased urination, unusual 

vocalization, and decreased grip strength. 

19 
(2) The testing for neurotoxicity required under this 

paragraph shall be conducted in accordance with 40 CFR Parts 

798.7100, 798.6400, and/or 798.3260. These guidelines may be 

modified or supplemented by EPA as required to ensure that the 

prescribed testing addresses the identified areas of concern. 

(g) Pulmonary Toxicity Testing. (1) A potential need for 

upper tier pulmonary toxicity testing will be indicated if the 

results of the Subacute Pulmonary Toxicity Study reguired under 

§79.56 demonstrate, in comparison with appropriate controls, any of 

the following responses: elevated enzyme or total protein levels, 

elevated numbers of cell types normally found in the lungs, a low 

index or lack of phagocytic activity, abnormal gross or 

histopathological findings. Persistence or delayed occurrence of 

toxic effects beyond the exposure period would also indicate 

positive results. 

(2) The testing for pulmonary toxicity required under this 

paragraph shall be conducted in accordance with 40 CFR Parts 

798.2450 and/or 798.3260. These guidelines may be modified or 

supplemented by EPA as required to ensure that the prescribed 

testing addresses the identified areas of concern. Pulmonary 

function measurements, host defense assays, immunotoxicity tests, 

and/or enzyme assays of lavage cells and fluids may also be 

required. 

(i) Other Tier 3 Testing. Other tests may be required at 

EPA's discretion in conjunction with or in addition to the studies 

identified elsewhere in this subsection. 

(1) A potential need for upper tier health effects testing 

related to hepatic toxicity, renal toxicity, endocrine toxicity, 

and/or other organ systems or endpoints not specifically addressed 

in Tier 2 may be demonstrated by findings in the Tier 1 literature 

search of adverse functional, physiologic, metabolic, or 

histopathologic effects of fuel or additive emissions to such other 

organ systems. In addition, findings in the Tier 1 emission 

characterization of significant levels of known toxicants to such 

other organ systems may also indicate a need for relevant health 

effects testing. The testing required under this paragraph may 

include tests conducted in accordance with 40 CFR Parts 798.2450 

and/or 798.3260. These guidelines may be modified or supplemented 

by EPA as required to ensure that the prescribed testing addresses 

the identified areas of concern. 

(2) Additional emissions characterization procedures may be 

required if uncertainty regarding the chemical identity or emission 

rate of fuel or additive emission species, as provided pursuant to 

the requirements of Tier 1, interferes with reasonable judgments as 

to the presence, concentration, and/or exposure to potentially 

toxic emission substances. The required tests may include 

repetition of Tier 1 emission characterization procedures on 

additional vehicles and/or other analytic procedures providing more 

precise identification or measurement of emissions. 

20 
P.23 

(3) Additional exposure studies may be required if uncertainty 

in the Tier 1 estimates of the level or extent of exposure to fuel 

or additive emissions interferes with reasonable estimates of the 

potential health risks presented by such emissions. The required 

studies may include higher level modeling or analytic approaches 

and/or exposure field studies. 

§79.45 Base fuel specifications. 

(a) The following subsections describe the method that shall 

be used to determine the specifications of a base fuel. EPA will 

apply this method in determining a base fuel for each of the 

unleaded gasoline, leaded gasoline, and diesel fuel families, and 

for determining M85 and MlOO base fuels for the methanol fuel 

family. 

(b) The fuel parameters most important in defining the fuel's 

physical and emissions characteristics shall be determined. 

(c) The cities from which fuel parameter survey data shall be 

acquired shall be determined. In general, all available data shall 

be used. 

(d) Fuel parameter and fuel consumption data from the most 

recent year for which such data is available shall be acquired.. 

Included shall be fuel surveys which report fuel parameter values 

by city, grade, and time period and fuel surveys which report fuel 

consumption by state, grade, and time period. 

(e) Fuel parameter data shall be weighted using fuel grade 

consumption data to produce fuel parameters that represent the 

average of the fuel grades in each city and time period. 

(f) State and surveyed-city populations shall be acquired. 

(g) One parameter value shall be determined for each state 

and each time period represented in the survey. 

(1) If the state does not contain any cities in the survey, 

it is not assigned a parameter value and is not represented in the 

parameter averaging procedure. 

(2) If the state contains only one city in the survey, the 

parameter value determined for that city in (e) is assigned to the 

state for each time period. 

(3) If the state contains more than one city in the survey, 

the state parameter value is determined by weighing parameter 

values determined in (e) for each city by the relative amount of 

fuel sold in that city. Relative city fuel sales are estimated by 

the fraction of population in each of the cities surveyed in that 

state in relation to the sum from all of the surveyed cities in 

that state. 

21 
P.24 

(h) Data on fuel sales shall be acquired for each state and 

each time period for which a parameter value was determined in (g) . 

(i) Fuel sales shall be summed from all represented states and 

all time periods surveyed. This sum is referred to as "Total 

Sales". 

(j) The per-state, per-time period fuel parameter values 

found in (g) shall be multiplied by the state fuel consumptions 

acquired in (h) . The products of these multiplications whall be 

summed over all surveyed cities and time periods (i.e., over the 

full year.) 

(k) The sum obtained in (j) shall be divided by the total 

sales found in (i) to find the average of each fuel parameter, and 

thereby the base fuel. 

(1) Designated fuel additives shall be added to the base fuel 

specifications as necessary. The additives to be used shall be 

determined, with participation of interested industries and groups, 

using the following method: 

(1) The objectives (purpose-in-use) of the additives shall be 

determined (for example, detergent, anti-oxidant, stabilizer). 

Only the objectives determined to be necessary shall be addressed 

with additives. 

(2) The existing additives that address the objectives shall 

be determined, along with their appropriate concentrations in-use. 

(3) One additive will be selected from the potential 

candidates for each objective. Those additives and only those 

additives shall be used as part of the base fuel, in the 

appropriate concentrations. 

§79.46 Fuel and Fuel Additive Grouping System. 

(a) Each manufacturer of a designated fuel or fuel additive 

must comply with the requirements of this subpart either on an 

individual basis or as a participant in a group of manufacturers of 

the same or similar fuels and fuel additives, as defined in this 

section. However, the notification requirements of §79.49(b) must 

be submitted individually for each fuel or fuel additive by the 

responsible manufacturer and are not subject to these grouping 

provisions. 

(b) Each manufacturer who chooses to enroll a fuel or fuel 

additive in a group of similar fuels and fuel additives as 

designated in this section may satisfy the requirements of this 

subpart through a group submission of jointly-sponsored testing and 

analysis conducted on a product which is representative of all 

products in that group, provided that the group representative is 

chosen according to the specifications in this section. 

22 
P.25 

(1) A given group's registration submission shall be 

considered applicable to all fuels and fuel additives enrolled in 

the group by their manufacturers. A manufacturer who has chosen to 

enroll a fuel or additive in a group may subsequently choose to 

retest and reregister such fuel or fuel additive on an independent 

basis; however, until such independent registration information has 

been received by EPA, the information initially submitted by the 

group on behalf of the manufacturer's fuel or fuel additive shall 

be considered applicable and valid for that fuel or fuel additive. 

(2) Manufacturers are responsible for determining the 

appropriate groups for their products according to the criteria in 

this section and for enrolling their products into those groups 

under industry-sponsored or other independent brokering 

arrangements. 

(3) Manufacturers who enroll a fuel or fuel additive into a 

group shall share the applicable costs according to appropriate 

arrangements established by the group. 

(c) In complying with the requirements of this subpart for a 

given fuel or fuel additive, notwithstanding the enrollment of such 

fuel or additive in a group, a manufacturer may make use of 

available information for any product which conforms to the same 

grouping criteria as the given product. If, for this purpose, a 

manufacturer wishes tp rely upon the information previously 

submitted by another manufacturer (or group of manufacturers) for 

registration of a similar product (or group of products), then the 

previous submitter(s) are entitled to reimbursement by the 

manufacturer for an appropriate portion of the applicable costs 

incurred to obtain and report such information. Such entitlement 

shall remain in effect for a period of five years following the 

date on which the original Tier 1 and Tier 2 information was 

submitted. 

(d) Basis for Classification. 

(1) The grouping system described in this section does not 

segregate fuels and fuel additives into separate groups. Rather, 

it reflects the fact that fuels and associated fuel additives are 

used in motor vehicles in conjunction with each other (i.e., as 

formulations containing both fuels and fuel additives). Thus, the 

same groups and grouping criteria are applicable both to fuels 

(which are generally formulated with a set of bulk additives) and 

to fuel additives (which must be mixed in fuel prior to their use 

in motor vehicles). 

(2) Fuels shall be classified pursuant to §79.46(e) and (f) 

into categories and groups of similar fuels and fuel additives 

according to the components and characteristics of such fuels in 

their original uncombusted liquid state, except in the case of 

gaseous fuels, which are classified on the basis of their 

components and characteristics in the original uncombusted gaseous 

state. 

23 
P.26 

(3) Fuel additives shall be classified pursuant to §79.46(e)-

(f) into categories and groups of similar fuels and fuel additives 

according to the components and characteristics of the uncombusted 

liquid additive/base fuel mixture which results when such additive 

is mixed in the designated base fuel at the maximum concentration 

recommended by the manufacturer of such additive pursuant to the 

requirements of §79.21(d). 

i 

(e) Grouping Scheme: The grouping system is based on a 

matrix of fuel/additive categories which are defined by the 

intersection of seven specified fuel families with three specified 

formulation classes (see Figure 1) . Some categories are defined in 

anticipation of future product designations under Subpart D. 

Within each category are one or more groups of similar fuels and 

fuel additives for which a group representative can be selected and 

joint registration information obtained and submitted. 

24 
FIGURE 1: FUEUADDITIVE CATEGORIES' 

FORMULATION 

CLASSES 

(1) 

BASEUNE 

FORMULATIONS 

(2) 

NON-BASEUNE 

FORMULATIONS 

(3) 

ATYPICAL 

FORMULATIONS 

•CONVENTIONAL' FUEL FAMUJES 

II 

UNLEADED GASOUNE 

BASEUNE 

UNLEADED GASOLINE 

CATEGORY 

ONE GROUP 

REPRESENTED BY 

UNLEADED 

BASE FUEL " 

NON-BASEUNE 

UNLEADED GASOLINE 

CATEGORY 

GROUPED ACCORDING 

TO PRESENCE OF 

NON-BASEUNE 

FACTORS 

ATYPICAL UNLEADED 

GASOUNE CATEGORY 

GROUPED ACCORDING 

TO ATYPICAL 

ELEMENTS 

AND OTHER 

CHARACTERISTICS 

LEADED GASOUNE 

(B) 

BASEUNE 

LEADED GASOLINE 

CATEGORY 

ONE GROUP 

REPRESENTED BY 

LEADED 

BASE FUEL 

NON-BASEUNE 

LEADED GASOUNE 

CATEGORY 

GROUPED ACCORDING 

TO PRESENCE OF 

NON-BASajNE 

FACTORS 

ATYPICAL LEADED 

GASOUNE CATEGORY 

GROUPED ACCORDING 

TO ATYPICAL 

ELEMENTS 

AND OTHER 

CHARACTERISTICS 

DIESEL 

<C) 

BASELINE 

DIESEL 

CATEGORY 

ONE GROUP 

REPRESENTED BY 

DIESEL 

BASE FUEL 

NON-BASEUNE 

DIESEL 

CATEGORY 

GROUPED ACCORDING 

TO PRESENCE OF 

NON-BASEUNE 

FACTORS 

ATYPICAL DIESEL 

CATEGORY 

GROUPED ACCORDING 

TO ATYPICAL 

ELEMENTS 

AND OTHER 

CHARACTERISTICS 

METHANOL 

<>-5OT- MaOH BY VOL) 

(D) 

BASEUNE 

METHANOL 

CATEGORY 

TWO GROUPS 

(MBO-M100AN0 

MSO-MBS) 

REPRESENTED BY 

M100 AND MBS 

BASE FUELS 

NON-BASEUNE 

METHANOL 

CATEGORY 

GROUPS 

TBA 

ATYPICAL METHANOL 

CATEGORY 

GROUPS 

TBA 

'ALTERNATIVE" FUEL FAMIUES 

ETHANOL 

(>-50% EtOH BY VOL) 

(E) 

BASELINE 

ETHANOL 

CATEGORY 

TWO GROUPS 

(E96-EI00AND 

ESO-E95) 

REPRESENTED BY 

E100 ANO ESS 

BASE FUELS 

NON-BASEUNE 

ETHANOL 

CATEGORY 

GROUPS 

TBA 

ATYPICAL ETHANOL 

CATEGORY 

GROUPS 

TBA 

METHANE 

(CNG. LNG) 

(F) 

BASEUNE 

METHANE 

CATEGORY 

TWO GROUPS 

(CNG ANO LNG) 

REPRESENTATED BY 

CNG ANO LNG 

BASE FUELS 

NON-BASEUNE 

METHANE 

CATEGORY 

GROUPS 

TBA 

ATYPICAL METHANE 

CATEGORY 

GROUPS 

TBA 

PROPANE 

(LPG) 

(Q) 

BASEUNE 

PROPANE 

CATEGORY 

ONE GROUP 

REPRESENTED BY 

LPG BASE FUEL 

NON-BASEUNE 

PROPANE 

CATEGORY 

GROUPS 

TBA 

ATYPICAL PROPANE 

CATEGORY 

GROUPS 

TBA 

• EACH CATEGORY CONTAINS ONE OR MORE FUEL/ADDITIVE GROUPS 

HEALTH EFFECTS TESTING INFORMATION IS REQUIRED FOR EACH SUCH FUEL/ADDITIVE GROUP. 

ro 

^i 
(1) Fuel families (columns A-G on Figure 1) are sets of fuels 

and fuel additives which share fundamental physical and chemical 

properties and engine/vehicle utilization characteristics. One or 

more base fuel formulations are specified for each fuel family 

pursuant to §79.45. Each fuel family includes fuels of the type 

referenced in the name of the family as well as bulk and 

aftermarket additives which are intended for use in those fuels. 

If an additive product is intended for use in more than one type of 

motor vehicle fuel, then it shall be classified in the applicable 

fuel family of which the most gallons of fuel are sold relative to 

the other applicable fuel families. The seven fuel classes and 

respective base fuels are described below. When applied to fuel 

additives, the criteria in these descriptions refer to the 

associated additive/base fuel mixture, pursuant to §79.46(d)(2). 

The base fuels referred to in these descriptions are as developed 

under §7 9.45. 

(i) "Conventional" Fuel Families (Fig. 1, columns A-C) : 

(A) The Unleaded Gasoline Family includes fuels and 

associated fuel additives composed of at least 50 percent unleaded 

gasoline. The base fuel for this family is the average of 

commercially-available unleaded gasoline fuels. 

(B) The Leaded Gasoline Family includes fuels and associated 

fuel additives composed of at least 50 percent leaded gasoline. 

The base fuel for this family is the average of commerciallyavailable leaded gasoline fuels. 

(C) The Diesel Family includes fuels and associated fuel 

additives composed of at least 50 percent diesel fuel. The base 

fuel for this family is the average of commercially-available 

diesel fuels. 

(ii) "Alternative" Fuel Families (Fig. 1, columns D-G): 

(A) The Methanol Family includes fuels and associated fuel 

additives composed of at least 50 percent methanol. Both MlOO and 

M85 base fuels will be defined for this family. 

(B) The Ethanol Family includes fuels and associated fuel 

additives composed of at least 50 percent ethanol. Both ElOO and 

E85 base fuels will be defined for this family. 

(C) The Methane Family includes compressed natural gas (CNG) 

and liquified natural gas (LNG) fuels and associated fuel 

additives. Both CNG and LNG base fuels will be defined for this 

family. 

(D) The Propane Family includes propane fuels and associated 

fuel additives. A liquified petroleum gas (LPG) base fuel will be 

defined for this family. 

(2) Formulation classes (Figure 1, rows 1-3) are subdivisions 

of fuel families which represent the degree to which fuels and fuel 

25 
P.29 

additives in the family resemble the base fuel(s) designated for 

the family. Three formulation classes are defined below. When 

applied to fuel additives, the criteria in these descriptions refer 

to the associated additive/base fuel mixture, pursuant to 

§79.46(d) (2) . 

(i) The Baseline Formulation Class consists of fuels and fuel 

additives which are similar to the base fuel(s) for their 

respective families, in that they contain no elements other than 

those found in the base fuel and, depending on the fuel family, 

conform to specified limitations on the amounts of certain elements 

or other physical/chemical characteristics. 

(ii) The Non-Baseline Formulation Class consists of fuels and 

fuel additives which conform to the same criteria as those 

specified for the Baseline Formulation Class except that their 

content of certain specified elements or compounds exceeds the 

limitations applicable to baseline formulations. 

(iii) The Atypical Formulation Class consists of fuels and 

fuel additives which contain elements other than those found in the 

base fuel for their respective fuel family or, depending on the 

family, exceed the specified limitations on the amount of certain 

elements other than oxygen. 

(iv) In determining the formulation class to which a fuel or 

fuel additive belongs, impurities present in trace amounts shall be 

ignored. Impurities are those substances which are present through 

contamination or which remain in the fuel or additive naturally 

after processing is completed. 

(3) Fuel/Additive Categories, depicted as the boxes in Figure 

1, are sets of products defined by the intersection of the seven 

fuel families with the applicable formulation classes. The defined 

categories follow below. When applied to fuel additives, the 

criteria in these descriptions refer to the applicable 

additive/base fuel mixture, pursuant to §79.46(d)(2). 

(i) The Baseline Unleaded Gasoline Category is defined as 

either (A) or (B) as follows: 

(A) Unleaded gasoline fuels and associated fuel additives 

which satisfy all of the following criteria: 

(1) Contain only carbon, hydrogen, oxygen, nitrogen, and/or 

sulfur, in the form of hydrocarbons, aliphatic ethers, or aliphatic 

alcohols. 

(2) Contain no more than 0.3 percent methanol by volume, 

unless an equal volume of butanol or higher molecular weight 

alcohol is also present, in which case the limit for methanol is 

2.75 percent by volume. 

(3) Contain less than 1.5 percent oxygen by weight. 

26 
P.30 

(4) Contain no more than 1,000 ppm sulfur. 

(5) Possess the characteristics of an unleaded gasoline as 

specified for at least one Seasonal and Geographical Volatility 

Class by the American Society for Testing and Materials (ASTM 

D4814). 

(B) Unleaded gasoline fuels and associated fuel additives 

which are substantially similar to unleaded gasoline as defined by 

EPA rule or have been waived by EPA under Clean Air Act Section 

211(f) authority. Fuels and fuel additive mixtures in this 

category satisfy all of the following criteria: 

(1) Contain only carbon, hydrogen, oxygen, nitrogen, and/or 

sulfur, in the form of hydrocarbons, aliphatic ethers, or aliphatic 

alcohols. 

(2) Contain no more than 0.3 percent methanol by volume, 

unless an equal volume of butanol or higher molecular weight 

alcohol is also present, in which case the limit for methanol is 

2.75 percent by volume, or unless waived to a higher level. 

(3) Contain less than 2.7 percent oxygen by weight, unless 

waived to a higher level for any given oxygenate compound. 

(4) Contain no more than 1,000 ppm sulfur. 

(5) Possess the characteristics of an unleaded gasoline as 

specified for at least one Seasonal and Geographical Volatility 

Class by the American Society for Testing and Materials (ASTM 

4814). 

(ii) The Non-Baseline Unleaded Gasoline category is comprised 

of fuels and associated additives which contain at least 50 percent 

unleaded gasoline and which conform to the above definition of the 

Baseline Unleaded Gasoline category except that they exceed the 

limits therein for oxygen and/or methanol content. Fuels which 

meet the criteria for the Baseline Unleaded Gasoline Category ((A) 

or (B) above) but whose physical characteristics or chemical 

composition (compounds used or amounts of particular compounds 

(e.g., oxygenates, aromatics) or chemical compound groups (e.g., 

total olefins, aromatics)) are substantially different than the 

base fuel, may be placed in the non-baseline category. 

(iii) The Atypical Unleaded Gasoline category is comprised of 

fuels and associated additives which contain at least 50 percent 

unleaded gasoline and contain one or more elements other than 

carbon, hydrogen, oxygen, nitrogen, and sulfur, and/or contain 

sulfur in excess of 1,000 ppm. 

(iv) The Baseline Leaded Gasoline category is comprised of 

(A) fuels which conform to the definition of the Baseline Unleaded 

Gasoline category ((A) above as finalized) except that they also 

contain lead (within amounts allowed by current regulation) and 

typical lead-scavenger halogen compounds, and (B) fuel additives 

27 
intended for use in such fuels which conform to the above 

definition of the Baseline Unleaded Gasoline Category ((A) above as 

finalized) except that they may contain lead (within amounts 

allowed by current regulation) and typical lead-scavenger halogen 

compounds. 

(v) The Non-Baseline Leaded Gasoline Category is comprised of 

fuels and associated additives which contain at least 50 percent 

leaded gasoline and which conform to the above definition of the 

Baseline Leaded Gasoline category except that they exceed the 

limits referenced therein for oxygen and/or methanol content. 

Fuels which meet the criteria for the Baseline Leaded Gasoline 

Category but whose physical characteristics or chemical composition 

(compounds used or amounts of particular compounds (e.g., 

oxygenates, aromatics) or chemical compound groups (e.g., total 

olefins, aromatics)) are substantially different than the base 

fuel, may be placed in the non-baseline category. 

(vi) The Atypical Leaded Gasoline category is comprised of 

fuels and associated additives which (A) contain at least 50 

percent leaded gasoline and (B) contain one or more elements other 

than carbon, hydrogen, oxygen, nitrogen, sulfur, lead, chlorine, 

and bromine, or sulfur in excess of 1,000 ppm, or lead in excess of 

amounts allowed by current regulation, or chlorine or bromine in 

compounds other than for use as lead scavengers. 

(vii) The Baseline Diesel category is comprised of fuels and 

associated fuel additives which satisfy all of the following 

criteria: 

(A) Conform to the published "substantially similar" 

definition applicable to diesel fuels (under development). 

(B) Contain no atypical elements other than in trace amounts. 

(C) Contain no more than 0.05 percent sulfur by weight. 

(D) Possess the characteristics of a diesel fuel as specified 

by the American Society for Testing and Materials (ASTM D975). 

(viii) The Non-Baseline Diesel Category is comprised of fuels 

and associated additives which contain at least 50 percent diesel 

fuel and which conform to the definition of the Baseline Diesel 

category except that they are not "substantially similar". Fuels 

which meet the criteria for the Baseline Diesel Category but whose 

physical characteristics or, chemical composition (compounds used or 

amounts of particular compounds (e.g., oxygenates, aromatics) or 

chemical compound groups (e.g., total aromatics)) are substantially 

different than the base fuel, may be placed in the non-baseline 

category. 

(ix) The Atypical Diesel category is comprised of fuels and 

associated additives which contain at least 50 percent diesel fuel 

and contain one or more elements other than carbon, hydrogen, 

28 
P.32 

oxygen, nitrogen, and sulfur, and/or contain sulfur in excess of 

0.05 weight percent. 

(x) The Baseline Methanol category is comprised of fuels and 

associated additives which contain at least 50 percent methanol and 

resemble the base fuels for the methanol fuel family. 

(xi) The Baseline Ethanol category is comprised of fuels and 

associated additives which contain at least 50 percent ethanol and 

resemble the base fuels for the ethanol fuel family. 

(xii) The Baseline Methane category is comprised of fuels 

and associated additives which contain at least 50 percent methane 

and resemble the base fuels for the methane fuel family. 

(xiii) The Baseline Propane category is comprised of fuels 

and associated additives which contain at least 50 percent propane 

and resemble the base fuel for the propane fuel family. 

(xiv) The Non-Baseline category in each "Alternative" fuel 

family consists of fuels and additives which have components 

similar to those of fuels and additives in the baseline category, 

but which exceed specific baseline limitations on the amount of 

certain components. 

(xv) The Atypical category in each "Alternative" fuel family 

consists of fuels and additives which contain elements or 

attributes other than those found in the base fuel for the 

respective family. 

(f) Fuel/Additive Groups are subsets of similar products 

within the fuel/additive categories defined above. If an additive 

is intended for use with fuels in more than one group within its 

category, then the additive shall be classified in the group 

containing fuels of which the most gallons are sold among all such 

applicable groups. The fuel/additive groups are described below. 

For each group defined within a Baseline category, a base fuel is 

designated to serve as the group representative. For each group 

defined within a Non-Baseline or Atypical category, a member fuel 

or fuel additive is selected to serve as the representative. 

(1) The Baseline Unleaded Gasoline, Baseline Leaded Gasoline, 

and Baseline Diesel categories each constitute one group. The base 

fuel for the associated fuel family shall serve as the respective 

representatives for these groups. 

(2) The Baseline Methanol category contains two groups. The 

first consists of fuels containing 96-100 percent methanol and 

associated fuel additives. The second consists of fuels containing 

50-95 percent methanol and associated fuel additives. The MlOO and 

M85 base fuels shall serve as representatives for the first and 

second group, respectively. 

(3) The Baseline Ethanol category contains two groups. The 

first group consists of fuels containing 96-100 percent ethanol and 

29 
P.33 

associated fuel additives. The second consists of fuels containing 

50-95 percent ethanol and associated fuel additives. The ElOO and 

E85 base fuels shall serve as representatives for the first and 

second group, respectively. 

(4) The Baseline Methane category contains two groups. The 

first group consists of CNG fuels and associated fuel additives. 

The second consists of LNG fuels and associated fuel additives. 

The CNG and LNG base fuels shall serve as the respective 

representatives for these groups. 

(5) The Baseline Propane category constitutes one group, 

which shall be represented by the LPG base fuel. 

(6) Within each of the Non-Baseline Unleaded Gasoline, NonBaseline Leaded Gasoline, and Non-Baseline Diesel categories, 

separate groups are defined for fuels and fuel additives which 

contain the same single differentiating compound (e.g., oxygenate, 

aromatic, etc.), and additional separate groups are defined for 

fuels and fuel additives which contain the same combination of two 

or more differentiating compounds. The representative for each of 

these groups shall be chosen as follows: 

(i) Within each group defined by the presence of a single 

compound of concern, the fuel or additive/base fuel mixture with 

the highest concentration of that compound shall be the designated 

representative. In the event that two or more products in the group 

contain the same and highest amount of that compound, then the 

representative shall be selected by a random method from these 

candidate products. 

(ii) Within each group defined by the combination of more 

than one compound of concern, the representative shall be the 

product within the group containing the highest total percent by 

weight of the compounds of concern. 

(A) In the event that two or more fuels or additive/base fuel 

mixtures within the group contain the same and highest percent 

oxygen then, among such candidate products, the designated 

representative shall be the product which has the greatest 

proportion of its total oxygen content contributed by the first 

applicable oxygenate compound to appear on the following list: 

methanol 

ethanol 

tert-butyl alcohol (including gasoline-grade tert-butyl 

alcohol [GTBA]) 

methyl-tert-butyl-ether (MTBE) 

tert-amyl-methyl-ether (TAME) 

ethyl-tert-butyl-ether (ETBE) 

isopropanol 

isopropyl ether 

other alcohol(s) or ether(s) 

30 
If two or more candidate products have the same and highest 

proportion of the oxygenate compound identified in paragraph (A), 

then the product among these remaining candidates which has the 

greatest proportion of the next applicable oxygenate compound on 

the list shall be the group representative. This decision-making 

process shall continue until a single product is determined to be 

the representative. If the same proportions of all applicable 

oxygenate compounds are present in two or more products with the 

highest total weight percent of oxygen, then the representative 

shall be chosen at random from such products. 

(B) In the event that two or more fuels or additive/fuel 

mixtures within the group contain the same and highest weight 

percent aromatics, then among such candidate products the 

designated product shall be the product with the greatest 

proportion of its aromatic content contributed by the heaviest 

weight aromatics. If two or more candidate products have the same 

and highest proportion of the heaviest weight aromatic, then the 

product among these remaining candidates which has the greatest 

proportion of the next heaviest weight aromatic shall be the group 

representative. This decision-making process shall continue until 

a single product is determined to be the group representative. If 

the same proportions of all aromatics are present in two or more 

compounds, than the representative shall be chosen at random from 

such products. 

(C) For groups with other compounds of concern where two or 

more forms are present, and for groups with more than one class of 

such compounds of concern, the group representative shall be 

determined by the Administrator after request by the group 

participants. 

(7) Within each of the Atypical categories, separate groups 

are defined according to the presence or absence in the fuel or 

fuel additive of any atypical element as well as the presence or 

absence of polymers and the presence or absence of oxygen in an 

amount equal to or greater than 1.5 (or 2.7) percent by weight. 

Each unique combination of atypical elements and additional 

specified variables constitutes a separate group. 

(i) Within each group containing only one atypical element, 

the fuel or additive/base fuel mixture with the highest 

concentration of that atypical element is the designated 

representative. In the event that two or more fuels or 

additive/base fuel mixtures in the group contain the same and 

highest concentration of the atypical element, then the 

representative shall be selected among these candidate products as 

follows: 

(A) If the products in the group do not contain polymers and 

do not contain oxygen in an amount equal to or greater than 1.5 (or 

2.7) percent, the representative shall be selected by a random 

method from the candidate products. 

31 
P.35 

(B) If the products in the group do contain polymers and/or 

oxygen in an amount equal to or greater than 1.5 (or 2.7) percent, 

then, among the candidate products, the representative shall be: 

(1) The product with the highest weight percent of oxygen, if 

no polymers are present 

(2) The product with the highest polymeric content, if the 

percent oxygen is less than 1.5 (or 2.7) percent 

(3) The product with the highest weight percent of oxygen and 

the highest polymeric content, if both variables are applicable to 

the group. Oxygen content shall take precedence over polymeric 

content in this determination. 

(ii) Within each group containing a combination of more than 

one atypical element, the designated representative shall be the 

product within the group which contains the highest total 

concentration of atypical elements. 

(A) In the event that two or more products within a given 

group contain the same and highest concentration of atypical 

elements then, among such candidate products, the designated 

representative shall be the product which has the highest 

concentration of the first applicable variable on the following 

list: 

(1) All "Metals" and combinations of "Metals" 

(2) All "Halogens" and combinations of "Halogens" 

(3) Other atypical elements 

(4) Oxygen \ 

(5) Polymeric content 

(B) If two or more products have the same and highest 

concentration of the variable identified in paragraph (A), then, 

among such candidate products, the product with the greatest 

concentration of the next highest variable on the list shall be the 

group representative. 

(C) This decision-making process shall continue until a 

single product is determined to be the representative. If the same 

and highest concentration of variables occurs in two or more 

products, then the representative shall be chosen by a random 

method from among such candidate products. 

(8) Upon request by group participants, the representative (s) 

for group testing may be selected by the Administrator based on 

information provided by the requestors. 

§79.47 Emission generation. 

This section specifies the equipment and procedures that must 

be used in generating the fuel and fuel additive emissions which 

are to be subjected to the emission characterization procedures 

32 
P.36^ 

specified in §79.42(a) and/or the biological tests specified in 

§79.50 of these regulations. 

(a) Vehicle and engine selection criteria. 

(1) The vehicle type, vehicle class, and vehicle subclass 

designated to generate emissions for a given fuel or additive shall 

be the type, class, and subclass that, in the previous three years, 

consumed the most gallons of fuel in the fuel family to which the 

given fuel or additive belongs. 

(2) Within this vehicle type, class, and subclass, the 

specific vehicles and engines acceptable for emission generation 

are those that represent the most common fuel metering system and 

emission control system design for the model year in which testing 

begins. These vehicles will be determined through analysis of the 

previous model year's vehicles within the given subclass. 

(i) The most common fuel metering system and the most common 

of the most important emission control system devices or 

characteristics with respect to emission reduction performance 

shall be determined through a survey of the previous model year's 

vehicle sales. In the case of light-duty gasoline powered 

vehicles, for example, some of these characteristics might be 

catalyst type, air injection, EGR, and feedback. 

(ii) Within the applicable subclass, the five highest selling 

vehicle models that contain the most common such equipment and 

characteristics shall be determined. Any of these five vehicle 

models of the current model year may be selected for emission 

generation. 

(iii) For cases in which the fuel or fuel additive undergoing 

testing is not commonly used or intended to be used in the vehicle 

type presribed by this selection procedure, vehicle selection is 

subject to the consent of the Administrator prior to the start of 

testing. 

(3) If any of the five selected vehicle models are not 

available for the current model year, emission generation shall be 

limited to the models among these five that are available. 

(4) If only five or fewer vehicle models of the given vehicle 

type are available for the current model year, all of these models 

shall be eligible to generate emissions. 

(5) All of the emissions required for testing a fuel or 

additive shall be generated by only one vehicle or engine. This 

includes evaporative and combustion emissions for emission 

characterization and biological testing. 

(6) All vehicles and engines used to generate emissions for 

characterization and biological testing must be new (i.e., never 

before titled) and placed into the program with less than 500 miles 

on the odometer or 12 hours on the engine chronometer. 

33 
(7) For the purpose of generating combustion emissions, 

light-duty vehicles shall be operated on chassis dynamometers and 

heavy-duty engines shall be operated on engine dynamometers with 

attached fuel and emission control systems. 

(8) Emissions generation using an additive/base fuel mixture 

must be performed using the same type of vehicle (i.e., same 

vehicle/engine make, model, and model year) used to generate the 

emissions from the base fuel only with which the comparison 

analysis shall be performed. 

(b) Fuel and fuel additive usage. 

(1) Fuels being tested shall be used in the test vehicles or 

engines at all times during mileage accumulation and emission 

generation and may be discontinued only when all phases of emission 

generation are completed. No other fuels or additives shall be 

used in the test vehicle or engine once mileage accumulation has 

begun until emission generation for emission characterization and 

biological testing purposes is completed. 

(2) Additives being tested shall be used in the test vehicle 

or engine at all times during mileage accumulation and emission 

generation and may be discontinued only when all phases of emission 

generation are completed. No other additives shall be used in the 

test vehicle or engine except those contained in the fuel used to 

make the additive/fuel mixture. The fuel component of the 

additive/fuel mixture shall be the specified base fuel of the fuel 

family to which the fuel additive belongs. 

(3) Additives which are recommended by the producer for use 

with more than one fuel family shall be tested in conjunction with 

the base fuel for the applicable family which contains fuels of 

which the most gallons are sold relative to the other applicable 

fuel families. 

(c) Vehicle conditioning. 

(1) Vehicles used to generate emissions shall be unmodified 

and shall be maintained as prescribed in the vehicle owner's 

manual. Repairs are to be performed only as is necessary. Routine 

maintenance performed solely for the purpose of emission 

improvement is not allowed. 

(2) Mileage accumulation. 

(i) The test vehicle or engine shall be operated for at least 

25,000 miles of mileage accumulation using the fuel or fuel 

additive being tested before it is used to generate emissions. 

(ii) Emission generation for emission characterization may 

take place at any time after the vehicle or engine accumulates 

25,000 miles using the fuel or additive being tested, except that 

additional mileage accumulation may be required for vehicles 

testing fuels or fuel additives containing atypical elements. 

34 
P.38 

Emission generation for biological testing shall take place after 

all mileage accumulation has been completed. 

(iii) Mileage accumulation may be accomplished using any 

appropriate method. 

(d) Combustion emission generation, collection, and sampling. 

(1) Generating combustion emissions for emission 

characterization. 

(i) Emissions shall be generated according to applicable 

portions of the Federal Test Procedure for the certification of new 

motor vehicles (40 CFR part 86 subpart B for light-duty vehicles 

and 40 CFR Part 86 Subpart N for heavy-duty vehicles) . For this 

purpose, the Federal Test Procedure shall be performed from the 

beginning of the procedure and continued through the section in 

which combustion emissions are generated (86.137-90 for light-duty 

vehicles and 86.1337-90 for heavy-duty engines). 

(ii) If the Federal Test Procedure in use at the time 

emission generation is performed includes artificial loading of 

emission canisters, the loading procedure shall be performed using 

a method which simulates the way the emissions are generated 

in-use. 

(iii) The collection of some types of emissions may require 

the use of equipment in addition to that specified in the Federal 

Test Procedure. Semi-volatile phase emission collection will 

require adding a porous polymer bed or similar equipment downstream 

from the particulate filter. These types of alterations to the 

Federal Test Procedure shall be acceptable. 

(iv) If the mass of particulate or semi-volatile phase 

emissions is not sufficient for characterization, the Federal Test 

Procedure may be performed again and the extracted fractions 

combined. Particulate emissions shall not be combined with semivolatile phase emissions. Emissions collected in different 

segments of the Federal Test Procedure shall not be combined. 

(v) Emissions shall be collected and sampled using methods 

appropriate for the type of emission being characterized and for 

the requirements of the emission characterization method. 

(vi) Equipment, procedural operations, and calibrations not 

necessary for emission generation for this program or for the 

specific emission being collected may be omitted from the Federal 

Test Procedure. 

(vii) The collection and characterization of background 

emissions is optional for this program. If background emissions are 

not characterized, emission characterization results will be 

attributed completely to the fuel or additive being tested. 

35 
P.39 

(viii) Emissions collected from each segment of the exhaust 

emission test shall be collected and characterized separately. 

(2) Generating whole combustion emissions for biological 

testing. 

(i) For biological tests requiring the use of whole 

combustion emissions, such emissions shall be generated 

continuously throughout the animal exposure periods, diluted by an 

amount appropriate for the test being performed as specified in 

§79.50(d)(3), and routed directly to the biological test chamber. 

(ii) The rate at which the whole combustion emissions enter 

the test chamber shall be determined by the number of animals in 

the test chamber and the specifications of the biological test. 

(iii) Emissions from a vehicle or engine may be used 

simultaneously for as many biological tests as desired. 

(iv) Equipment used to dilute and transport whole combustion 

emissions shall not alter the concentration or composition of the 

emissions, to the extent possible. 

(v) Automated systems may be used in place of human drivers 

if the systems operate within the prescribed tolerances of the 

driving cycle. 

(vi) Light-duty dynamometers shall be calibrated prior to the 

start of a biological test (40 CFR 86.118-78), verified weekly (40 

CFR 86.118-78), and recalibrated as required. Heavy-duty 

dynamometers shall be calibrated and checked prior to the start of 

a biological test (40 CFR 86.1318-84), recalibrated every two weeks 

(40 CFR 86.1318-84 (a)) and checked as stated in 40 CFR 86.1318-

84(b) and (c) . 

(vii) The entire system used to dilute and transport whole 

combustion emissions (i.e., from exhaust pipe to outlet in the 

biological testing chamber) shall be verified prior to use for this 

program and verified after every week of operation (40 CFR 

86.119-90 for light-duty vehicles and 86.1319-90 for heavy-duty 

engines). 

(viii) In order to allow for unforeseeable problems with the 

emission generation or dilution equipment, emission generation may 

be interrupted for up to four hours on up to two occasions for each 

of the Tier 2 biological tests. The amount of time for which 

emission generation was interrupted shall subsequently be added 

after the equipment problem is corrected. If the equipment problem 

causes more than four consecutive hours of emission generation to 

be interrupted, the testing shall be void. 

(ix) Emission generation procedure. The following procedure 

shall be performed every day whole emissions are generated. 

36 
P.40 

(A) The fuel tank of the vehicle or engine used to generate 

emissions shall be refueled with the test fuel or additive/base 

fuel mixture. 

(B) The vehicle or engine shall undergo a cold soak 

temperature stabilization as specified in the Federal Test 

Procedure (40 CFR 86). 

(C) The dilution/vapor transport system for the biological 

testing system will be opened or started so that the vehicle's or 

engine's emissions are presented to the test animals as soon as 

emission generation begins. 

(D) The vehicle/engine shall be started and operated over the 

Urban Dynamometer Driving Schedule/Engine Dynamometer Schedule (40 

CFR 86 App. I) appropriate for the vehicle type being used. The 

tolerances of the driving cycle shall be two times those of the 

Federal Test Procedure and must be met 95 percent of the time. 

(E) The cycle shall be repeated as many times as required for 

the biological test session. A one minute vehicle or engine idle 

shall be performed between cycles. 

(F) If refueling is required before emission generation is 

completed, the engine shall be turned off, refueled, and operation 

restarted within 15 minutes of turning off the engine. 

(3) Generating particulate and semi-volatile emissions for 

biological testing. 

(i) For biological tests requiring the use of the particulate 

and semi-volatile fraction of combustion emissions, the emissions 

shall be generated by operating one vehicle or engine over the 

Federal Test Procedure, collected on particulate filters and porous 

polymer beds or their equivalent, and extracted from the collection 

equipment for use in biological tests. These emissions used for 

biological testing may be generated and used at different locations 

provided that they are handled as prescribed in §79.47(d)(3)(vi). 

(ii) The Federal Test Procedure appropriate for the vehicle 

or engine used for generating emissions (40 CFR 86) shall be 

performed through the exhaust emissions test. Particulate and 

semi-volatile emissions shall be collected during the exhaust 

emission portion of the test procedure. The use of one particulate 

filter to collect particulate emissions from all segments of the 

Federal Test Procedure is acceptable. Similarly, the use of one 

porous polymer bed to collect semi-volatile emissions is 

acceptable. If emissions from different segments of the Federal 

test procedure are collected separately, the extracts of all 

segments are to be combined. After the particulate and semivolatile emissions have been collected the Federal Test Procedure 

may be discontinued. 

(iii) If the Federal Test Procedure in use at the time 

emission generation is performed includes artificial loading of 

37 
P.41 

emission canisters, the loading procedure shall be performed using 

a method which simulates the way the emissions are generated 

in-use. 

(iv) The amount pf particulate and semi-volatile emissions 

required for biological tests shall be determined by the 

particulate emission characteristics of the vehicle and the 

requirements of the biological test protocol. If the mass of the 

collected particulate emissions from one performance of particulate 

emission collection is insufficient for the required biological 

test(s), the collection procedure shall be repeated and the 

extracts combined. 

i 

(v) The method used to collect particulate matter shall not 

be limited to that found in the Federal Test Procedure. A CVS shall 

be used but it may have more than one sample probe and different 

filter(s) than specified in the Federal Test Procedure. 

(vi) Particulate matter may be stored on the filter in a 

sealed container, or the soluble organic fraction may be extracted 

and stored in a separate sealed container. Both the particulate 

and the extract shall be shielded from ultraviolet light and stored 

at -20°C or less. Particulate emissions shall be tested no later 

than six months from the date they were generated. The soluble 

organic fraction of semi-volatile emissions shall be extracted 

immediately and tested within six months of being generated. The 

extract shall be stored in a sealed container which is shielded 

from ultraviolet light and stored at -20°C or less. Particulate 

and semi-volatile phase emission collection, handling and 

extraction methods shall not alter the composition of the collected 

material, to the extent possible. 

(vii) The extraction method shall be determined by the 

specifications of the biological test for which particulate 

emissions are used. 

(e) Evaporative emission generation, collection and sampling. 

(1) Generating evaporative emissions for emission 

characterization. 

(i) The Federal Test Procedure (40 CFR 86) appropriate for 

the vehicle type selected shall be performed from the beginning of 

the procedure and continued to the start of the section in which 

diurnal breathing loSs emissions are measured. Equipment, 

procedural operations, and .calibrations not necessary for emission 

generation for this program or for the emissions being collected 

may be omitted from the Federal Test Procedure. 

(ii) The vehicle's evaporative emission control collection 

equipment (e.g., canister) shall be disconnected and left 

unplugged. 

38 
P.42 

(iii) One diurnal heat build shall be performed in an 

evaporative emissions SHED in the manner specified by the Federal 

Test Procedure. 

(iv) Collection of diurnal heat build emissions shall begin 

within one minute of completion of the diurnal heat build. 

(v) Emissions shall be collected and sampled using equipment 

and methods appropriate for use with the compounds being 

characterized and the requirements of the emission characterization 

analysis. 

(vi) The vehicle's evaporative emission control system shall 

be reconnected and the Federal Test Procedure reentered at the 

start of the diurnal breathing loss section. The Federal Test 

Procedure shall be continued to the start of the section in which 

hot soak emissions are measured. 

(vii) The vehicle's evaporative emission control collection 

equipment shall be disconnected and left unplugged. 

(viii) The hot soak shall be performed in an evaporative 

emissions SHED in the manner specified by the Federal Test 

Procedure. 

(ix) Collection of hot soak emissions shall begin immediately 

upon completion of the hot soak. 

(x) The collection and characterization of background 

emissions is optional for this program. If background emissions are 

not characterized, emission characterization results will be 

attributed completely to the fuel or additive being tested. 

(2) Generating evaporative emissions for biological testing. 

(i) Evaporative emissions shall be generated using an 

evaporation chamber. A fixed amount (40 percent of the evaporation 

chamber volume) of the fuel or additive/base fuel mixture being 

tested shall be placed in the evaporation chamber and evaporated in 

the air inside the evaporation chamber at fixed temperature and 

pressure. The evaporated fuel or additive/base fuel mixture in the 

vapor space of the evaporation chamber shall be withdrawn at a 

constant rate, diluted with air if required pursuant to 

§79.50(d)(3), and directed immediately to the biological testing 

chamber. The rate at which emissions are removed shall be 

determined by the number and type of animals being exposed and by 

the specifications of the biological testing protocol. 

(ii) The fuel or additive/base fuel mixture used to generate 

emissions in the evaporation chamber shall be renewed daily in 

order to keep the emissions constant. 

(iii) In order to allow for unforeseeable problems with the 

emission generation or dilution equipment, emission generation may 

be interrupted for up to four hours on up to two occasions for each 

39 
P.43 

of the Tier 2 biological tests. The amount of time for which 

emission generation was interrupted shall subsequently be added 

after the equipment problem is corrected. If the equipment problem 

causes more than four consecutive hours of emission generation to 

be interrupted, the testing shall be void. 

(iv) Evaporation Chamber Description. 

(A) The evaporation chamber shall be made from materials 

compatible with the fuels and additives being tested. 

(B) The chamber shall be filled to 40 +/-5 percent of its 

interior volume with the fuel or additive/base fuel mixture being 

tested, with the remainder of the volume containing air. 

(C) The concentration of the evaporated fuel or additive/base 

fuel mixture in the vapor space of the evaporation chamber during 

the time emissions are being withdrawn for testing shall never vary 

by more than 10 percent from the equilibrium concentration in the 

vapor space of emissions generated from fresh fuel or additive/base 

fuel in the chamber. 

(D) The size of the evaporation chamber shall be determined 

by the rate at which evaporative emissions are withdrawn and the 

rate at which the fuel or the additive/base fuel mixture 

evaporates. 

(E) The temperature of the fuel or additive/base fuel mixture 

in the evaporation chamber and the vapors used for testing shall be 

between 80 and 90 F. The vapors shall maintain this temperature up 

to the point in the system where the vapors are diluted. 

(F) The pressure in the vapor space of the evaporation 

chamber and the dilution and sampling apparatus is to stay within 

10 percent of ambient atmospheric pressure. 

(G) The vapor space of the evaporation chamber shall be well 

mixed throughout the time emissions are being withdrawn for 

testing. 

(H) There are to be no controls or equipment on the 

evaporation chamber system that change the concentration or 

composition of the vapors generated for testing. 

(f) In the case of fuels and fuel additives containing 

atypical elements, prior to the generation, collection, and/or use 

of combustion emissions in emission characterization or biological 

tests, the selected vehicle shall accumulate mileage until the 

atypical element emissions reach steady state or until the vehicle 

or engine has accumulated mileage equal to or greater than 80 

percent of the useful life of the vehicle or engine used for 

generating emissions in the manner specified in §79.47(c)(2). 

Steady state of all atypical elements must occur simultaneously for 

the atypical element emissions of a fuel or additive to be in 

steady state. 

40 
§79.48 Special Provisions 

(a) Relabeled Additives. Sellers of relabeled additives are 

not required to comply with the provisions of §§79.42, 79.43 or 

79.44 provided (a) the additive was previously registered pursuant 

to this subpart by the original manufacturer and (b) the seller has 

evidence of that previous registration. Sellers of relabeled 

additives are required to comply with §79.49(b). 

(b) Low Vapor Pressure Fuels and Additives. Fuels which are 

not "designated evaporative fuels" and fuel additives which are not 

"designated evaporative fuel additives" pursuant to the definitions 

in this subpart need not undergo the emission characterization or 

health effects tests specified in this subpart for evaporative 

emissions. 

(c) Availability of Information. All information submitted 

pursuant to §§79.42(c), 79.43 or 79.44 regarding the testing of 

fuels and fuel additives registered pursuant to this subpart is 

public information and shall be made available to the public by EPA 

upon request. A reasonable fee may be charged by EPA for copying 

such materials. Any manufacturer who believes that any information 

supplied pursuant to this subpart is a protected business secret 

shall make such claim to EPA in writing when making his submission 

and shall segregate any such information; provided no health and 

welfare testing shall be considered confidential. If EPA agrees 

with the manufacturer, the availability to the public of such 

information shall be governed by Part 2 of this chapter. 

(d) Emission Control Svstem Testing. Notwithstanding 

anything in §79.31(b) to the contrary, if any information submitted 

in accordance with this subpart or any other information available 

to EPA shows that a fuel or fuel additive may have a deleterious 

effect on the performance of any emission control system or device 

currently in use or which has been developed to a point where in a 

reasonable time it would be in general use were such effect 

avoided, EPA may, in its judgment, require testing to determine 

whether such effects in fact exist. Such testing will be required 

in accordance with such protocols and schedules as the 

Administrator shall reasonably require and shall be paid for by the 

fuel or fuel additive manufacturer. 

§79.49 Reporting Requirements 

(a) Timing 

(1) The information specified in §§79.49(b) and (c) shall be 

submitted at the conclusion of activities performed in compliance 

with Tiers 1 and 2 under the provisions of §§79.42 and 79.43, 

according to the time constraints specified in §§79.41(c)-(d). 

41 
P.45 

(2) The information specified in §79.49(d) shall be submitted 

at the conclusion of activities performed in compliance with Tier 

3 under the provisions of §79.44. 

(b) Notification. Each manufacturer of a designated fuel or 

fuel additive shall submit the following in regard to such fuel or 

fuel additive: 

(1) The information specified in Subpart B §79.11 or Subpart 

C §79.21. If such information has already been submitted to EPA in 

compliance with Subparts B or C, and if such previous information 

is accurate and up-to-date, the manufacturer may instead submit a 

statement attesting to the validity of such previous information. 

(2) Annual production volume in gallons per year. For fuels 

and fuel additives already in production, the submitted figure 

shall reflect the most recent annual production volume. For 

products not yet in production, this shall be the best estimate of 

expected annual volume after the fifth year of production. 

(3) The density in pounds per gallon. 

(4) If applicable, the fuel/additive group in which the fuel 

or fuel additive has been enrolled, pursuant to the provisions of 

§79.46. 

(5) If the product is a relabeled additive, the manufacturer 

and brand name of the original product. 

(c) Summarv Report. Each manufacturer of a designated fuel 

or fuel additive, or each group of such manufacturers pursuant to 

the provisions of §79.46, shall submit a summary report regarding 

such fuels and/or fuel additives. This report shall contain the 

following six parts. 

(1) Group Identification: grouping criteria and names and 

manufacturers of all member fuels and additives, if the summary 

report is a group submission. If the summary report represents a 

single fuel or additive, this requirement does not apply. 

(2) Executive Summary: text overview of the significant 

results and conclusions obtained as a result of completing the 

requirements of Tiers 1 and 2. 

(3) Summary of Tier 1: 

(i) Literature Search. Pursuant to §79.42(c), this part 

shall include a text summary of the methods and results of the 

literature search, including the following: 

(A) Identification of person(s) performing the literature 

search, 

(B) Description of data bases accessed, search strategy used, 

search period, and terms included in data base search, 

42 
P.46 

(C) Summary of significant results and conclusions with 

respect to the effects of the emissions of the subject fuel or fuel 

additive on the public health and welfare, 

(D) The extent to which the literature search has produced 

adequate information comparable to that which would otherwise be 

obtained through the performance of applicable emission 

characterization requirements under §79.42 (a) and/or health effects 

testing requirements under §79.43, including justifications and 

specific references. 

(ii) Emission Characterization. Pursuant to §79.42(a), this 

part shall include: 

(A) Name, address, and telephone number of the laboratory 

performing the characterization, 

(B) Name and description of analytic methods used for 

characterization. 

(iii) Data Modeling. Pursuant to §79.42(b), this part shall 

include: 

(A) Identification of person(s) performing the analyses, 

(B) Identification of models or other analytic methods used, 

including applicable references, and descriptions of the principles 

and limitations of these methods, 

(C) Summary of results and conclusions. 

(4) Summarv of Tier 2. For each health effects test 

performed pursuant to the provisions of §79.43, this part shall 

contain the following summary information: 

(i) Name, address, and telephone number of each laboratory 

which performed the testing, 

(ii) Summary of procedures (including quality assurance, 

quality control and compliance with Good Laboratory Practice 

Standards), findings, and conclusions, 

(iii) Description of any problems and their resolution. 

(5) Conclusions: identification of needs for further testing 

or justification as to why current testing and/or available 

information is adequate. 

(6) Appendices: detailed documentation related to the 

summary information described above, including the following five 

appendices: 

(i) Literature search appendices shall contain: 

43 
P.47 

(A) Printed copies of data base outputs, including reference 

lists and associated abstracts, 

(B) Summary tables organized by health or environmental 

endpoint and type of emission (e.g., combustion, evaporation, 

individual emission product), in the format specified in Table IV 

of the Draft Notice of Proposed Rule titled, "Procedures and Data 

Requirements for Petitions Submitted Under Section 112 (b) of the 

Clean Air Act as Amended in 1990" (Central Docket Section A-130, 

Environmental Protection Agency, Docket No. A-90-38). 

(C) Complete documentation and/or reprints of articles for 

any previous study relied upon for satisfying emission 

characterization and/or Tier 2 test requirements, pursuant to 

§79.50(c) (3) (i) (C), 

(D) Full reports for unpublished/in-house studies. 

(ii) Emissions characterization appendices shall contain: 

(A) Complete laboratory reports, including documentation of 

calibration and verification procedures, 

(B) Documentation of the emissions generation procedures used, 

(C) Lists of speciated emission products and their emission 

rates reported in units of grams/mile. 

(iii) Data modeling appendices shall contain detailed 

documentation of calculations and results. 

(iv) Tier 2 appendices shall contain, for each test 

performed: 

(A) Complete protocol used, 

(B) Documentation of emission generation procedures, 

(C) Complete laboratory report in compliance with the 

reporting standards in 40 CFR Part 792 Subpart J, including 

detailed test results and conclusions, 

(v) Laboratory certification information, personnel 

credentials, and statements of compliance with the Good Laboratory 

Practices Standards specified in 40 CFR Part 792. 

(d) Tier 3 Report. Subject to applicability as specified in 

§7 9.44, each manufacturer of a designated fuel or fuel additive, or 

each group of such manufacturers pursuant to the provisions of 

§79.46, shall submit the following information with respect to each 

Tier 3 test conducted for such fuels or fuel additives: 

(1) The test objectives, including the reasons why such 

testing was required in addition to that conducted in compliance 

with Tiers 1 and 2, 

44 
P.48 

(2) Name, address, and telephone number of the laboratories 

which conducted the testing, 

(3) Summary of test procedures, results and conclusions, 

(4) Complete documentation of test protocols and emission 

generation procedures, complete laboratory reports in compliance 

with the reporting standards of 40 CFR Part 792 Subpart J, detailed 

test results and conclusions, and descriptions of any problems 

encountered and their resolution, and 

(5) Laboratory certification information, personnel 

credentials, and statements of compliance with the Good Laboratory 

Practices Standards specified in 40 CFR Part 792. 

§79.50 Vehicle Emissions Inhalation Exposure Guideline. 

(a) Purpose. This guideline provides additional information 

on methodologies required to conduct inhalation exposures in health 

effects tests of vehicle combustion and evaporative emissions from 

fuels or additive/base fuel mixtures. Where this guideline and the 

individual health testing guidelines in §§79.51 through 79.57 

specify differing values for the same test parameter, the 

specifications in the individual health testing guideline shall 

prevail. 

(b) Definitions. 

(1) An acute inhalation study is a single exposure by 

inhalation over a short period of time (at least 4 hours and less 

than 24 hours), followed by at least 14 days of observation. 

(2) The aerodynamic diameter applies to the size of aerosol 

particles. It is the diameter of a sphere of unit density that has 

the same settling velocity as the particle of the test substance. 

It is used to compare particles of different sizes, densities and 

shapes, and to predict where in the respiratory tract such 

particles may be deposited. 

(3) A chronic inhalation study is a prolonged and repeated 

exposure by inhalation for the life span of the test animal; 

technically, two years ih the rat. 

(4) Concentration refers to an exposure level. Exposure is 

expressed as weight or volume of test substance per volume of air 

(mg/m3 for aerosols) or as parts per million (ppm) for vapors and 

gases over a given time period. 

(5) Cumulative toxicity is the adverse effects of repeated 

doses/exposures occurring as a result of prolonged action on, or 

increased concentration of, the administered test substance or its 

metabolites in the susceptible tissues. 

45 
P.49 

(6) Inhalable diameter refers to that aerodynamic diameter of 

a particle which is considered to be inhalable for the organism. 

It is used to refer to particles which are capable of being inhaled 

and may be deposited anywhere within the respiratory tract from the 

trachea to the alveoli. 

(7) The mass median aerodynamic diameter (MMAD) is the 

calculated aerodynamic diameter, which divides the particles of an 

aerosol in half based on the mass of the particles. Fifty percent 

of the particles in mass will be larger than the median diameter, 

and fifty percent will be smaller than the median diameter. MMAD 

describes the particle distribution of any aerosol based on the 

weight and size of the particles. MMAD and the geometric standard 

deviation describe the particle-size distribution. 

(8) A material safety data sheet (MSDS) contains information 

on the physical, chemical, and hazardous characteristics of a given 

chemical. 

(9) Reynolds number is a dimensionless number that is 

proportional to the ratio of inertial forces to frictional forces 

acting on a fluid. It quantitatively provides a measure of whether 

flow is laminar or turbulent. A fluid traveling through a pipe is 

fully developed into a laminar flow for a Reynolds number less than 

2000, and fully developed into a turbulent flow for a Reynolds 

number greater than 4000. 

(10) Subacute inhalation toxicity is the adverse effects 

occurring as a result of the repeated daily exposure of 

experimental animals to a chemical by inhalation for part (less 

than 10 percent) of a lifespan. 

(11) A subchronic inhalation study is a repeated exposure by 

inhalation for part (approximately 10 percent) of a life span; 

technically, 90 days for rodents. 

(12) A toxic effect is an adverse change in the structure or 

function of an experimental animal as a result of exposure to a 

chemical substance. 

(c) Principles and design criteria of inhalation exposure 

systems. Proper conduct of inhalation toxicity studies of the 

emissions of fuels and additive/fuel mixtures requires that the 

exposure system be designed to ensure the controlled generation of 

the exposure atmosphere, the adequate dilution of the test 

emissions, delivery of the.diluted exposure atmosphere to the test 

animals, and use of appropriate exposure chamber systems selected 

to meet criteria for a given exposure study. 

(1) Emissions generation. Emissions shall be generated 

according to the specifications in §79.47. Additional general 

information regarding the hardware and maintenance of a vehicle 

emissions generation system for inhalation exposures can be found 

in "General Health Testing Guideline for Inhalation Exposure to 

46 
P.50 

Fuels and Fuel Additives", (Cheng and Barr, 1991), available in 

public docket A-90-07. 

(2) Dilution and delivery systems. The dilution of emissions 

simulates the conditions of exhaust emissions released to the 

atmosphere from operational motor vehicles and the formation of 

particle species is strongly dependent on the dilution rate. 

Dilution also provides control of the emissions concentration 

delivered to the exposure system. 

(i) The delivery system is the means used to transport the 

emissions from the generation system to the exposure system. The 

dilution system is generally a component of the delivery system. 

(ii) Flow requirements. Exhaust emissions shall be diluted 

with clean filtered air at a flow rate in the range of 8:1 or 

higher (Hinners, et al., 1979). 

(A) The conduit for dilute emissions should be of such 

dimensions as to provide residence times for the emissions in the 

order of <1 second to several seconds before the emissions are 

introduced to the chamber system. As a result of high flow rates 

in the emission dilution section, it may be necessary to extract a 

fraction of the diluted emissions for delivery to the chamber 

system and additional dilution. The remaining emissions are 

normally exhausted to the atmosphere outside of the exposure 

facility. 

(B) Dimensions of the first-stage-dilution section should be 

such that, at a minimum, the flow Reynolds number is 70,000 or 

greater (Mokler, et al., 1984). This will maintain highly 

turbulent flow conditions so that there is complete mixing of the 

exhaust emissions. 

(iii) Materials. In designing the dilution and delivery 

systems, the use of plastic, PVC, or similar pipe and tubing shall 

be avoided. Metal tubing and pipe are recommended for all dilution 

and delivery systems. Copper, brass, and aluminum pipe and tubing 

should be avoided if there exists a possibility of chemical 

reaction occurring between emissions and tubing. Stainless steel 

pipe and tubing are recommended as the best choices. 

(iv) Wall losses. The delivery system shall be designed to 

minimize wall losses. This can be done by sizing the tubing or 

pipe to maintain laminar ,flow of the diluted emissions to the 

exposure chamber. A flow Reynolds number of 1000-3000 will ensure 

minimal wall losses. Also, the length of and number and degree of 

bends in the delivery lines to the exposure chamber system should 

be minimized. 

(v) Whole-body exposure vs. nose-only exposure delivery 

systems. Flow rates through whole-body chamber systems are of the 

order of 50 liters per minute to 500 liters per minute. Nose-only 

systems are of the order of less than 50 liters per minute. To 

47 
P.51 

maintain laminar flow conditions, the principles described in 

section (iv) apply to both systems. 

(3) Exposure chamber systems. There are two basic types of 

dynamic inhalation exposure chambers: whole-body chambers and noseonly /head-only exposure chambers. (Cheng and Moss, 1989). 

(i) Whole-body chambers. 

(A) The flow rate through a chamber should be maintained at 

10 to 15 air changes per hour. 

(B) The chambers are usually maintained at a slightly 

negative pressure (0.5 to 1.5 inch of water) to prevent leakage of 

test substance into the exposure room. 

(C) The exposure chamber should be designed in such a way as 

to provide uniform distribution of exposure concentrations in all 

compartments (Cheng et al., 1989). 

(D) Animals are housed in separate compartments inside the 

chamber, where the whole surface area of an animal is exposed to 

the test material. The spaces required for different animal species 

should follow the Guide for the Care and Use of Laboratory Animals 

(the Guide), U.S. Department of Health and Human Services, 1985. 

In general, the volume of animal bodies occupy less than 5% of the 

chamber volume. 

(ii) Head/nose-only exposure chambers. In head/nose-only 

exposure chambers, only the head portion (oronasal) of the animal 

is exposed to the test material. 

(A) The chamber volume and flow rates are much less than in 

the whole-body exposure chambers because the subjects are usually 

restrained in a tube holder where the animal's breathing can be 

easily monitored. The head/nose-only exposure chamber is suitable 

for short-term exposures or when use of a small amount of test 

material is required. 

(iii) Since whole-body exposure appears to be the least 

stressful mode of exposure, it is the preferred method. In 

general, head/nose only exposure, which is sometimes used to avoid 

concurrent exposure by the dermal or oral routes, i.e., grooming, 

is not recommended because of the stress accompanying the restraining of the animals. However, there may be specific instances where 

it may be more appropriate than whole-body exposure. The tester 

shall provide justification/reasoning for its selection. 

(d) Inhalation exposure procedures. 

(1) Animal selection. 

(i) A mammalian species shall be used for testing. Any 

variety of rodent species may be used, with the rat being the 

preferred species. Commonly used laboratory strains shall be used. 

48 
(ii) Young adult animals, at least ten weeks old for the rat 

and mouse, shall be used. At the commencement of the study, the 

weight variation of animals used should not exceed ± 20 percent of 

the mean weight for each sex. Animals should be randomly assigned 

to treatment and control groups according to their weight. 

(iii) An equal number of male and female animals shall be 

used in each dose level. Females shall be nulliparous and 

nonpregnant. 

(iv) The number of animals used at each concentration level 

and in the control group(s) depend on the type of study, number of 

biological end points used in the toxicity evaluation, the predetermined sensitivity of detection and power of significance of 

the study, and the animal species. For an acute study, at least 

five animals of each sex shall be used in each test group. For 

both the subacute and subchronic studies, at least 10 rodents of 

each sex shall be used in each test group. For a chronic study, at 

least 20 male and 20 female rodents shall be used in each test 

group. 

(A) If interim sacrifices are planned, the number of animals 

shall be increased by the number of animals scheduled to be 

sacrificed during the course of the study. 

(B) For a chronic study, the number of animals at the 

termination of the study must be adequate for a meaningful and 

valid statistical evaluation of chronic effects. 

(v) A concurrent control group is required. This group shall 

be exposed to clean, filtered air under conditions identical to 

those used for the group exposed to the test atmosphere. Where a 

base fuel is used to help generate an appropriate concentration of 

the product of interest in the test atmosphere, a base fuel-only 

control group shall be used. It is recommended that air vs. base 

fuel-only exposures be compared in preliminary range-finding 

studies. If there is no substantial difference, filtered air 

exposure itself would be an appropriate control. If base fuel-only 

and filtered air-only exposures yield different results, both base 

fuel-only and filtered air-exposed control groups are required. 

(vi) The same species/strain will be used to make comparisons 

between base fuel only and base fuel-additive mixture studies. If 

another mammalian species is used, the tester shall provide 

justification/reasoning for its selection. 

(2) Animal handling and care. 

(i) A key element in the conduct of inhalation exposure 

studies is the proper handling and care of the test animals. 

Therefore, it is strongly recommended that the exposure conditions 

conform strictly with the conditions for housing and animal care 

and use set forth in the Guide. The animal facility, itself, must 

49 
P.53. 

r 

be operated in compliance with the Animal Welfare Act as amended 

and in compliance with all pertinent USDA regulations. 

(ii) In whole-body-exposure chambers, animals shall be housed 

in individual caging. The minimum cage size per animal will be in 

accordance with instructions set forth in the Guide. 

(iii) Chambers will be cleaned and maintained in accordance 

with guidelines set forth in the Guide. 

(A) Observations should be made daily with appropriate 

actions taken to minimize loss of animals to the study (e.g., 

necropsy or refrigeration of animals found dead and isolation or 

sacrifice of weak or moribund animals). Exposure systems using 

head/nose-only exposure chambers require no special daily chamber 

maintenance. Chambers should be inspected to ensure that they are 

clean, and that there are no obstructions in the chamber which 

would restrict air flow to the animals. Whole-body exposure 

chambers will be inspected on a minimum of twice daily, once before 

exposures and once after exposures. 

(B) Signs of toxicity shall be recorded as they are observed, 

including the time of onset, degree, and duration. 

(C) Cage-side observations shall include, but not be limited 

to: changes in skin and fur, eye and mucous membranes, as well as 

respiratory, autonomic, and central nervous systems, somatomotor 

activity, and behavioral pattern. Particular attention should be 

directed to observation of tremors, convulsions, salivation, 

diarrhea, lethargy, sleep, and coma. 

(iv) Food and water will be withheld from animals for 

head/nose-only exposure systems. For whole-body-exposure systems, 

food and water will be available ad libitum at all times when the 

generation system is not operating. During operation of the 

generation system, food will be withheld to avoid possible 

contamination by emissions. Water must be provided at all times. 

(3) Concentration levels and selection. 

(i) In acute and subacute toxicity tests, three exposure 

concentrations and a control group should be used and spaced 

appropriately to produce test groups with a range of toxic effects 

and mortality rates. The data should be sufficient to produce a 

concentration-response curve and permit an acceptable estimation of 

the median lethal concentration. The concentration-response data 

should also be sufficient to determine a No Observed Adverse Effect 

Level (NOAEL), unless the result of a limit test precludes such a 

finding. 

(A) The highest concentration should result in toxic effects 

but not produce an incidence of fatalities which would prevent a 

meaningful evaluation of the study. 

50 
(B) The lowest concentration should not produce toxic effects 

which are directly attributable to the test exposure. Where there 

is a useful estimation of human exposure, the lowest concentration 

should exceed this. 

(C) The intermediate concentration level(s) should produce 

minimal observable toxic effects. If more than one intermediate 

concentration level is used, the concentrations should be spaced to 

produce a gradation of toxic effects. 

(D) In the low, intermediate, and control groups, the 

incidence of fatalities shall be low, or absent, to permit a 

meaningful evaluation of the results. 

(ii) At least three concentration levels with a control shall 

be used in subchronic and chronic toxicity tests, as it is 

desirable to have a concentration-response relationship as well as 

a NOAEL. Concentrations shall be spaced appropriately to produce 

test groups with a range of toxic effects. The criteria for 

selecting dose level have been published (40 CFR §§798.2450 and 

798.3260). 

(4) Exposure chamber environmental conditions. The following 

environmental conditions in the exposure chamber are critical to 

the maintenance of the test animals: flow, temperature, relative 

humidity, and lighting. 

(i) Filtered and conditioned air should be used to dilute the 

exhaust emissions during exposure and during nonexposure periods to 

maintain environmental conditions that are free of trace gases, 

dusts, and microorganisms on the test animals. Ten to fifteen 

(chamber volume) air changes per hour will be provided at all times 

to whole-body-exposure chambers. The minimum air flow rate for 

head/nose-only exposure chambers will be a function of the number 

of animals and the average minute volume of the animals: 

Q«-tBix.__i(Ij/

min)= 2 x number of animals x average minute volume 

(Cheng and Moss, 1989). 

(ii) Temperature is one of the most important environmental 

parameters. Recommended ranges of temperature for various species 

are given in the Guide. The recommended temperature ranges will be 

used for establishing temperature conditions of whole-body-exposure 

chambers. For rodents in whole-body-exposure chambers, the 

recommended temperature is 22°C +/- 2° and for rabbits, it is 20°C 

+/- 3°. Temperature ranges have not been established for 

head/nose-only tubes; however, recommended maximum temperature 

limits have been established at the Inhalation Toxicology Research 

Institute (Barr, 1988). Maximum temperature for rats and mice in 

head/nose-only tubes is 23°C. 

(iii) Relative humidity. The relative humidity in the 

chamber air is important for heat balance and should be maintained 

between 40%-60%, but in certain instances, this may not be 

51 
P.55 

practicable. The Guide recommends a 30%-70% relative humidity 

range for rodents in exposure chambers. 

(iv) Lighting. Light intensity of 30 foot candles at 3 ft. 

from the floor of the exposure facility is recommended (Rao, 1986). 

(5) Exposure Conditions. Study animals shall be exposed to 

the test atmosphere (s) on a repeated basis for at least 6 hours per 

day and 7 days per week for the exposure period. 

(6) Exposure atmosphere. 

(i) The exposure atmosphere shall be held as constant as is 

practicable and must be monitored continuously or intermittently, 

depending on the method of analysis, to ensure that exposure levels 

are at the target values or within stated limits during the 

exposure period. Sampling methodology will be determined based on 

the type of generation system and the type of exposure chamber 

system specified for the exposure study. 

(A) Integrated samples of aerosol concentration should be 

taken daily during the exposure from a single representative sample 

port in the chamber near the breathing zone of the animals. Gas 

samples should be taken daily, but not less than once a week to 

determine concentrations (ppm) of individual components including 

CO, C02, NOx, S02, and fuel vapors. 

(B) At a minimum, vapor concentrations of the evaporative 

emissions in the chamber should be determined hourly. The sample 

should be taken from a single, representative sampling port. 

(C) To ensure that animals in different locations of the 

chamber receive a similar exposure atmosphere, distribution of 

aerosol or vapor concentration in exposure chambers can be 

determined without animals during the developmental phase of the 

study by measurement from numerous positions throughout each 

chamber. It will be compared to that of a fixed reference position 

within each chamber. Subsequently, all test data will be recorded 

from the reference position. For head/nose-only exposure chambers, 

it may not be possible to monitor the chamber distribution during 

the exposure, because the exposure ports contain animals. 

(D) During the development of the emissions generating 

system, particle size analysis shall be performed to establish the 

stability of aerosol concentrations with respect to particle size. 

During exposure, analysis shall be conducted as often as necessary 

to determine the consistency of particle size distribution. 

(E) Chamber rise and fall times. The time required for the 

exposure concentration to reach 90% (T90) of the stable 

concentration after the generator is turned on, and the fall time 

when the chamber concentration decreases to 10% of the stable 

concentration after the generation system is stopped should be 

determined in the developmental phase of the study. Time-integrated samples collected for calculating exposure concentrations should 

52 
be taken after the rise time. The daily exposure time is exclusive 

of T90 or the fall time. 

(ii) Instrumentation used for a given study will be 

determined based on the type of generation system and the type of 

exposure chamber system specified for the exposure study. 

(A) For combustion exhaust studies, time-integrated samples 

using a filter as a collection substrate is recommended. 

Gravimetric determination of the aerosol concentration (mg/m3) 

should be made. Samples shall be taken as often as is necessary to 

determine the variance of the aerosol mass concentration. Other 

aerosol instruments, such as photometers, may be used to determine 

the stability of aerosol concentration in the chamber. Combustion 

gases should be sampled by collecting exposure air in bags and then 

analyzing the collected air sample to determine major components of 

the combustion gas using gas analyzers. Exposure chambers can also 

be connected to gas analyzers directly by using sampling lines and 

switching valves. Samples can be taken more frequently using the 

latter method. 

(B) For evaporative emission studies, concentration of fuel 

vapors can usually be determined by using a gas chromatograph (GC) 

and/or infrared spectrometry (IR). Grab samples can be taken from 

the chamber by using bubble samplers with the appropriate solvent 

to collect the vapors, or by collecting a small volume of air in a 

syringe. Intermediate or continuous monitoring of the chamber 

concentration is also possible by connecting the chamber with a GC 

or IR detector. 

(7) Monitoring chamber environmental conditions. Monitoring 

may be performed by a computer system or exposure system operating 

personnel. 

(i) The flow-metering device used for the exposure chambers 

must be a continuous monitoring device, and actual flow 

measurements must be recorded at least every 30 minutes. Accuracy 

must be ±5% of full scale range. Measurement of air flow through 

the exposure chamber may be accomplished using any device that has 

sufficient range to accurately measure the air flow for the given 

chamber. Types of flow metering devices include rotameters, 

orifice meters, venturi meters, critical orifices, and 

turbinemeters (Benedict, 1984; Spitzer, 1984) . Pressure measurement may be accomplished using manometers, electronic pressure 

transducers, magnehelics, or similar devices (Gillum, 1982). 

Accuracy of the pressure device must be ± 5% of full scale range. 

Pressure measurements must be continuous and recorded at least 

every 30 minutes. 

(ii) Temperature. The temperature of exposure chambers must 

be monitored continuously and recorded at least every 30 minutes. 

Temperature may be measured using thermometers, RTD's, 

thermocouples, thermistors, or other devices (Benedict, 1984). It 

is necessary to incorporate an alarm system into the temperature 

monitoring system. The exposure operators must be notified by the 

53 
P.57 

alarm system when the chamber temperature exceeds 26.7°C (80°F). 

The exposure system must be discontinued and emergency procedures 

enacted to immediately reduce temperatures or remove test animals 

from high temperature environment when chamber temperatures exceed 

29°C. Accuracy of the temperature monitoring device will be ±1°C 

for the temperature range of20-30°C. 

(iii) Relative humidity. The relative humidity of exposure 

chambers must be monitored continuously and recorded at least every 

30 minutes. Relative humidity may be measured using various 

devices (Chaddock, 1985). 

(iv) Lighting shall be measured quarterly, or once at the 

beginning, middle, and end of the study for shorter studies. 

(v) Noise level in the exposure chamber (s) shall be measured 

quarterly, or once at the beginning, middle, and end of the study 

for shorter studies. 

(vi) Oxygen content is critical, especially in nose-only 

chamber systems, and should generally be greater than or equal to 

19% in the test cages. An oxygen sensor shall be located at a 

single position in the test chamber and a lower alarm limit of 18% 

shall be used to activate an alarm system. 

(8) Safety procedures and requirements. 

(i) It is mandatory that the upper explosive limit (UEL) and 

lower explosive limit (LEL) for the fuel and/or fuel additive(s) 

that are being tested be determined. These limits can be found in 

the material safety data sheets (MSDS) for each substance and in 

various reference texts. The air concentration of the fuel or 

additive-base fuel mixture in the generation system, 

dilution/delivery system, and the exposure chamber system shall be 

calculated to ensure that explosive limits are not present. The 

test system shall minimize the probability of and the consequences 

associated with fire and/or explosion in a manner similar to that 

presented in the National Toxicology Program (NIH/NIEHS) General 

Statement of Work, "Minimum Health and Safety Requirements for 

Inhalation Studies" (§ II.B.6). Consistent with these 

requirements, the air concentraion of a potentially flammable or 

explosive test substance should remain below 50 percent of its LEL 

during testing. 

(ii) Storage, handling, and use of fuel or additive-base fuel 

mixture shall follow guidelines given in Occupational Safety and 

Health Standards for General Industry (29 CFR, §1910.106, 1989). 

(iii) Monitoring for carbon monoxide (CO) levels is mandatory 

for combustion systems. CO shall be continuously monitored in the 

immediate area of the engine/vehicle system and in the exposure 

chamber. 

(iv) Air samples shall be taken quarterly in the immediate 

area of the vapor generation system and the exposure chamber 

54 
P.58 

system, or once at the beginning, middle, and end of the study for 

shorter studies. These samples shall be analyzed by methods 

described in section (d) (6) ((ii) (B). 

(v) With the presence of fuels and/or fuel additives, all 

electrical and electronic equipment must be grounded. Also, the 

dilution/delivery system and chamber exposure system must be 

grounded. Guidelines for grounding are given in Occupational 

Safety and Health Standards for General Industry (29 CFR, 

§1910.304, 1989). 

(9) Quality control and quality assurance procedures. 

(i) Standard operating procedures (SOP). SOPs for exposure 

operations, sampling instruments, animal handling, and analytical 

methods should be written during the developmental phase of the 

study. 

(ii) Training of exposure operators should be performed and 

documented. 

(iii) Flow meters, sampling instruments, and balances used in 

the inhalation experiments shall be calibrated with standards 

during the developmental phase to determine the sensitivity, 

detection limits, and linearity. During the exposure period, 

instruments should be checked for calibration and documented to 

ensure that each instrument still functions properly. 

(iv) The mean exposure concentration should be within 10% of 

the target concentration on greater than or equal to 90% of 

exposure days. The coefficient of variation should be within 25% 

on greater than or equal to 90% of exposure days. 

(v) The spatial variation of the chamber concentration should 

be less than 8%. If a higher spatial variation is observed during 

the developmental phase, then air mixing in the chamber shall be 

increased. In any case, animal cage assignments shall be rotated 

daily within their respective chambers to minimize positional 

effects and insure a uniform exposure during the study. 

(e) Data and reporting. Data shall be summarized in tabular 

form, showing for each group the number of animals at the start of 

the test, the number of animals showing lesions, the types of 

lesions, and the percentage of animals displaying each type of 

lesion. 

(1) Treatment of results. All observed results, quantitative 

and incidental, shall be evaluated by an appropriate statistical 

method. Any generally accepted statistical method may be used; the 

statistical methods should be selected during the design of the 

study. 

(2) Evaluation of results. The findings of an inhalation 

toxicity study should be evaluated in conjunction with the findings 

of preceding studies and considered in terms of the observed toxic 

55 
P.59 

effects and the necropsy and histopathological findings. The 

evaluation will include the relationship between the concentration 

of the test substance and the duration of exposure, and the 

severity, of abnormalities, gross lesion, identified target organs, 

body weight changes, effects on mortality and any other general or 

specific toxic effects. 

(3) Test conditions. 

(i) The exposure apparatus shall be described, including: 

(A) The engine design and type, the dynamometer, the cooling 

system, the computer control system, and the dilution system, for 

engine exhaust emissions, or 

(B) The generator type, the generator design, and the 

dilution system, for evaporative emissions. 

Other test conditions, such as the source of air, fuel type, 

treatment of exhaust air, design of exposure chamber and the method 

of housing animals in a test chamber shall be described. 

(ii) The equipment for measuring temperature, humidity, 

particulate aerosol concentrations and size distribution, gas 

analyzers, fuel vapor concentrations, chamber distribution, and 

rise and fall time shall be described. 

(iii) Daily exposure results. The daily record should 

document the date, the start and stop times of the exposure, number 

of samples taken during the day, daily concentrations, calibration 

of instruments, and problems encountered during the exposure. The 

daily exposure data shall be signed by the exposure operator and 

reviewed and signed by the exposure supervisor responsible for the 

study. 

(4) Exposure data shall be tabulated and presented with mean 

values and a measure of variability (e.g., standard deviation), and 

shall include: 

(i) Airflow rates through the inhalation equipment. 

(ii) Temperature and humidity of air. 

(iii) Chamber concentrations in the chamber breathing zone. 

(iv) Concentration of combustion exhaust gases in the chamber 

breathing zone. 

(v) Particle size distribution (median diameter and geometric 

standard deviation). 

(vi) Rise and fall time. 

(vii) Chamber concentration during the non-exposure period. 

56 
P.60 

(viii) Distribution of test substance in the chamber. 

(5) Animal data. Tabulation of toxic response data by 

species, strain, sex and exposure level for: 

(i) Number of animals exposed. 

(ii) Number of animals showing signs of toxicity. 

(iii) Number of animals dying. 

(g) References. For additional background information on 

this exposure guideline, consult the following references: 

(1) Barr, E.B. (1988) Operational Limits for Temperature and 

Percent Oxygen During HM Nose-Only Exposures — Emergency 

Procedures [interoffice memorandum]. Albuquerque, NM: Lovelace 

Inhalation Toxicology Research Institute; May 13. 

(2) Barr, E.B.; Cheng, Y.S.; Mauderly, J.L. (1990) 

Determination of Oxygen Depletion in a Nose-Only Exposure Chamber. 

Presented at: 1990 American Association for Aerosol Research; June; 

Philadelphia, PA: American Association for Aerosol Research; 

abstract no. P2el. 

(3) Barrow, C.S. (1989) Generation and Characterization of 

Gases and Vapors. In: McClellan, R.O., Henderson, R.F. ed. Concepts 

in Inhalation Toxicology. New York, NY: Hemisphere Publishing 

Corp., 63-84. 

(4) Benedict, R. (1984) Fundamentals of Temperature, Pressure, 

and Flow Measurements. 3rd ed. John Wiley & Sons. New York, NY. 

(5) Cannon, W.C ; Blanton,E.F.; McDonald,K.E. The Flow-Past 

Chamber. (1983) An Improved Nose-Only Exposure System for 

Rodents. Am. Ind. Hyg. Assoc. J. 44 : 923-928. 

(6) Chaddock, J.B. ed. (1985) Moisture and humidity. 

Measurement and Control in Science and Industry: Proc. of the 1985 

Int'l Symposium on Moisture and Humidity; April 1985; Washington, 

D.C. Research Triangle Park, NC: Instrument Society of America. 

(7) Cheng,Y.S.; Barr,E.B.; Carpenter,R.L.; Benson,J.M.; 

Hobbs,C.H. (1989) Improvement of Aerosol Distribution in Whole-Body 

Inhalation Exposure Chambers. Inhal. Toxicol. 1: 153-166. 

(8) Cheng,Y.S.; Moss,O.R. (1989) Inhalation Exposure Systems. 

In: McClellan, R.O.; Henderson,R.F. ed. Concepts in Inhalation 

Toxicology. New York, NY: Hemisphere Publishing Corp., 19-62. 

(9) Cheng, Y.S.; Yeh, H.C ; Mauderly, J.L.; Mokler, B.V. 

(1984) Characterization of Diesel Exhaust in a Chronic Inhalation 

Study. Am. Ind. Hyg. Assoc. J. 45: 547-555. 

57 
P.61 

(10) Gillum, D.R. (1982) Industrial Pressure Measurement. 

Research Triangle Park, NC: Instrument Society of America. 

(11) Hinners, R.G.; Burkart, J.K.; Malanchuk, M. (1979) 

Animal Exposure Facility for Diesel Exhaust Studies. 

(12) Kittelson, D.B.; Dolan, D.F. (1979) Diesel Exhaust 

Aerosols. 

(12) Mokler, B.V.; Archibeque, F.A.; Beethe, R.L.; Kelly, 

C.P.J.; Lopez, J.A.; Mauderly, J.L.; Stafford, D.L. (1984) Diesel 

Exhaust Exposure System for Animal Studies. Fundamental and 

Applied Toxicology 4: 270-277. 

(13) Moore, W.; et al. (1978) Preliminary finding on the 

Deposition and Retention of Automotive Diesel Particulate in Rat 

Lungs. Proc. of Annual Meeting of the Air Pollution Control Assn, 

3, paper 78-33.7. 

(14) Raabe O.G., Bennick, J.E., Light, M.E., Hobbs, C.H., 

Thomas, R.L., Tillery,M.I. (1973) An Improved Apparatus for Acute 

Inhalation Exposure of Rodents to Radioactive Aerosols. Toxicol & 

Applied Pharmaco.; 1973; 26: 264-273. 

(15) Rao, G.N. (1986) Significance of Environmental Factors 

on the Test System. In: Hoover, B.K.; Baldwin, J.K.; Uelner, A.F.; 

Whitmire, C.E.; Davies, C.L.; Bristol, D.W. ed. Managing conduct 

and data quality of toxicology studies. Raleigh, NC: Princeton 

Scientific Publishing Co.,Inc.: 173-185. 

(16) Spitzer, D.W. (1984) Industrial Flow Measurement. 

Research Triangle Park, NC: Instrument Society of America. 

(17) 40 CFR Part 798 (1990) Health effects testing 

guidelines. 

(18) 29 CFR Part 1910 (1989) Occupational safety and health 

standards for general industry. 

(19) Federal Register, V42, No. 101, p. 26748, 1977. 

(20) Guide for the Care and Use of Laboratory Animals (1985) 

U.S. Department of Health and Human Services, Pub. No. [NIH] 85-23. 

§79.51 S*lmonmllm typhimurium reverse mutation (Ames) assay. 

(a) Purpose. The Salmonella typhimurium histidine (his) 

reversion system is a microbial assay which measures his" —> his+ 

reversion induced by chemicals which cause base changes or 

frameshift mutations in the genome of this organism. 

(b) Definitions. 

58 
P.62 

(1) A reverse mutation assay in Salmonella typhimurium 

detects mutation in a gene of a histidine-requiring strain to 

produce a histidine independent strain of this organism. 

(2) Base pair mutagens are agents which cause a base change 

in the DNA. In a reversion assay, this change may occur at the site 

of the original mutation or at a second site in the chromosome. 

(3) Frameshift mutagens are agents which cause the addition 

or deletion of single or multiple base pairs in the DNA molecule. 

(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 

particulate extract from the filter and the organic extract from 

the resin. 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 and compared to the number of 

spontaneous revertants in a base fuel-only and/or unexposed control 

culture. 

(2) Description. Several methods for performing the test 

have been described. Among those used are: 

(i) Direct plate incorporation method, 

(ii) Preincubation method, 

(iii) Azo-reduction method, and 

(iv) Microsuspension method. 

The procedures described here are for the direct plate incorporation method and the azo-reduction method. 

(3) Strain selection. 

(i) Designation. 

(A) 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. The choice of the 

particular strain is left to the discretion of the researcher, but 

59 
P.63 

TA98/1,8-DNPs or other suitable Rosenkranz nitro-reductase 

resistant strains will be considered acceptable. 

(B) The use of other strains in addition to the five tester 

strains is left to the discretion of the investigator. 

(ii) Preparation and storage. Recognized methods of stock 

culture preparation and storage should be used. The requirement of 

histidine for growth should be demonstrated for each strain. Other 

phenotypic characteristics should be checked using such methods as 

crystal violet sensitivity and resistance to ampicillin. Spontaneous reversion frequency should be in the range expected either as 

reported in the literature or as established in the laboratory by 

historical control values. 

(iii) Bacterial growth. Fresh cultures of bacteria should be 

grown up to the late exponential or early stationary phase of 

growth (approximately 108-109 cells per ml) . 

(4) Metabolic activation. Bacteria should be exposed to the 

test substance both in the presence and absence of an appropriate 

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 prepared from the livers of untreated hamsters is 

preferred. For this method, the cofactor supplement should 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 and/or base fuel only) 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. 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. 

(D) TA98/1,8-DNPs: benzo(a)pyrene (with activation). 

(iii) Positive controls to ensure the efficacy of the 

activation system. The positive control reference substance for 

tests including a metabolic activation system should be selected on 

the basis of the type of activation system used in the test. 

60 

. 
P.64 

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 should include positive controls from the same class of 

compounds as the test agent wherever possible. 

(6) Test chemicals. 

(i) Emissions from test products and positive control 

reference chemicals should be dissolved or suspended in an 

appropriate solvent/vehicle and samples prepared from this material 

for use in the assay. Final concentration of the vehicle should 

not interfere with cell viability or growth rate. 

(A) The paper by Claxton and Huising, et. al. (1980) is 

useful for preparing extracts from particulate matter (soluble 

organic fraction) and is recommended. 

(B) The paper by Stump (1982) is useful for preparing 

extracts of semi-volatile organic compounds from sorbent resins and 

is recommended. 

(ii) 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 chemical 

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 compounds should be tested up to the limits of 

solubility. For freely soluble nontoxic chemicals, the upper test 

chemical concentration should 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 amounts of test 

substance shall be tested 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. 

(1) Direct plate incorporation method. For this test without 

metabolic activation, test sample and 0.1 ml of a fresh bacterial 

culture should be added to 2.0 ml of overlay agar. For tests with 

61 
P.65 

metabolic activation, 0.5 ml of activation mixture containing an 

adequate amount of postmitochondrial fraction should be added to 

the agar overlay after the addition of test chemical and bacteria. 

Contents of each tube shall be 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. 

(2) Azo-reduction method. For this test with metabolic 

activation, 0.5 ml of S - 9 mix containing 150 |il of S - 9 and 0.1 

ml of bacterial culture should be added to a test tube kept on ice. 

One-tenth milliliter of chemical should be added, and the tubes 

should be incubated with shaking at 30° C for 30 min. At the end 

of the incubation period, 2.0 ml of agar should 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. 

(3) For tests without metabolic activation, 0.5 ml of buffer 

should be used in place of the 0.5 ml of S - 9 mix. All other 

procedures shall be the same as those used for the test with 

metabolic activation. 

(4) Other methods. Other methods may also be appropriate. 

(5) Media. An appropriate selective medium with an adequate 

overlay agar shall be used. 

(6) 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°C 

(7) Number of cultures. All plating should 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 and, quantitatively, as revertants 

per mile(kilometer) for each replicate and dose. The numbers of 

revertant colonies and revertants per mile (or kilometer) on both 

negative (base fuel-only and/or untreated) and positive control 

plates shall also be presented. Individual plate counts, the mean 

number of revertant colonies per plate, revertants per mile (or 

kilometer) and standard deviation shall be presented for test 

chemical and positive and negative (untreated and/or vehicle) 

controls. 

(2) Statistical evaluation. Data should be evaluated by 

appropriate statistical methods. 

(3) Interpretation of results. 

62 
P.66 

(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 should 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 Part 792, Subpart J, the following 

specific information shall be reported: 

(i) Sampling method(s) used and manner in which cells are 

exposed to sample 

(ii) Bacterial strains used. 

(iii) Metabolic activation system used (source, amount and 

cofactor); details of preparations of S - 9 mix. 

(iv) Dose levels and rationale for selection of dose. 

(v) Positive and negative controls. 

(vi) Individual plate counts, mean number of revertant 

colonies per plate, number of revertants per mile (or kilometer), 

and standard deviation. 

(vii) Dose-response relationship, if applicable. 

(g) References. For additional background information on 

this test guideline the following references should be consulted: 

(1) 40 CFR §798.5265 (1990) The salmonella typhimurium 

reverse mutation assay, pp 512-15. 

63 
P.67 

(2) Ames, B.N., McCann, J., Yamasaki, E. (1975) Methods for 

Detecting Carcinogens and Mutagens with the Salmonella Mammalian 

Microsome Mutagenicity Test. Mutation Research 31:347-364. 

(3) Brusick, D.J., Young, R.R., Jagannath, D.R. (1984) The 

Use of Short-term Genotoxic Bioassays in the Evaluation of 

Unregulated Automobile Emissions. U.S. EPA., Off. Res. & Dev. 

[Rep.] EPA-600/S1-84-011, pp 1-5. 

(4) Huisingh, J.L., et al. (1980) 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. EPA600/9-80-057b, pp 788-800. 

(5) Claxton, L.D., Kohan, M., Austin, A.C , Evanc, C (1982) 

U.S. EPA, Off. Res. Dev. [Rep.] EPA-600/2-82-008, pp 1-148 

(6) de Serres, F.J., Shelby, M.D. (1979) The Salmonella 

Mutagenicity Assay: Recommendations. Science 203:563-565. 

(7) Jones, E., Richold, M., May, J.H., and Saje, A. (1985) 

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. 

(8) Maron, D., and Ames, B.N. (1983) Revised Methods for the 

Salmonella Mutagenicity Test. Mutation Research, 113:173-212. 

(9) Prival, M.J., and Mitchell, V.D. (1982) 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. 

(10) Rosenkranz, H.S., et al. (1980) Nitropyrenes: Isolation, 

Identification, and Reduction of Mutagenic Impurities in Carbon 

Black and Toners. Science 209:1039-43. 

(11) Stump, F., Snow, R., et al. (1982) Trapping gaseous 

Hydrocarbons for Mutagenic Testing. SAE Tech. Paper No. 820776. 

(12) Vogel, H.J., Bonner, D.M. (1956) Acetylornithinase of E. 

coli: Partial Purification and Some Properties. J. Biol. Chem. 

218:97-106. 

§79.52 In vivo micronucleus assay. 

(a) Purpose. 

(1) The micronucleus test is a mammalian in vivo cytogenetic 

test which detects damage of the chromosomes or mitotic apparatus 

by chemicals. Normochromatic erythrocytes in the peripheral blood 

or bone marrow of rodents are used in this assay. In the bone 

64 
P.68 

marrow, as the erythroblast develops into an erythrocyte, the main 

nucleus is extruded and may leave a micronucleus in the cytoplasm. 

The visualization of micronuclei is facilitated in these cells 

because they lack a nucleus. Micronuclei form under normal 

conditions. The assay is based on an increase in the frequency of 

micronucleated normochromatic erythrocytes in the blood or bone 

marrow of treated animals. 

(b) Definition. 

(1) Micronuclei are small particles consisting of acentric 

fragments of chromosomes or entire chromosomes, which lag behind at 

anaphase of cell division. After telophase, these fragments may 

not be included in the nuclei of daughter cells and form single or 

multiple micronuclei in the cytoplasm. 

(2) Normochromatic erythrocyte is a typical erythrocyte, or 

red blood cell, which is stainable generally only with an acidic 

stain. It is contrasted with a polychromatic erythrocyte, which is 

stainable by various types of stains. 

(c) Reference substances. Not applicable. 

(d) Test method. 

(1) Principle of the test method. 

(i) Several groups of experimental animals are exposed daily 

for at least 6 hours/day for a period of 42 days by inhalation 

route to the test substance in graduated concentrations, one 

concentration being used per group. At the conclusion of the 

exposure period, a peripheral blood sample is taken, for example, 

from the tail, and normochromatic erythrocytes are stained for 

micronuclei. If rats are used, individual animals are sacrificed 

and the femoral bone marrow is extracted. Bone marrow is smeared 

onto glass slides, dried, and stained for micronuclei. Normochromatic erythrocytes are scored for micronuclei under the microscope. 

(2) Animal selection. 

(i) Species and strain. The rat is the recommended test 

animal; however, any appropriate mammalian species may be used. 

Examples of commonly used rodent species are mice, rats, and 

hamsters. 

(ii) Number and sex. At least five female and five male 

animals per experimental and control group shall be used. The use 

of a single sex or a smaller number of animals should be justified. 

(3) Positive control group. A single dose of a compound 

known to produce micronuclei in vivo is adequate as a positive 

control if it shows a significant response at any one time point. 

(4) Concentration levels. To select the appropriate 

concentration dose levels, a pilot or trial study may be advisable. 

65 
P.69 

For an initial assessment, one concentration of the test substance 

may be used, the maximum tolerated dose or that producing some 

indication of toxicity, e.g., a drop in the ratio of polychromatic 

to normochromatic erythrocytes. A concentration of 50 to 80% of an 

LD50 would be a suitable guide and additional concentration levels 

may be used. 

(5) Inhalation exposure. The general conduct of this study 

shall be in accordance with the vehicle emissions inhalation 

exposure guideline in section 7 9.50 of this subpart. 

(e) Test performance. 

(1) Preparation of slides and sampling times. 

(i) At the end of the exposure period, twenty-four hours 

after the last exposure, one femur will be removed and placed in 

fetal bovine serum. The bone marrow is removed, processed, and two 

smears per animal are made on coded glass microscope slides. The 

slides are stained according to the acridine-orange (AO) staining 

protocol, fluorescence-plus-Giemsa, or other appropriate stain and 

examined microscopically. A DNA-specific stain should be used for 

the rat. Chromosome preparations are made following standard 

procedures. 

(2) Analysis. Slides shall be coded for study and animal 

identification only before microscopic analysis. At least 1,000 

normochromatic erythrocytes per animal shall be scored for the 

incidence of micronuclei. Sexes will be analyzed separately. 

(f) Data and report. 

(1) Treatment of results. In addition to the reporting 

requirements specified under 40 CFR Part 792 Subpart J, data will 

be reported in accordance with the vehicle emissions inhalation 

exposure guideline in section 79.50 of this subpart. The final 

test report must include the criteria for scoring micronuclei. 

Individual data shall be presented in a tabular form including 

positive and negative, i.e., base fuel-only, controls and experimental groups. The number of normochromatic erythrocytes scored, 

the number of micronucleated normochromatic erythrocytes, the 

percentage of micronucleated cells, and, if applicable, the 

percentage of micronucleated erythrocytes shall be listed separately for each experimental and control animal. Absolute numbers 

shall be included if percentages are reported. 

(2) Interpretation of results. 

(i) There are several criteria for determining a positive 

response, one of which is a statistically significant dose-related 

increase in the number of micronucleated normochromatic erythrocytes. Another criterion may be based upon detection of a 

reproducible and statistically significant positive response for at 

least one of the test substance concentrations. 

66 
P.70 

(ii) A test substance which does not produce either a 

statistically significant dose-related increase in the number of 

micronucleated normochromatic erythrocytes or a statistically 

significant and reproducible positive response at any one of the 

test points is considered nonmutagenic in this system. 

(3) Test evaluation. 

(i) The results of the micronucleus test provide information 

on the ability of a chemical to induce micronuclei in 

normochromatic erythrocytes of the test species under the conditions of the test. This damage may have been the result of 

chromosomal damage or damage to the mitotic apparatus. 

(ii) Negative results indicate that under the test conditions 

the test substance does not produce micronuclei in the bone marrow 

or peripheral blood, as appropriate, of the test species. 

(g) Test report. In addition to the reporting recommendations as specified under 40 CFR Part 792, Subpart J, and in section 

79.50(e) of this subpart, the following specific information shall 

be reported: 

(1) Test atmosphere preparation, concentration levels used, 

rationale for concentration selection. 

(2) Rationale for and description of treatment and sampling 

schedules, toxicity data, negative and positive controls. 

(3) Historical control data (negative and positive), if 

available. 

(4) Details of the protocol used for slide preparation. 

(5) Criteria for identifying micronucleated erythrocytes. 

(6) Statistical methodology chosen for test analysis. 

(h) References. For additional background information on 

this test guideline the following references should be consulted: 

(1) 40 CFR §798.5395 (1990) In Vivo Mammalian Bone Marrow 

Cytogenetics Tests: Micronucleus Assay, pp 525-27. 

(2) Cihak, R. (1979) Evaluation of Benzidine by the Micronucleus Test. Mutation Research, 67:383-84. 

(3) Cole, R.J., et al. (1981) Short-term Tests for 

Transplacentally Active Carcinogens. 1. Micronucleus Formation in 

Fetal and Maternal Mouse Erythroblasts. Mutation Res., 80:141-157. 

(4) Evans, H.J. (1976) Cytological Methods for Detecting 

Chemical Mutagens. In: Chemical Mutagens: Principles and Methods 

for Their Detection, Vol. 4. A. Hollaender (Ed.) Plenum Press, New 

York and London, pp 1-29. 

67 
P.71 

(5) Heddle, J.A., et al. (1983) The Induction of Micronuclei 

as a Measure of Genotoxicity. A Report of the U.S. Environmental 

Protection Agency Gene-Tox Program. Mutation Res., 123:61-118. 

(6) Kliesch, U., Danford, N., and Adler, I.D. Micronucleus 

Test and Bone-Marrow Chromosome Analysis. A Comparison of 2 

Methods In Vivo for Evaluating Chemically Induced Chromosomal 

Alterations. Mutation Research, 80:321-332. 

(7) Preston, J.R. et al. (1981) Mammalian In Vivo and In 

Vitro Cytogenetics Assays: Report of the Gene-Tox Program. 

Mutation Research, 87:143-188. 

(8) Schmid, W. (1975) The Micronucleus Test. Mutation Res., 

31:9-15. 

(9) Tice, R.R., Luke, C.A., and Shelby, M.D. (1987) Methyl 

Isocyanate: An Evaluation of In Vivo Cytogenetic Activity. 

Environmental Mutagenesis, 9:37-58. 

§79.53 In vivo sister chromatid exchange assay. 

(a) Purpose. The sister chromatid exchange (SCE) assay 

detects the ability of a chemical to enhance the exchange of DNA 

between two sister chromatids of a duplicating chromosome. The 

most commonly used assays employ mammalian tissues, bone marrow, 

peripheral blood or spleen lymphocytes, from species such as mice, 

rats or hamsters. Human lymphocytes may also be used. 

(b) Definition. 

(1) *'C-metaphase'' refers to a state of arrested cell growth 

typically seen after treatment with a spindle inhibitor, i.e., 

colchicine. 

(2) Sister chromatid exchanges represent reciprocal 

interchanges of the two chromatid arms within a single chromosome. 

These exchanges are visualized during the metaphase portion of the 

cell cycle and presumably require enzymatic incision, translocation 

and ligation of at least two DNA helices. 

(c) Reference substances. Not applicable. 

(d) Test method. 

(1) Principle of the test method. Several groups of 

experimental animals are exposed daily for a minimum of 6 hours per 

day by inhalation route to the test substance in graduated 

concentrations, one concentration being used per group, for a 

period of 42 days. Primary cultures are prepared from the cells of 

the lung and blood lymphocytes isolated from each study animal. 

Bromo-deoxyridine (BrdU) is added to each plate of cells and a 

spindle inhibitor, e.g., colchicine or colcemid, is administered 

68 
P.72 

prior to harvest. At harvest, cells are fixed and stained before 

scoring for SCE. 

(2) Description. The method described here employs both 

primary lung cells and peripheral blood lymphocytes (PBL) of 

laboratory rodents exposed to the test atmosphere. 

(i) Following the exposure period, all test animals are 

anaesthetized and heart punctures are performed. Lymphocytes are 

isolated over a Ficoll gradient and replicate cell cultures are 

started for each animal. The cells are treated with bromodeoxyuridine (BrdU) and, the following day, a spindle inhibitor 

(e.g., colchicine/colcemid) is added to arrest cell growth in 

c-metaphase. Cells are harvested and chromosome preparations are 

fixed, differentially stained, and scored for SCE. 

(ii) After heart puncture, the lungs are washed of blood and 

they are instilled with a solution of trypsin, EDTA, and 

collagenase. The lungs, with fluid, are removed, minced, and a 

cell pellet is centrifuged out of the resulting supernatant. Cells 

in complete medium are then seeded onto coverslips in culture 

dishes and BrdU is added. After two days of incubation, a spindle 

inhibitor is added and the cells are returned to the incubator. 

Cells are then harvested, fixed, and stained for SCE's. 

(3) Animal selection. 

(i) Species and strain. The rat is the recommended test 

animal; however, any appropriate mammalian species may be used. 

Examples of commonly used rodent species include mice, rats, and 

hamsters. 

(ii) Number and sex. At least five female and five male 

animals per experimental and control group should be used. The use 

of a single sex or different number of animals should be justified. 

(4) Positive control group. A compound known to produce SCE 

in vivo should be employed as the positive control. 

(5) Test chemicals. Concentration levels. For an initial 

assessment, one concentration of the test substance may be used, 

the maximum tolerated dose or that producing some indication of 

toxicity as evidenced by animal morbidity (including death) or 

target cell toxicity. A concentration of 50-80% of an LD50 would 

be a suitable guide and additional concentration levels may be 

used. 

(6) Observation period. Duration of observation shall be for 

at least 42 days. 

(7) Inhalation exposure. The general conduct of this study 

shall be in accordance with the vehicle emissions inhalation 

exposure guideline in section 79.50 of this subpart. 

(e) Test performance. 

69 
(1) Treatment. 

(i) At the conclusion of the exposure period, after a 

recovery period of approximately 20 hours, all test animals are 

anaesthetized and heart punctures are performed. Lymphocytes are 

isolated over a Ficoll gradient and replicate cell cultures are 

started for each animal. After 21 hours, the cells are treated 

with BrdU and returned to incubation. The following day, a spindle 

inhibitor (e.g., colchicine/Colcemid) is added to arrest cell 

growth in c-metaphase. Cells are harvested 4 hours later and 

chromosome preparations from second-division metaphase cells are 

fixed in methanol:acetic acid, stained, and scored for SCE. 

(ii) After heart puncture, the lungs are isolated, washed of 

blood, and a solution of trypsin, EDTA, and collagenase is 

instilled into the lungs. After 5 minutes, the lungs, with fluid, 

are removed, minced, and incubated for 1.5 to 2 hours. A cell 

pellet is centrifuged out of the resulting supernatant. Cells in 

complete medium are then seeded onto coverslips in culture dishes, 

four to five replicates from each animal, and BrdU is added. After 

two days of incubation, a spindle inhibitor is added and the cells 

are returned to the incubator for an additional 3 hours. Cells are 

then harvested, fixed, and stained for SCE's. 

(2) Staining method. Staining of slides to reveal SCEs can 

be performed according to any of several protocols. However, the 

fluorescence plus Giemsa method is recommended. 

(3) Number of cells scored. 

(i) A minimum of 25 well-stained, second-division metaphase 

cells should be scored for each animal for each cell type. 

(ii) 100 consecutive metaphase cells should be scored for the 

number of first, second, and third division metaphases. 

(iii) 1000 consecutive PBL's should be scored for the number 

of metaphase cells present. 

(iv) The number of cells to be analyzed per animal should be 

based upon the number of animals used, the negative control 

frequency, the pre-determined sensitivity and the power chosen for 

the test. Slides should be coded before microscopic analysis. 

(f) Data and report. 

(1) Treatment of results. In addition to the reporting 

requirements specified under 40 CFR Part 792 Subpart J, data will 

be reported in accordance with the vehicle emissions inhalation 

toxicity guideline as published in subsection (a) of this section. 

Specifically, data should be presented in tabular form, providing 

scores for both the number of SCE for each metaphase and the number 

of SCE per chromosome for each metaphase. Differences among 

animals within each group should be considered before making 

comparisons between treated and control groups. 

70 
(2) Statistical evaluation. Data should be evaluated by 

appropriate statistical methods. 

(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 SCE. Another criterion may be based upon 

detection of a reproducible and statistically significant positive 

response for at least one of the test points. 

(ii) A test substance which does not produce either a 

statistically significant dose-related increase in the number of 

SCE or a statistically significant and reproducible positive 

response at any one of the test points is considered not to induce 

rearrangements of DNA segments in this system. 

(iii) Both biological and statistical significance should be 

considered together in the evaluation. 

(4) Test evaluation. 

(i) A positive result in the in vivo SCE assay for either, or 

both, the lung or lymphocyte cultures indicate that under the test 

conditions the test substance induces reciprocal interchanges of 

DNA in duplicating chromosomes from lung or lymphocyte cells of the 

test species. 

(ii) Negative results indicate that under the test conditions 

the test substance does not induce reciprocal interchanges in lung 

or lymphocyte cells of the test species. 

(5) Test report. In addition to the reporting recommendations as specified under 40 CFR Part 792, Subpart J and in section 

79.50(e) of this subpart, the following specific information should 

be reported: 

(i) Concentrations used, rationale for concentration 

selection, negative and positive controls. 

(ii) Toxic response data by concentration. 

(iii) Route and schedule of administration of both test 

atmosphere and BrdU. 

(iv) Time of harvest.after administration of BrdU. 

(v) Identity of spindle inhibitor, its concentration and 

duration of treatment. 

(vi) Details of the protocol used for slide preparation. 

(vii) Criteria for scoring SCE. 

71 
P.75 

(viii) Replicative index, i.e., [% 1st division + (2 X % 2nd 

division) + (3 X % 3rd division) metaphases]/IOO. 

(ix) Mitotic activity, i.e., # of metaphases/1000 cells. 

(g) References. For additional background information on 

this test guideline the following references should be consulted: 

(1) 40 CFR §798.5915 (1990) In Vivo Sister Chromatid Exchange 

Assay, pp 542-44. 

(2) Campbell, J.A., et al. (1986) Sister Chromatid Exchange 

Analysis in Cultured Primary Lung, Liver, and Kidney Cells of Mice 

Following In Vivo Exposure to Vinyl Carbamate. In Vitro Cellular 

and Developmental Biology, 22 (8):443-448. 

(3) Kato, H. (1974) Spontaneous Sister Chromatid Exchanges 

Detected by a BudR-Labeling Method. Nature, 251:70-72. 

(4) Kligerman, A.D., et al. (1987) Sister Chromatid Exchange 

Analysis in Lung and Peripheral Blood Lymphocytes of Mice Exposed 

to Methyl Isocyanate by Inhalation. Environ. Mutagenesis 9:29-36. 

(5) Latt, S.A., et al. (1981) Sister Chromatid Exchanges: A 

report of the U.S. EPA Gene-Tox Program. Mutation Res., 87:17-62. 

(6) Wollf, S. and Perry, P. (1974) Differential Giemsa 

Staining of Sister Chromatids and the Study of Sister Chromatid 

Exchanges Without Autoradiography. Chromosoma, 48:341-53. 

§79.54 Inhalation developmental toxicity study. 

(a) Purpose. In the assessment and evaluation of the toxic 

characteristics of an inhalable material such as a gas, volatile 

substance, or aerosol/particulate, determination of the potential 

developmental toxicity is important. The inhalation developmental 

toxicity study is designed to provide information on the potential 

hazard to the unborn which may arise from exposure of the mother 

during pregnancy. 

(b) Definition. Developmental toxicity is the capability of 

an agent to induce in utero death, structural, or functional 

abnormalities, or growth retardation after contact with the 

pregnant animal. 

(c) Principle of the test method. The test substance is 

administered in graduated concentrations, for at least that part of 

the pregnancy covering the major period of organogenesis, to 

several groups of pregnant experimental animals, one exposure level 

being used per group. Shortly before the expected date of 

delivery, the pregnant females are sacrificed, the uteri removed, 

and the contents examined for embryonic or fetal deaths, and live 

fetuses. 

72 
P.76 

(d) Test procedures. 

(1) Animal selection. 

(i) Species and strain. Testing shall be performed in at 

least two mammalian species. Commonly used species include the 

rat, mouse, rabbit, and hamster. If other mammalian species are 

used, the tester shall provide justification/reasoning for their 

selection. Commonly used laboratory strains shall be employed. 

The strain shall not have low fecundity and shall preferably be 

characterized for its sensitivity to developmental toxins. 

i 

(ii) Age. Young adult animals, at least 10 weeks old, shall 

be used. 

(iii) Sex. Pregnant female animals shall be used at each 

exposure level. [ 

(iv) Number of animals. At least 20 pregnant test animals 

are required at each exposure level. The objective is to ensure 

that sufficient pups are produced to permit meaningful evaluation 

of the potential developmental toxicity of the test substance. 

(2) Concentration levels and concentration selection. 

(i) To select the appropriate dose levels, a pilot or trial 

study may be advisable. Since pregnant animals have an increased 

minute ventilation as compared to non-pregnant animals, it is 

recommended that the trial study be conducted in pregnant animals. 

Similarly, since presumably the minute ventilation will vary with 

progression of pregnancy, the animals should be exposed during the 

same period of gestation as in the main study. It is not always 

necessary, though, to carry out a trial study in pregnant animals. 

Comparisons between the results of a trial study in non-pregnant 

animals, and the main study in pregnant animals will demonstrate 

whether or not the test substance is more toxic in pregnant 

animals. In the trial study, the concentration producing embryonic 

or fetal lethalities or maternal toxicity should be determined. 

(ii) Unless limited by the physical/chemical nature or 

biological properties of the substance, the highest concentration 

level shall induce some overt maternal toxicity such as reduced 

body weight or body weight gain, but not more than 10 percent 

maternal deaths. 

(iii) The lowest concentration level should not produce any 

grossly observable evidence of either maternal or developmental 

toxicity. 

(3) Observation period. Day 0 in the test is the day on 

which a vaginal plug or sperm are observed. The exposure period 

shall cover the period of major organogenesis. This may be taken 

as days 6 to 15 for rat and mouse, 6 to 14 for hamster, or 6 to 18 

for rabbit. 

73 
(4) Inhalation exposure. The general conduct of this study 

shall be in accordance with the vehicle emissions inhalation 

exposure guideline in section 79.50 of this subpart. Directions 

specific to this study are: 

(i) Where a chamber is used, its design should minimize 

crowding of the test animals. This is best accomplished by 

individual caging. 

(ii) Pregnant animals shall not be subjected to beyond the 

minimum amount of stress. Since whole-body exposure appears to be 

the least stressful mode of exposure, it is the method preferred. 

In general, oronasal or head-only exposure, which are sometimes 

used to avoid concurrent exposure by the dermal or oral routes, are 

not recommended because of the associated stress accompanying the 

restraining of the animals. However, there may be specific 

instances where it may be more appropriate than whole-body 

exposure. The tester shall provide justification/reasoning for its 

selection. 

(iii) Measurements should be made at least every other day of 

food consumption for all animals in the study. 

(iv) Animals shall be weighed at least every other day. 

(v) Females showing signs of abortion or premature delivery 

shall be sacrificed and subjected to a thorough macroscopic 

examination. 

(7) Gross necropsy. 

(i) At the time of sacrifice or death during the study, the 

dam shall be examined macroscopically for any structural 

abnormalities or pathological changes which may have influenced the 

pregnancy. 

(ii) Immediately after sacrifice or death, the uterus shall 

be removed, weighed, and the contents examined for embryonic or 

fetal deaths and the number of viable fetuses. Gravid uterine 

weights should not be obtained from dead animals if autolysis or 

where decomposition has occurred. The degree of resorption shall 

be described in order to help estimate the relative time of death. 

(iii) The number of corpora lutea shall be determined for all 

species except mice. 

(iv) The sex of the fetuses shall be determined and they 

shall be weighed individually, the weights recorded, and the mean 

fetal weight derived. 

(v) Following removal, each fetus shall be examined 

externally. 

(vi) For rats, mice and hamsters, one-half of each litter 

shall be prepared and examined for skeletal anomalies, and the 

74 
P.78 

remaining part of each litter shall be prepared and examined for 

soft tissue anomalies using appropriate methods. 

(vii) For rabbits, each fetus shall be examined by careful 

dissection for visceral anomalies and then examined for skeletal 

anomalies. 

(e) Data reporting and evaluation. 

(1) Treatment of results. In addition to the reporting 

requirements specified under 40 CFR Part 792 Subpart J, data will 

be reported in accordance with section 79.50(e) of this subpart. 

The final test report must also include the following information: 

(i) The number of pregnant animals, 

(ii) The number and percentages of live fetuses, and 

(iii) The number of fetuses with any soft tissue or skeletal 

abnormalities. 

(iv) Other animal data. 

(A) Date of death during the study or whether animals 

survived to termination. 

(B) Date of onset and duration of each abnormal sign and its 

subsequent course. 

(C) Feed, body weight and uterine weight data. 

(D) Pregnancy and litter data. 

(E) Fetal data (live/dead, sex, soft tissue and skeletal 

defects, resorptions). 

(2) Evaluation of results. The findings of a developmental 

toxicity study shall be evaluated in terms of the observed effects 

and the exposure levels producing effects. It is necessary to 

consider the historical developmental toxicity data on the 

species/strain tested. 

(f) References. For additional background information on 

this test guideline the following references should be consulted: 

(1) 40 CFR §798.4350 (1990) Inhalation Developmental Toxicity 

Study, pp 485-89. 

(2) Department of Health and Welfare (1975) The Testing of 

Chemicals for Carcinogenicity, Mutagenicity and Teratogenicity. 

Minister of Health and Welfare, Canada. 

(3) National Academy of Sciences (1977) Principles and 

Procedures for Evaluating the Toxicity of Household Substances. A 

report prepared by the Committee for the Revision of NAS Public. 

75 
P.79 

1138, under the auspices of the Committee on Toxicology, National 

Research Council, National Academy of Sciences, Washington, DC. 

(4) World Health Organization (1967) Principles for the 

Testing of Drugs for Teratogenicity. WHO Technical Report Series 

No. 364. World Health Organization, Geneva. 

§79.55 Reproduction and Fertility Screen. 

(a) Purpose. 

(1) The reproduction and fertility screen is an in vivo study 

which has been designed to provide information on health hazards to 

the reproductive organs and effects on gonadal function, 

conception, and parturition which are likely to arise from a 

repeated inhalation exposure to a test substance over a period of 

42 days. The study includes mating a group of study animals to 

provide information about the effects of the test atmosphere on 

neonatal morbidity, mortality, and preliminary data on teratogenesis. The test is not capable of determining those effects that 

have a long latency period for development (e.g., carcinogenicity 

and life shortening) but it can be of use in selecting dose levels 

for chronic studies and for establishing safety criteria for human 

exposure. 

(b) Definitions. 

(1) Sperm measures, including epididymal sperm counts, and 

assessment of sperm motility and morphology, are evaluated as 

indicators of chemically-induced damage in the testis and/or 

epididymis. 

(2) Vaginal cytology evaluation uses vaginal smears from 

these animals to determine the regularity of the estrous cycle and 

the effects of toxic chemicals on reproductive function. Common 

cell types found in the smears correlate well with the various 

stages of the estrous cycle and to changes occurring in the 

reproductive tract. 

(3) Estrous cycle refers to the periodic recurrence of the 

biological phases of the female reproductive system which prepare 

the animal for conception and the development of offspring. The 

phases of the estrous cycle for a particular animal can be 

characterized by the general condition of the cells present in the 

vagina and the presence or absence of various cell types. 

(c) Principle of the test method. 

(1) For a three week period before exposure starts, female 

animals undergo daily vaginal lavages and the wet smears are 

examined to identify and cull those animals which are acyclic. 

Exposure begins and all animals are exposed by inhalation to 

graduated concentrations of the test atmosphere for at least six 

hours per day for 42 days. Daily wet vaginal smears continue to be 

76 
P.80 

examined from all females, including those designated for mating 

and pregnancy, through the end of exposure period. After 

sufficient numbers of females are found pregnant, males will begin 

to be analyzed for sperm measures and both males and non-pregnant 

females will have tissues taken and fixed for histopathologic 

examination. 

(2) One testis and epididymis are collected and weighed from 

each anesthetized male animal for sperm measures. Using fluid from 

the cauda epididymis, sperm motility will be assessed. A 

suspension of spermatozoa is made from a weighed section of the 

cauda and the sperm count determined. A quantity of this 

suspension is also stained and dried to categorize the morphology 

of intact sperm. 

(3) At the end of the exposure period, females selected for 

the fertility trial will be mated and exposure to the test 

atmosphere will resume. One day before parturition, exposure 

ceases. Gross observations are made on the pups at birth and four 

days later. Dams, which have not delivered by five days after the 

anticipated date of parturition, are sacrificed and reproductive 

tissues are collected for gross examination and histopathology. 

(d) Limit test. If a test at one dose level of the highest 

concentration that can be achieved while maintaining a particle 

size distribution with a mass median aerodynamic diameter (MMAD) of 

4 micrometers (^lm) or less, using the procedures described in 

subsection (a) of this section, produces no observable toxic 

effects and if toxicity would not be expected based upon data of 

structurally related compounds, then a full study using three dose 

levels might not be necessary. Expected human exposure though may 

indicate the need for a higher dose level. 

(e) Test procedures. 

(1) Animal selection. 

(i) Species and strain. The mouse is the preferred species. 

If another mammalian species is used, the tester should provide 

justification/reasoning for its selection. Strains with low 

fecundity should not be used. 

(ii) Animals should be approximately 10 weeks old at the 

start of the exposure period. 

(iii) Number and sex. Each test and control group should 

begin the exposure period with at least 20 males and at least 20 

females. 

(2) Exposure, mating, and delivery schedule. 

(i) Females are mated at the end of the exposure period. 

(ii) For the fertility trial, Day 0 of the pregnancy is the 

day on which a vaginal plug or sperm are observed. A minimum of 

77 
P.81 

ten pregnant females will be followed through to parturition or Day 

25 of the dam's pregnancy. 

(iii) Observation of dams and their pups shall continue until 

the fourth day post partum. Histopathological examinations will 

start four days after parturition or on Day 25 of the dam's 

pregnancy. 

(iv) Sperm measures and histopathology on all males will 

begin after a sufficient number of females are found to be pregnant 

or when a male has been eliminated as a sire. 

(3) Observation period. Duration of observation shall be for 

at least 42 days. 

(4) Inhalation exposure. The general conduct of this study 

shall be in accordance with the vehicle emissions inhalation 

exposure guideline in section 79.50 of this subpart. Directions 

specific to this study are: 

(i) Where a chamber is used for the fertility study, its 

design should minimize crowding of the test animals. This is best 

accomplished by individual caging. 

(ii) Pregnant animals shall not be subjected to beyond the 

minimum amount of stress. Since whole-body exposure appears to be 

the least stressful mode of exposure, it is the method preferred. 

In general oronasal or head-only exposure, which is sometimes used 

to avoid concurrent exposure by the dermal or oral routes, i.e., 

grooming, is not recommended because of the associated stress 

accompanying the restraining of the animals. However, there may be 

specific instances where it may be more appropriate than whole-body 

exposure. The tester shall provide justification/reasoning for its 

selection. 

(iii) Measurements should be made at least every other day of 

food consumption for all animals in the study. 

(iv) Animals shall be weighed at least every other day in the 

study. 

(v) Females showing signs of abortion or premature delivery 

shall be sacrificed and subjected to a thorough macroscopic 

examination for any structural abnormalities or pathological 

changes which may have influenced the pregnancy. 

(f) Test performance'. 

(1) Sperm measures. The procedures cited here follow those 

used by Morrissey, et al., 1988. On the last day of exposure to 

the test atmosphere, male test subjects should be weighed. They 

are weighed again after mating and anesthetized. The left testis 

and epididymis is removed and weighed for each animal and sperm 

measures assessment is begun. Sperm motility should be assessed 

within four minutes of organ removal by two independent observers, 

78 
each counting five microscope fields of 10-20 sperm per field. 

Fluid from the cauda is suspended in Tyrode's buffer and sperm are 

considered motile if they show any movement at all. Results are 

reported as the total number of motile sperm divided by the sum of 

motile and non-motile sperm expressed as a percentage. A count of 

spermatozoa shall be determined for each animal using a known 

dilution of tissue suspension in phosphate-buffered saline from a 

weighed section of the cauda epididymis. After 15 minutes of 

incubation, 0.5 milliliter of suspension is immobilized or killed 

and further diluted for counting on a hemocytometer. An aliquot of 

this tissue suspension should then be stained with 1% Eosin Y 

stain, dried, and evaluated for sperm head morphology (Wyrobek and 

Bruce, 1975). The remaining testis and epididymis are removed, as 

are other tissues, fixed and sectioned for viewing under a light 

microscope. 

(2) Vaginal cytology. A freshly prepared vaginal lavage 

smear should be examined wet and evaluated each day of the exposure 

period, exclusive of mating and pregnancy days, from all females. 

At the end of the exposure period, female test subjects should be 

weighed and the uterus and ovaries removed. These, and other 

tissues, are fixed and sectioned for viewing under a light 

microscope. 

(3) Mating procedure. 

(i) For each mating, either a 1:1 (1 male to 1 female) or a 

1:2 (1 male to 2 females) method may be used. Females should be 

placed with males from the same concentration level until pregnancy 

occurs or 1 week has elapsed. If mating has not occurred after 1 

week, the female should be placed with a different male from the 

same concentration level. Paired matings should be clearly 

identified. 

(ii) Those pairs that fail to mate should be evaluated in the 

course of the study to determine the cause of the apparent 

infertility. This may involve such procedures as additional 

opportunities to mate with proven fertile males or females, 

histological examination of the reproductive organs, and 

examination of the estrous or spermatogenic cycles. 

(iii) Each day, the females should be examined for presence 

of sperm or vaginal plugs. Day 0 of pregnancy is defined as the 

day vaginal plugs or sperm are found. 

(iv) After evidence of copulation, pregnant animals should be 

caged separately in delivery or maternity cages and provided with 

nesting materials. 

(4) Observation of animals. 

(i) Clinical observations will be made at least once daily. 

Observations will include, but are not limited to, examination of 

the hair, skin, eyes, mucous membranes, respiratory system, 

circulatory system, autonomic and central nervous systems, feces, 

79 
P.83 

urine, and general activity. Post-partum neonatal observations 

will be conducted similarly. Physical and behavioral abnormalities 

shall be recorded as they are observed, including the time of 

onset, degree, and duration. 

(ii) Males and females shall be weighed on the first day of 

exposure and 2-3 times per week thereafter. 

(iii) The duration of gestation should be calculated from day 

0 of pregnancy. Signs of difficult or prolonged parturition should 

be recorded. 

(iv) All pups will be examined, without handling, as soon as 

possible after birth to determine the number of viable and 

stillborn pups. The day on which the dam has completely finished 

delivering will be considered post-partum Day 0. Litters will be 

examined for abnormalities on Day 0 and Day 4 post-partum. The 

number of pups (alive and dead) will be counted and sexed. Litters 

should be weighed at birth, or soon thereafter, and at Day 4. In 

all cases, litter weights should be calculated from the weights of 

the individual pups. 

(v) Animals that die or are euthanized because of morbidity 

will be necropsied as soon as possible. 

(g) Data and reporting. 

(1) Treatment of results. All observed results, quantitative 

and incidental, shall be evaluated by an appropriate statistical 

method. The specific methods, including consideration of 

statistical power, should be selected during the design of the 

study. 

(i) Gross necropsy. 

(A) All animals (except offspring) should be subjected to a 

full necropsy which includes examination of the external surface of 

the body, all orifices, and the cranial, thoracic, and abdominal 

cavities and their contents. Special attention should be directed 

to the organs of the reproductive system. 

(B) At least the liver, kidneys, adrenals, pituitary, and 

gonads, including uterus, ovaries, testes, epididymides and seminal 

vesicles (with coagulating glands) should be weighed wet, as soon 

as possible after dissection to avoid drying. 

(C) The following organs and tissues, or representative 

samples thereof, should be preserved in a suitable medium for 

future histopathological examination: All gross lesions; liver; 

kidneys/adrenals; vagina; uterus; cervix; ovaries; testes; 

epididymides; prostate; seminal vesicles. 

(D) Dead or moribund pups should be examined macroscopically 

for external abnormalities 

80 
P.84 

(ii) Histopathology. The following histopathology should be 

performed: 

(A) Full histopathology on the organs and tissues, listed 

above, of all animals (except offspring) in the control and high 

dose groups, and all animals (except offspring) that died or were 

euthanized during the study. If abnormalities or equivocal results 

are seen in any of these tissues, the same tissues from lower 

concentration groups will be examined. 

(B) All gross lesions in all animals. 

(C) Ovaries from any animal which fails to complete its 

pregnancy will be examined and its uterus will be examined for 

implantation sites and corpora lutea, if possible. As suggested 

under mating procedures, reproductive organs of animals suspected 

of infertility may be subjected to microscopic examination. 

(2) Interpretation of results. 

(i) There are several criteria for determining a positive 

result; for example, a statistically significant dose-related 

decrease in the number and/or motility of sperm or an increase in 

the niamber and/or type of sperm abnormalities for treated subjects 

over control subjects. Other criterion, e.g., length of the 

estrous cycle or time spent in any one stage of the estrous cycle, 

may be based upon detection of a reproducible and statistically 

significant positive response for that evaluation parameter. 

(ii) A test substance which does not produce either a 

statistically significant dose-related change in sperm measures or 

length of estrous cycle or a statistically significant and 

reproducible positive response at any one of the test points is 

considered not to induce reproductive organ toxicity in this test 

system. 

(iii) In any study which demonstrates an absence of toxic 

effects, further investigation to establish absorption and 

bioavailability of the test substance should be considered. 

(3) Evaluation of the study results, 

(i) Sperm morphology. 

(A) Positive results in the sperm morphology evaluation 

indicate that under the test conditions the test substance induces 

male reproductive organ toxicity ih the test species. 

(B) Negative results indicate that under the test conditions 

the test substance does not induce male reproductive organ toxicity 

in the test species. 

(ii) Vaginal cytology. 

81 
P.85 

(A) Positive results in the vaginal cytology evaluation 

indicate that under the test conditions the test substance induces 

female reproductive organ toxicity in the test species. 

(B) Negative results indicate that under the test conditions 

the test substance does not induce female reproductive organ 

toxicity in the test species. 

(h) Test report. In addition to the reporting requirements 

as specified under Good Laboratory Practice Standards, 40 CFR Part 

792, Subpart J and in section 79.50(e) of this subpart, the 

following specific information shall be reported: 

(1) Individual animal data. 

(i) Time of death during the study or whether animals 

survived to termination. 

(ii) Time of observation of each abnormal sign and its 

subsequent course. 

(iii) Body weight data. 

(iv) Necropsy findings. 

(v) Male test subjects. 

(A) Testicle weight, and body weight:testicle weight ratio. 

(B) Degree of motility of cauda epididymal sperm. 

(C) Morphology of cauda epididymal sperm categorized for head 

shape and number, body type, tail shape and number, and/or other 

abnormalities, including percentage change from normal, if 

observed. 

(D) Sperm count per milligram of cauda epididymis. 

(E) Detailed description of all histopathological findings, 

especially for the testes and the epididymides. 

(vi) Female test subjects. 

(A) Beginning and ending collection dates for vaginal cell 

smears. 

(B) Determination of mean cycle length. 

(C) Percentage of time spent in each stage of cycle. 

(D) Detailed description of all histopathological findings, 

especially for the uterus and ovaries. 

(2) Additional information from fertility study. 

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(i) Toxic response data by sex and dose, including fertility, 

gestation, and viability indices, and length of gestation. 

(ii) Toxic or other effects on reproduction, offspring, or 

postnatal growth. 

(iii) Time of observation of each abnormal sign and its 

subsequent course. 

(iv) Body weight data for parental animals and offspring. 

(3) Type of stain/fixative and procedures used in preparing 

tissue samples. 

(4) Statistical treatment of the study results. 

(j) References. For additional background information on 

this test guideline the following references should be consulted: 

(1) 40 CFR §798.2675 (1990) Oral Toxicity with Satellite 

Reproduction and Fertility Study, pp 460-66. 

(2) 40 CFR §798.4420 (1990) Preliminary Developmental 

Screen, pp 489-91. 

(3) Ficsor, G. and L.C. Ginsburg (1980) The Effect of 

Hydroxyurea and Mitomycin C on Sperm Motility in Mice. Mutation 

Res., 70:383-87. 

(4) Leblond, CP . and Y. Clermont (1952) Definition of the 

Stages of the Cycle of the Seminiferous Epithelium of the Rat. 

Ann. N. Y. Acad. Sci., 55:548-73. 

(5) Morrissey, R. E., et al. (1988) Evaluation of Rodent 

Sperm, Vaginal Cytology, and Reproductive Organ Weight Data from 

National Toxicology Program 13-week Studies. Fundam. Appl. 

Toxicol., 11:343-358. 

(6) National Toxicology Program: General Statement of Work 

for the Conduct of Toxicity and Carcinogenicity Studies in 

Laboratory Animals (revised April, 1987/modifications through 

January, 1990) Appendix "G", U.S. Dept. of Health and Human 

Services, Public Health Service, National Toxicology Program. 

(7) Stockard, C.R., and G.N. Papanicolaou (1917) The 

Existence of a Typical Oestrous Cycle in the Guinea Pig With a 

Study of its Historical and'Physiological Changes. Am. Jour. Anat., 

22: 225-83. 

(8) Wyrobek, A.J., and W.R. Bruce. (1975) Chemical Induction 

of Sperm Abnormalities ih Mice. Proc. Nat. Sci., 72(11): 4425-29. 

§79.56 Sub-Acute Pulmonary Toxicity Study. 

83 
(a) Purpose. This subacute inhalation study has been 

designed to permit the determination of toxic pulmonary effects 

associated with repeated exposure to a gas, volatile substance, or 

aerosol/particulate for a period of 42 days. The test is not 

capable of determining those effects that have a long latency 

period for development (e.g., carcinogenicity and life shortening). 

It provides information on health hazards likely to arise from 

repeated exposures by the inhalation route over a limited period of 

time. It will provide information on a target organ, the lungs, 

and can be of use in selecting dose levels for chronic studies and 

for establishing safety criteria for human exposure. Hazards of 

inhaled substances are influenced by their inherent toxicity and by 

physical factors such as volatility and particle size. 

(b) Definitions. Not applicable. 

(c) Principle of the test method. Several groups of 

experimental animals are exposed daily for a defined period to the 

test substance in graduated concentrations, one concentration being 

used per group, for a period of 42 days. During the period of 

administration, the animals are observed daily to detect signs of 

toxicity. After one week's exposure, some test animals are 

examined for acute effects of high exposure. Animals which die 

during the test are necropsied. At the conclusion of the exposure 

period, lungs/tissues/cells are harvested from surviving animals 

and evaluated with appropriate histopathological and other 

procedures. 

(d) Limit test. If a test at one dose level of the highest 

concentration that can be achieved while maintaining a particle 

size distribution with a mass median diameter (MMAD) of 4 

micrometers (|im) or less, using the procedures described in 

subsection (a) of this section, produces no observable toxic 

effects and if toxicity would not be expected based upon data of 

structurally related compounds, then a full study using three dose 

levels might not be necessary. Expected human exposure though may 

indicate the need for a higher dose level. 

(e) Test procedures. 

(1) Animal selection. 

(i) Species and strain. The rat is recommended, but any 

rodent species may be used. If another mammalian species is used, 

the tester shall provide justification/reasoning for its selection. 

(ii) Age. Young adult animals, at least ten weeks of age, 

shall be used. 

(iii) Number and sex. At least 10 animals of each sex will 

be used per concentration or control group. 

(2) Satellite groups. 

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(i) A satellite group of 10 animals treated at the highest 

concentration level for the study and ten unexposed control animals 

(if mice are used, 10 animals per sex per group) shall be 

sacrificed after one week of exposure and broncho-alveolar lavage 

will be performed on all test subjects. Total and differential 

cell counts will be performed and alveolar macrophages will be 

cultured for a phagocytic activity assay. The levels of several 

pulmonary enzymes will be determined. 

(ii) A second satellite group of 10 animals may be treated 

with the highest concentration level for 42 days and observed for 

reversibility, persistence, or delayed occurrence of toxic effects 

for a post-treatment period of not less than 28 days. 

(3) Observation period. Duration of observation shall be for 

at least 42 days. 

(4) Inhalation exposure. The general conduct of this study 

shall be in accordance with the vehicle emissions inhalation 

exposure guideline in section 79.50 of this subpart. 

(5) Observation of animals. Animals Shall be weighed weekly. 

Feed consumption shall also be determined weekly. 

(6) Clinical Procedures. 

(i) At the end of the first week of the exposure period, the 

following procedures shall be conducted on a satellite group of 

animals. 

(A) Broncho-alveolar lavage. The following is a brief 

description of a lung lavage procedure after Schlesinger, 1987. 

Animals are anesthetized, exsanguinated, and the lungs are 

irrigated with saline fluid. The fluid is retained and centrifuged 

to form cell pellets (two mouse lung lavages from the same sex 

shall be pooled to form one sample) . Red blood cells are lysed 

from the cell pellets and the remaining cells, predominantly 

alveolar macrophages (AM), are resuspended in medium. The bronchoalveolar lavage fluid (BALF) is likewise retained for biochemical 

analysis. 

(B) Phagocytic activity assay. Several methods for 

performing a phagocytic activity assay have been described. Among 

those used are: 

(1) Alveolar macrophage/sheep red blood cells method (Benson, 

et al., 1988) . 

(2) Alveolar macrophage/fluorescent polystyrene beads (AM/FPB) 

method (Burleson, et al., 1987). 

The procedures described here are for the AM/FPB method. AM are 

resuspended in medium and incubated with FPB for 2 hours. At the 

end of the incubation period, cell suspensions are centrifuged onto 

glass slides and fixed. The slides are placed in methylene chloride 

85 
P.89 

for 15-30 seconds to dissolve uningested and cell-associated 

particles. The cells having ingested beads are then quantified 

under a fluorescence microscope. 

(C) Cell suspensions from the initial lavage fluids are 

evaluated for total and differential cell counts. 

(D) Biochemical determinations. BALF is evaluated for 

lactate dehydrogenase (LDH), beta-glucuronidase (BG), and total 

protein (TP). 

(ii) The following organs and tissues, or representative 

samples thereof, shall be preserved in a suitable medium for 

possible future histopathological examination: 

(A) Lungs, which shall be treated with a suitable fixative to 

ensure that lung structure is maintained (both perfusion-underpressure and inflation-fixed fixatives are considered to be 

effective techniques). 

(B) Nasopharyngeal and tracheal tissues. 

(7) Histopathology. 

(i) A full histopathological examination will be conducted on 

the respiratory tracts of 42-day exposed animals in the control and 

high dose groups. The lungs of animals who are in the low and 

intermediate dose groups shall be subjected to examination, 

primarily, for evidence of inflammation since this provides a 

convenient assessment of the state of health of the animals. 

(ii) For the satellite groups, histopathology may be 

performed on pulmonary organs when a particular concentration is 

identified as showing effects in the sub-acute study treatment 

groups. 

(f) Data and reporting. 

(1) Treatment of results. All observed results, quantitative 

and incidental, should be evaluated by an appropriate statistical 

method. The specific methods, including consideration of 

statistical power, should be selected during the design of the 

study. 

(2) Test report. In addition to the reporting requirements 

as specified under Good Laboratory Practice Standards, 40 CFR Part 

792, Subpart J and in section 79.50(e) of this subpart, the 

following specific information shall be reported: 

(i) Group animal data. Tabulation of toxic response data by 

species, strain, sex, and exposure level for: 

(A) Number of animals exposed. 

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P.90 

(B) Number of animals showing signs of pulmonary and/or other 

toxicity. 

(C) Number of dead animals, 

(ii) Individual animal data. 

(A) Date of death during the study or whether animals 

survived to termination. 

(B) Date of observation of each abnormal sign and its 

subsequent course. 

(C) Individual body weight data, and group average body 

weight data vs. time. 

(D) Phagocytic index, i.e., the number of macrophages 

containing at least one particle, and the phagocytic capacity, 

i.e., the numbers of particles per phagocytizing cell. 

(E) Levels of LDH, BG, and TP. 

(F) Types and total numbers of cells identified in lavage 

cells. 

(G) Lung weights, including trachea, and detailed description 

of all histopathological findings. 

(H) Necropsy findings. 

(I) Statistical treatment of results where appropriate. 

(g) References. For additional background information on 

this test guideline the following references should be consulted: 

(1) 40 CFR §798.2450 (1990) Inhalation toxicity, pp 451-56. 

(2) Benson, J.M., et al. (1988) In-Vitro Toxicity of Nickel 

Compounds. J. Toxicology and Environmental Health, 24:374-80. 

(3) Benson, J.M., et al. (1989) Biochemical Responses of Rat 

and Mouse Lung to Inhaled Nickel Compounds. Toxicol., 57:255-66. 

(4) Burleson, G. R., et al. (1987) Poly(I):Poly(C)-Enhanced 

Alveolar and Peritoneal Macrophage Phagocytosis: Quantification by 

a New Method Utilizing Fluorescent Beads. Proceedings of the 

Society for Experimental Biology and Medicine, 184:468-476. 

(5) Schlesinger, R.B. (1987) Functional Assessment of Rabbit 

Alveolar Macrophages Following Intermittent Inhalation Exposures to 

Sulfuric Acid Mist. Fundamental and Applied Toxicology, 8:328-334. 

(6) Wolff, R.K., et al. (1987) Alterations in Particle 

Accumulation and Clearance in Lungs of Rats Chronically Exposed to 

Diesel Exhaust. Fundamental and Applied Toxicology, 9:154-66. 

87 
P.91 

§79.57 Functional observational neurotoxicity battery. 

(a) Purpose. 

(1) In the assessment and evaluation of the potential human 

health effects of a gas, semi-volatile substance, aerosol, or 

particulate, it is appropriate to test for neurotoxic effects. The 

functional observational neurotoxicity battery (FONB) consists of 

non-invasive procedures designed to detect gross functional 

deficits in animals associated with repeated subacute inhalation 

exposure to a test substance for a period of 42 days. It is also 

used to better quantify behavioral and neurological effects 

detected in other studies. This battery of tests is not intended 

to provide a detailed evaluation of neurotoxicity. It is designed 

to be used in conjunction with other neuropathologic evaluation 

and/or general toxicity testing. Additional functional tests may 

be necessary to assess completely the neurotoxic potential of a 

chemical. 

(b) Definition. Neurotoxicity is any adverse effect on the 

structure or function of the nervous system related to exposure to 

a chemical substance. 

(c) Principle of the test method. Groups of laboratory 

rodents breathe the test substance in air for a period of 42 days. 

Each group receives one of several graded concentrations of the 

test substance for at least six hours per day on a daily basis. 

During the study period, the animals are observed under carefully 

standardized conditions with sufficient frequency to ensure the 

detection of behavioral and/or neurologic abnormalities, if 

present. Various functions that could be affected by 

neurotoxicants are assessed during each observation period. The 

exposure levels at which significant neurotoxic effects are 

produced are compared to one another and to those levels that 

produce other toxic effects. 

(d) Limit test. If a test at one dose level of the highest 

concentration that can be achieved while maintaining a particle 

size distribution with a mass median diameter (MMAD) of 4 

micrometers {\im) or less, using the procedures described in 

subsection (a) of this section, produces no observable toxic 

effects and if toxicity would not be expected based upon data of 

structurally related compounds, then a full study using three dose 

levels might not be necessary. However, expected human exposure 

may indicate the need for a higher dose level. 

(e) Test procedures. 

(1) Animal selection. 

(i) Species and strain. The laboratory rat is recommended 

and a standard strain should be used. Although information will 

generally be lacking, whenever possible the choice of species 

should take into consideration such factors as the comparative 

88 
P.92 

metabolism of the chemical and species sensitivity to the toxic 

effects of the test substance, as evidenced by the results of other 

studies. 

(ii) Combined studies. 

(A) The potential for combined studies should also be 

considered. When exposed to the same test atmosphere, subjects 

used to measure endpoints other than neurotoxicity may be used if 

none of the requirements of either study are violated by the 

combination. Animals assigned the neurotoxicity study should be 

designated for behavioral testing alone. 

(2) Control groups. 

(i) A concurrent (unexposed) control group is required. 

(ii) Positive control data from the laboratory performing the 

testing shall provide evidence of the ability of the procedures 

used to detect major neurotoxic endpoints such as limb weakness or 

paralysis (e.g., repeated exposure to acrylamide), tremor (e.g., 

p,p'DDT), and autonomic signs (e.g., carbaryl). Positive control 

data shall be collected at the time of the test study unless the 

laboratory can demonstrate the adequacy of historical data for the 

planned study. 

(ii) A satellite group may be treated with the high 

concentration level for the duration of exposure and observed for 

recovery from, persistence, or delayed occurrence of toxic effects. 

The post-treatment period of followup observations should be kept 

for at least 28 days. 

(3) Concentration levels and selection. At least 3 

concentrations equally spaced on a log scale (e.g., *i log units) 

over a range of at least 1 log unit shall be used in addition to a 

zero concentration and a vehicle administration only, i.e., base 

fuel, as required. The data should be sufficient to produce a 

concentration-effect curve. 

(i) The highest concentration shall produce 1) clear 

behavioral effects (e.g., change in reactivity to external 

stimulation) or 2) life-threatening toxicity. 

(ii) The data from the lower concentrations must show either 

1) graded concentration-dependent effects at 2 concentration levels 

or 2) no effects at 2 concentration levels, respectively. 

(4) Inhalation exposure. The general conduct of this study 

shall be in accordance with the vehicle emissions inhalation 

exposure guideline in section 79.50 of this subpart. 

(5) Study conduct. All animals in a given study should be 

observed carefully by trained observers who are unaware of the 

animal's treatment, using standardized procedures to minimize 

observer variability. Where possible, it is advisable that the 

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same observer be used to evaluate the animals in a given study. If 

this is not possible, some demonstration of inter-observer 

reliability is required. All animals should be observed within 72 

hours prior to initiation of exposure. Subsequent observations 

should be made with sufficient frequency to ensure the detection of 

behavioral and/or neurologic abnormalities, if present. Testing 

should be done prior to exposures on any day. At a minimum, 

observations should be made after the first (i.e., prior to 

exposure on Day 2 of exposure), seventh (i.e., prior to exposure on 

Day 8 of exposure), fourteenth (i.e., prior to exposure on Day 15 

of exposure), and forty-second (i.e., on the morning after the last 

exposure) exposure. The animals should be removed from the home 

cage to a standard arena for observation. Effort should be made to 

ensure that variations in the test conditions are minimal and are 

not systematically related to treatment. Among the variables that 

can affect behavior are sound level, temperature, humidity, 

lighting, odors, time of day, and environmental distractions. 

Explicit, operationally defined scales for each measure of the 

battery are to be used. The development of objective quantitative 

measures of the observational endpoints specified is encouraged. 

Examples of observational procedures using defined protocols may be 

found in Irwin (1968), Gad (1982), and Moser et. al. (1988). The 

FONB shall include a thorough description of the subject's 

appearance, behavior, and functional integrity. This shall be 

assessed through: observations in the home cage; while the test 

subject is moving freely in an open field; and through manipulative 

tests. Testing should proceed from the least to the most 

interactive with the subject. Scoring criteria, or explicitly 

defined scales, shall be developed for those measures which involve 

subjective ranking. 

(6) Animals shall be weighed at least weekly and on each test 

day during the exposure period. Feed consumption shall also be 

determined weekly if abnormal body weight changes are observed. 

(7) The following is a minimal list of measures that shall be 

noted: 

(i) Assessment of signs of autonomic function, including but 

not limited to: 

(A) ranking of the degree of lacrimation and salivation, with 

a range of severity scores from none to severe; 

(B) presence or absence of piloerection and exophthalmus; 

(C) ranking or count of urination and defecation, including 

polyuria and diarrhea. This is most easily conducted during the 

open field assessment; 

(D) pupillary function such as constriction of the pupil in 

response to light or a measure of pupil size; 

(E) degree of palpebral closure, e.g., ptosis. 

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P.94 

(ii) Description, incidence, and severity of any convulsions, 

tremors, or abnormal motor movements, both in the home cage and in 

the open field. 

(iii) Ranking of the subject's reactivity to general stimuli 

such as removal from the cage or handling, with a range of severity 

scores from no reaction to hyperreactivity. 

(iv) Ranking of the subject's arousal level or state of 

alertness during observations of the unperturbed subject in the 

open field, with a range of severity scores from coma to 

hyperalertness. 

(v) Descriptions and incidence of posture and gait 

abnormalities observed in the home cage and open field. 

(vi) Ranking of any gait abnormalities with a range of 

severity scores from none to severe. 

(vii) Forelimb and hind limb grip strength measured using an 

objective procedure, e.g., that described by Meyer et al. (1979). 

(viii) Quantitative measure of landing foot splay; the 

procedure described by Edwards and Parker (1977) is recommended. 

(ix) Sensorimotor responses to stimuli of different 

modalities will be used to detect gross sensory deficits. Pain 

perception may be assessed by a ranking or measure of the reaction 

to a tail-pinch, tail-flick, or hot-plate. The response to a 

sudden sound, e.g., click or snap, may be used to assess audition. 

(x) Body weight. 

(xi) Description and incidence of any unusual or abnormal 

behaviors, excessive or repetitive actions (stereotypies), 

emaciation, dehydration, hypotonia or hypertonia, altered fur 

appearance, red or crusty deposits around the eyes, nose, or mouth, 

and any other observations that may facilitate interpretation of 

the data. 

(8) Additional measures. Other measures may also be included 

and the development and validation of new tests is encouraged. 

Further information on the neurobehavioral integrity of the subject 

may be provided by: 

(i) Count of rearing.activity on the open field; 

(ii) Ranking of righting ability; 

(iii) Body temperature; 

(iv) Excessive or spontaneous vocalizations; 

(v) Alterations in rate and ease of respiration, e.g., rales 

or dyspnea; 

91 
(vi) Sensorimotor responses to visual or proprioceptive 

stimuli. 

(9) When a satellite group is used, grip strength and an 

assessment of sensory function shall be performed on animals in 

concentration groups identified as showing effects in the treated 

groups. 

(f) Data reporting and evaluation. In addition to the 

reporting requirements specified under 40 CFR Part 792 Subpart J, 

data will be reported in accordance with section 79.50(e) of this 

subpart. The final test report must include the following 

information: 

(1) Description of equipment and test methods. 

(i) A detailed description of the procedures used to 

standardize observation, including the arena and scoring criteria. 

(ii) Positive control data from the laboratory performing the 

test that demonstrate the sensitivity of the procedures being used. 

Historical data may be used if all essential aspects of the 

experimental protocol are the same. 

(2) Results. 

(i) The data for each animal must show, in tabular form by 

test group concentration level, the following: 

(A) Its identification number; 

(B) Its body weight and score on each sign at each 

observation time, the time and cause of death (if appropriate). 

(ii) Summary data for each test group concentration level 

must include: 

(A) The number of animals at the start of the test; 

(B) The number of animals showing each observation score at 

each observation time; 

(C) The mean and standard deviation for each continuous 

endpoint at each observation time. 

(D) Results of statistical analyses for each measure, where 

appropriate. 

(3) Evaluation of data. The findings of the FONB should be 

evaluated in the context of preceding and/or concurrent toxicity 

studies and any correlative histopathological findings. The 

evaluation shall include the relationship between the concentration 

of the test substance and the presence or absence, incidence and 

severity, of any neurotoxic effects. The evaluation should include 

appropriate statistical analyses; for example, parametric tests for 

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V 

V 

continuous data and non-parametric tests for the remainder. Choice 

of analyses should consider tests appropriate to the experimental 

design, including repeated measures. There may be many acceptable 

ways to analyze data. The report must include concentration-effect 

curves for observations and measures. 

(g) References. For additional background information on 

this test guideline the following references should be consulted: 

(1) 40 CFR §798.6050 (1991) Functional Observational Battery, 

pp 546-49. 

(2) Edwards, P.M., Parker, V.H. (1977) A Simple, Sensitive 

and Objective Method for Early Assessment of Acrylamide Neuropathy 

in Rats. Toxicology and Applied Pharmacology, 40:589-591 

(3) Gad, S.A. (1982) A Neuromuscular Screen for Use in 

Industrial Toxicology. J. Toxicol. Environ. Health 9:691-704. 

(4) Irwin, S. (1968) Comprehensive Observational Assessment: 

Ia. A Systematic Quantitative Procedure for Assessing the 

Behavioral and Physiologic State of the Mouse. Psychopharmacologia, 

13:222-257. 

(5) MacFarland, H.N. (1976) Respiratory Toxicology. In: 

Essays in Toxicology, Vol. 7. W.J. Hayes (Ed). Academic Press, 

New York. pp 121-154. 

(6) Meyer, O.A., Tilson, H.A., Byrd, W.C , Riley, M.T. (1979) 

A Method for the Routine Assessment of Fore- and Hindlimb Grip 

Strength of Rats and Mice. Neurobehavioral Toxicology, 1:233-236. 

(7) Moser, V.C , McCormick, J.P., Creason, J.P., MacPhail, 

R.C. (1988) Comparison of chlordimeform and carbaryl using a 

functional observational battery. "Fund. Appl. Toxicol., 11:189-206. 

(8) Sette, W. (1991) Pesticide Assessment Guidelines, 

Subdivision F, Neurotoxicity Test Guidelines. U.S. EPA. Report No. 

540/09-91-123 (NTIS #PB91-154617). 

93