43820 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

ENVIRONMENTAL PROTECTION

AGENCY

40 CFR Part 799

[OPPTS–42193; FRL–5719–5]

RIN 2070–AB76

Toxic Substances Control Act Test

Guidelines

AGENCY: Environmental Protection

Agency (EPA).

ACTION: Final rule.

SUMMARY: This rule establishes 11 Toxic

Substances Control Act (TSCA) health

effects test guidelines in the Code of

Federal Regulations (CFR).

Establishment of these guidelines is

necessary to ensure enforceable test

standards in test rules promulgated

under section 4 of TSCA. Codification of

this series of TSCA test guidelines does

not by itself impose obligations upon

any person. Obligations are only

imposed when these guidelines are

cross-referenced in a test rule

promulgated under section 4 of TSCA.

DATES: This rule is effective on August

15, 1997.

FOR FURTHER INFORMATION CONTACT:

Susan Hazen, Director, Environmental

Assistance Division (7408), Office of

Pollution Prevention and Toxics,

Environmental Protection Agency, Rm.

E-543B, 401 M St., SW., Washington, DC

20460; telephone: (202) 554–1404; TDD:

(202) 554–0551; e-mail: TSCAHotline@epamail.epa.gov. For specific

information regarding this action or

related activities, please contact Roger

Nelson, Chemical Control Division,

OPPT; telephone: (202) 260–8163; email: nelson.roger@epamail.epa.gov.

SUPPLEMENTARY INFORMATION: This final

rule establishes a new series of TSCA

test guidelines in the CFR.

I. Introduction

Section 4(b)(1)(B) of TSCA requires

that test rules promulgated under the

authority of TSCA section 4 include

‘‘standards for the development of test

data for such substance or mixture * *

*.’’ Test rules promulgated under TSCA

section 4 must specify the standards for

the development of data. Standards

established in test rules for the

development of data must specify how

the study is to be conducted, what data

will be collected, and how the data will

be analyzed. The Agency has found that

these specifications to a large degree can

be standardized into a common set of

protocols, or, as the Agency terms them,

‘‘guidelines.’’ These guidelines are

organized by testing endpoint. Each test

standard can modify these guidelines as

needed for an individual test substance.

The Agency uses a system where

standardized guidelines are organized

by testing endpoint and codified in a

subpart of this part. When a test rule is

promulgated, the test standard specified

in the test rule cross-references the

guideline for the bulk of the testing

requirements. In this context, the public

is given notice of, and an opportunity to

comment on, the guidelines as they are

applied in chemical-specific test rules.

This approach eliminates the need to

repeat the same test specifications for

each substance-specific test rule since

most of the specifications for testing do

not change across substances. The test

specifications in a guideline can be

varied, when necessary, to the specific

requirements of a test rule by language

in the test rule itself.

In 1985, the Agency established a set

of TSCA test guidelines in 40 CFR parts

795 through 798 (50 FR 39252,

September 27, 1985). These guidelines

were established as standardized

protocols for laboratory testing of an

effect or characteristic deemed

important for the evaluation of health or

environmental hazards of a chemical.

Standardized guidelines are necessary

for the establishment of enforceable test

standards in test rules promulgated

under section 4 of TSCA.

The Agency has over time amended

and improved these guidelines (52 FR

19072, May 20, 1987). In order to reduce

the text of the CFR, the Agency deleted

those guidelines which had not been

cited in any test rules (60 FR 31917,

June 19, 1995 (FRL–4955–2)).

II. OPPTS Harmonized Test Guidelines

EPA is undertaking a comprehensive

modification, or harmonization, of its

pesticides and toxics guidelines for

testing of health effects, environmental

effects, and chemical fate. The rationale

for this harmonization is to incorporate

state of the art science, and to minimize

variations among the protocols

contained in:

1. Test guidelines developed by the

EPA Office of Pesticide Programs (OPP),

which appeared in publications of the

National Technical Information Service.

2. The series of TSCA test guidelines

established in 1985, which are

contained in 40 CFR parts 795, 796, 797,

and 798.

3. Guidelines published by the

Organization for Economic Cooperation

and Development (OECD).

Harmonization operates as follows:

EPA scientists develop guidelines (or

modify existing guidelines) for specific

endpoints. The new or rewritten

guidelines are reviewed by other

Agency experts and, in some instances,

presented at domestic and international

colloquia to solicit the views of

recognized experts and the regulated

community. The draft harmonized

guidelines are made available as public

drafts. A notice is published in the

Federal Register announcing their

availability and soliciting public

comment.

Seven of the 11 health effects test

guidelines that are being codified in

subpart H of 40 CFR part 799 have their

origin in this harmonization process. A

notice was published in the Federal

Register of June 20, 1996, (61 FR 31522

(FRL–5367–7)) announcing the

availability of the proposed test

guidelines for Series 870—Health

Effects Test Guidelines and soliciting

public comment. Comments were

received, and a meeting of the Agency’s

Federal Insecticide, Fungicide, and

Rodenticide Act (FIFRA) Scientific

Advisory Panel (SAP) was held on

October 29 and 30, 1996. The SAP, an

advisory committee consisting of

scientific experts both inside and

outside the U.S. Government, reviewed

the guidelines and made comments. The

Agency reviewed these comments in

developing the harmonized health

effects guidelines.

Four of the 11 guidelines

(§ § 799.9510, 799.9530, 799.9538, and

799.9539) were initially developed by

the OECD.

III. TSCA Test Guidelines

Harmonization has resulted in

significantly improved guidelines.

However, creating a single set of

guidelines which can be used by both

OPP, in its administration of the FIFRA

and the Federal Food, Drug and

Cosmetic Act (FFDCA), and the Office of

Pollution Prevention and Toxics

(OPPT), which administers TSCA

presented certain challenges.

Under FIFRA, test guidelines are used

in an interactive process between the

Agency and registrants seeking

registration of pesticides or food residue

tolerances. Flexibility to tailor required

testing to individual circumstances is

critical, and the Agency has

considerable discretion to determine

whether submitted test results are

adequate to support the requested

action. Under this scheme, registrants

have an intrinsic motivation to conduct

well-grounded testing. Thus, pesticide

testing protocols tend to have few

absolute requirements specifying the

details of the conduct of the testing.

By contrast the Agency is required

under section 4 of TSCA to impose

prescriptive test requirements by notice

and comment rulemaking. Rules
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43821

promulgated under section 4 of TSCA

specify classes of affected parties,

usually manufacturers and processors of

the chemical being specified for testing,

rather than interacting with companies

on an individual basis. These

rulemakings typically take years to

complete. Without initiating another

rulemaking process, the Agency has the

ability to require further testing only if

the tests were not conducted in

accordance with the procedures

specified in the test rule. In addition,

the Agency has an alternative process of

negotiating TSCA testing requirements

via enforceable consent agreements

(ECAs), but these agreements require the

consent of all the parties involved.

Under TSCA section 4 enforceable

test standards, much in the conduct of

these test protocols is left to the

judgment of those professionals

conducting the testing. EPA believes

that certain provisions must be

mandatory whenever the guidelines are

cross-referenced in specific test rules.

Therefore, the Agency has used the

OPPTS harmonized health effects test

guidelines developed using the public

notice and comment process described

in Unit II. of this preamble as well as

certain OECD guidelines to create the

TSCA-specific test guidelines which are

the subject of this rule. Future TSCA

section 4 test rules will cross-reference

part 799 guidelines rather than the

older, 1985 non-harmonized guidelines

in 40 CFR parts 795 through 798. The

only significant difference between the

TSCA test guidelines and the OPPTS

harmonized test guidelines is that

certain recommended procedures in the

OPPTS harmonized test guidelines are

made mandatory (i.e., the guideline

states that they ‘‘shall’’ be carried out).

IV. Codification in 40 CFR Part 799

The Agency had originally planned

not to publish the guidelines in the CFR,

but to instead make the guidelines

available via other means (such as the

Internet) and reference the guidelines in

specific test rules using the

incorporation by reference procedures

provided by 5 U.S.C. 552(a)(1)(E) and 1

CFR part 51. In the Federal Register

document proposing the TSCA section 4

test rule for 21 hazardous air pollutant

substances (HAPs) (61 FR 33178, 33187,

June 26, 1996 (FRL–4869–1)), the

Agency stated that it was considering

using incorporation by reference.

Subsequently, however, the Director of

the Office of Federal Register advised

EPA that the planned TSCA section 4

process for guideline incorporation was

not eligible for incorporation by

reference under 1 CFR part 51.

Therefore, the Agency finds it necessary

to codify a separate set of TSCA test

guidelines into the CFR. As discussed in

this preamble, the TSCA guidelines are

essentially those resulting from the

harmonization process with minor

changes to promote enforceability. EPA

has elected to codify these new

guidelines in part 799 so as to

distinguish them from the preharmonization guidelines in 40 CFR

parts 795 through 798.

These guidelines will be placed in a

new subpart H of part 799. In addition,

EPA plans to reserve additional subparts

of part 799 for test guidelines, so that

the structure of part 799 would be as

follows:

Subpart A—General Provisions

Subpart B—Specific Chemical Test

Rules

Subpart C—Testing Consent Orders

Subpart D—Multichemical Test Rules

Subpart E—G [Reserved]

Subpart H—Health Effects Test

Guidelines

The TSCA test guidelines currently in

40 CFR parts 795 through 798 will be

retained for so long as there exist test

rules whose data reimbursement periods

under TSCA section 4(c) have not

expired and which cross-reference the

guidelines.

This table identifies the TSCA test

guideline number with its comparable

OPPTS harmonized test guideline

public draft.

TABLE 1.—TSCA Test Guidelines Cross-Referenced to the OPPTS Harmonized Test Guidelines

Guideline title TSCA 40 CFR section OPPTS harmonized test guideline (public draft)

TSCA acute inhalation toxicity with histopathology ...................................... 799.9135 ..................................... 870.1350

TSCA subchronic inhalation toxicity .............................................................. 799.9346 ..................................... 870.3465

TSCA prenatal developmental toxicity .......................................................... 799.9370 ..................................... 870.3700

TSCA reproduction and fertility effects ......................................................... 799.9380 ..................................... 870.3800

TSCA carcinogenicity .................................................................................... 799.9420 ..................................... 870.4200

TSCA bacterial reverse mutation test ........................................................... 1799.9510 ................................... 1OECD 471

and 472

TSCA in vitro mammalian cell gene mutation test ........................................ 1799.9530 ................................... 1OECD 476

TSCA mammalian bone marrow chromosomal aberration test .................... 1799.9538 ................................... 1OECD 475

TSCA mammalian erythrocyte micronucleus test ......................................... 1799.9539 ................................... 1OECD 474

TSCA neurotoxicity screening battery ........................................................... 799.9620 ..................................... 870.6200

TSCA immunotoxicity .................................................................................... 799.9780 ..................................... 870.7800

1The four TSCA genetic toxicity testing guidelines were adopted from the OECD guideline series and not the OPPTS public drafts.

Codification of these guidelines does

not itself impose any obligations on any

person. Obligations are imposed only

when the guidelines are crossreferenced in individual TSCA section 4

rulemakings. When cross-referenced in

such test rules, the pertinent TSCA

guidelines serve as test standards for

only these particular section 4 rules.

EPA may propose modifications to the

various guidelines as they are utilized

for chemical-specific test rules. In each

chemical-specific test rule, the proposed

test standards and any modifications

thereto will be subject to public notice

and comment.

V. Guideline by Guideline Discussion

In this unit is a summary of the

significant changes made to the 11

harmonized guidelines proposed on

June 20, 1996, which are being

published in this document.

A. Section 799.9135 TSCA Acute

Inhalation Toxicity with Histopathology

1. EPA dropped the requirement for a

1-hour (hr) exposure test. The Agency

recognizes that such a technically

difficult test would not be likely to yield

useful information due to complicating

factors such as biological rhythms and

inapplicability to insoluble or

chemically inactive particulates.

Instead, EPA is requiring a 4-hr

exposure point with a trigger for an 8-

hr exposure point. Test sponsors have
43822 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

the option to extrapolate from shorterterm exposures.

2. EPA dropped the requirement for

performing histopathology in all

animals and substituted a triggered

approach (wherein gross pathology will

be performed only when the frequency

and severity of adverse effects for dosed

animals are greater than those for

control animals in the study).

3. EPA dropped the requirement of a

breathing zone purity determination as

unnecessary since the Agency now

believes that standard inhalation

toxicology will provide the purity

measurement of the test substances.

4. EPA requires only a single control

group in some circumstances. If both 4-

and 8-hr exposures are being conducted

in the study, then there would be a

single control at the 8-hr exposure

provided adequate historical control

data show no changes in histopathology

or bronchoalveolar lavage between

controls for these test periods. If the 8-

hr exposure is being performed as a

result of the 4-hr trigger, there would

need to be control groups for both 4-

and 8-hr exposure groups.

5. EPA redefined the test exposure to

4 hrs of exposure to the target

concentration as defined by an average

of plus or minus 5% for gases and plus

or minus 11% for particles. This

redefinition establishes exposure

tolerances, which better assures known

test concentration than the original

provision which only allowed for test

exposure after the test chamber reached

equilibrium.

6. EPA now distinguishes air change

requirements between nose-only

exposure (300 milliter (mL)/minutes

(min)/animal) and whole-body exposure

(at least 12 to 15 air changes per hr).

7. EPA changed its description of the

respiratory histopathology requirements

to ensure that inflated state and fixed

pressure with infusion fixation are used

to prepare the lungs for examination.

8. EPA added the requirement to

specify the anatomical location where

the four sections are to be taken for

nasal histopathology.

B. Section 799.9346 TSCA Subchronic

Inhalation Toxicity

1. EPA changed the terms used for

certain weekly observations from

‘‘motor activity’’ to ‘‘level of activity’’

and from ‘‘grip strength’’ to ‘‘altered

strength’’ to reinforce the point that

these observations need not be

automated.

2. ‘‘Dose’’ and ‘‘dose level’’ were

changed to ‘‘concentration’’ and

‘‘dosing’’ was changed to ‘‘exposure’’ to

reflect that this is an inhalation study.

C. Section 799.9370 TSCA Prenatal

Developmental Toxicity

EPA made no significant changes to

this guideline.

D. Section 799.9380 TSCA Reproduction

and Fertility Effects

1. EPA added the requirement for a

triggered quantitative evaluation of

primordial follicles from qualitative

evidence of a possible treatment-related

effect. While the Agency recognizes that

there are issues concerning the validity

of existing methods used to screen

ovarian-primordial follicle counts, the

Agency believes that the necessity to

identify early senescence in females

outweighs these concerns. EPA

considers data about the effects of

chemical substances on effects such as

early female senescence to be essential

to protecting human health.

2. EPA reduced the requirement for

taking organ weights for pups already

opened for necropsy. The guideline only

requires organ weight data from one

randomly selected pup/sex/litter rather

than the three pups specified in the

public draft. The Agency believes that

collection of organ weight data from one

pup/sex/litter rather than three will

reduce burdens without compromising

the ability to detect a treatment-related

effect on brain, spleen, or thymus

weight. The random selection is to be

made from the population of pups

already opened for necropsy.

3. EPA reduced the requirement that

20 adult animals per sex per exposure

group be examined for histopathology to

10 animals (randomly chosen) per sex

per exposure group. This reduction was

made because there would be little

additional statistical value in examining

more than 10 animals per sex per group.

Since the guideline still requires that

gross necropsy and organ weight data be

collected for all parental animals and

that the weighed organs be preserved,

questions about interpretation of

marginal histopathological effects can

be resolved by evaluation of the tissues

from these animals.

4. EPA dropped the requirement of

histopathology of developmental

anomalies observed macroscopically in

F1 and F2 weanlings. Since the intent

of this requirement was to confirm the

nature of the lesions already identified

macroscopically, the Agency believes

that the added value of the information

would not be worth the cost of the

evaluation.

E. Section 799.9420 TSCA

Carcinogenicity

1. EPA revised the guideline to allow

5-day per week dosing for both gavage

and capsule administration. This change

was made to eliminate the disparity

between the original 7-day specification

for capsules and 5 days for gavage since

there was no justification for this

disparity.

2. EPA changed the terms used for

certain weekly observations from

‘‘motor activity’’ to ‘‘level of activity’’

and from ‘‘grip strength’’ to ‘‘altered

strength’’ to reinforce the point that

these observations need not be

automated.

3. The requirement for the

immunotoxicity screen has been

deleted. The Agency agreed that the

immunotoxicity screen conducted at

study termination would provide little

meaningful information on the potential

toxicity of the chemical on the immune

function system due to the geriatric

changes in the animals.

4. EPA deleted the requirement for the

weighing of spleens because their

weight would be unacceptably variable

due to the amount of blood lost during

the exsanguination process. (The

weighing of spleens is still a

requirement in the immunotoxicity

guideline).

F. Genetic Toxicity Testing

1. Section 799.9510 TSCA Bacterial

Reverse Mutation Test.

2. Section 799.9530 TSCA In Vitro

Mammalian Cell Gene Mutation Test.

3. Section 799.9538 TSCA

Mammalian Bone Marrow

Chromosomal Aberration Test.

4. Section 799.9539 TSCA

Mammalian Erythrocyte Micronucleus

Test.

EPA is incorporating these genetic

toxicity guidelines directly from the

OECD versions. The Agency made

format changes in order to ensure

consistency with the TSCA test

guidelines format. The Agency actively

participated in international discussions

regarding the development of these

guidelines. EPA participated in the

review of the OECD drafts. EPA believes

that because these OECD guidelines

were developed with international

scientific input through the OECD

guideline development process, they

provide state-of-the-art guidance which

is equivalent to and more broadly

accepted than that in the OPPTS

harmonized test guidelines public drafts

published on June 20, 1996. The process

EPA used in developing the four TSCA

genetic toxicity test guidelines is

described in reference 5 of Unit VI. of

this preamble.
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43823

G. Section 799.9620 TSCA Neurotoxicity

Screening Battery

EPA made no significant changes to

the public draft of this guideline

although EPA made two clarifications to

address SAP concerns. Clarifications to

the positive control treatment were

made to indicate that such testing need

not be done as frequently as every 12

months. Examples were eliminated to

clarify EPA’s position that permanently

injurious chemicals are not necessary,

though EPA continues to believe that

chemical exposures are appropriate

H. Section 799.9780 TSCA

Immunotoxicity

1. EPA incorporated the

recommendation of the SAP that the

requirement for flow cytometric analysis

of lymphocyte and Natural Killer (NK)

cell phenotypes be eliminated. A test for

the primary antibody (IgM) response to

sheep red blood cell (PFC) or enzyme

linked immunosorbent assay (ELISA)

would still be required. The guideline

now sets the required exposure time for

the anti-sheep red blood cells (SRBC)

assay at 28 days, thus providing

information on the effects of the test

material on non-specific immunity.

2. EPA adopted the SAP

recommendation to delete the ‘‘optional

immunotoxicity screen’’ because

lymphocyte phenotyping by flow

cytometry should be an option.

3. EPA added the requirement that

appropriate species-specific monoclonal

antibodies be used in the phenotyping

assay. The Agency accepts the SAP

recommendation that this will allow

sufficient flexibility to allow for future

advances in flow cytometry and

antibody marker technology.

4. EPA adopted the SAP

recommendation that a minimum of

eight animals per treatment group be

used in order to yield a sufficient

statistical power to detect a 20% change

based upon the inter-animal variation

usually encountered in these assays.

5. EPA added the intraperitoneal

route of exposure to the guideline in

response to the SAP comment that this

is an acceptable method for

immunization with SRBCs.

6. EPA adopted the SAP

recommendation that testing

laboratories need not perform a positive

control after every experiment. Instead,

it is sufficient to include this control

every 6 months or whenever new

reagents are titrated.

VI. Public Record

The official record for this

rulemaking, as well as the public

version, has been established for this

rulemaking under docket control

number OPPTS–42193 (including

comments and data submitted

electronically). This record contains the

basic information considered by EPA in

developing this rule. EPA will

supplement this record as necessary.

A public version of this record,

including printed, paper versions of

electronic comments, which does not

include any information claimed as

Confidential Business Information (CBI),

is available for inspection from 12 noon

to 4 p.m., Monday through Friday,

except legal holidays. The public record

is located in the TSCA Nonconfidential

Information Center, Rm. NE-B607, 401

M St., SW., Washington, DC 20460.

The record includes the following

information:

1. Public drafts of seven OPPTS

harmonized health effects guidelines.

2. Four OECD genetic toxicity test

guidelines.

3. References contained in TSCA

health effects test guidelines

promulgated in this document.

4. Final report of the FIFRA Scientific

Advisory Panel meeting, held October

29–30, 1996.

5. USEPA. Memorandum, Angela

Auletta to Roger Nelson. HAPs Rule:

OECD Process for Update of Genetic

Toxicity Test Guidelines. March 10,

1997.

VII. Regulatory Assessment

Requirements

A. Waiver of Notice of Proposed

Rulemaking and Delay in Effective Date

Because the test guidelines codified in

this document have no substantive

effect on any person without further

rulemaking, and such rulemaking would

be conducted under public notice and

comment procedures, EPA finds that

public notice and comment are

unnecessary for this action. Thus, this

rule may be promulgated without prior

opportunity for public notice and

comment, pursuant to the

Administrative Procedure Act, 5 U.S.C.

553(b)(3)(B), and may be made effective

immediately, without a 30-day delay,

pursuant to 5 U.S.C. 553(d)(3).

B. Executive Order 12866, Executive

Order 12898, and Executive Order

13045

This action is not subject to Executive

Order 12866 (58 FR 51735, October 4,

1993) since, as explained in Units I. and

IV. of this preamble, the guidelines are

not intended to have the force and effect

of law until they are cross-referenced in

future test rules through public notice

and comment procedures that establish

those rules. For the same reason, this

action is not considered under

Executive Order 12898 (59 FR 7629,

February 16, 1994) as having a

disproportionately high and adverse

human health or environmental effect

on minority populations and lowincome populations. In addition, the

action is not subject to Executive Order

13045 ‘‘Protection of Children From

Environmental Health Risks and Safety

Risk’’ (62 FR 19885, April 23, 1997)

since it is neither economically

significant under Executive Order 12866

nor does it concern an environmental

health risk or safety risk that an agency

has reason to believe may

disproportionately affect children.

C. Paperwork Reduction Act

This rule does not contain

information collection requirements that

necessitate the approval of OMB under

the Paperwork Reduction Act of 1980

(44 U.S.C. 3501 et seq.).

D. Regulatory Flexibility Act

The guidelines codified in this

document do not constitute a rule for

which EPA must publish a general

notice of proposed rulemaking under 5

U.S.C. 553(b). Therefore, sections 603

and 604 of the Regulatory Flexibility

Act, 5 U.S.C. 603 and 604 do not apply

to this action.

E. Unfunded Mandates Reform Act

Title II of the Unfunded Mandates

Reform Act of 1995 (UMRA), Pub. L.

104–4, which establishes requirements

for Federal agencies to assess the effects

of certain regulatory actions on State,

local, and tribal governments and the

private sector, does not apply. This

action contains neither a private sector

nor an intergovernmental mandate

because it does not impose an

enforceable duty on anyone.

Furthermore, a written statement is not

required under section 202 of UMRA

because section 202 only applies to

rules for which a general notice of

proposed rulemaking was published,

and no such notice was issued for this

rule.

F. Submission to Congress and the

General Accounting Office

This action is not a major rule as

defined by 5 U.S.C. 804(2). Pursuant to

5 U.S.C. 801(a)(1)(A), EPA has

submitted a report containing this rule

and other required information to the

U.S. Senate, the U.S. House of

Representatives, and the Comptroller

General of the General Accounting

Office prior to its publication in today’s

Federal Register.
43824 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

List of Subjects in 40 CFR Part 799

Environmental protection, Chemicals,

Hazardous substances, Health,

Reporting and recordkeeping

requirements.

Dated: August 7, 1997.

Lynn R. Goldman,

Assistant Administrator for Prevention,

Pesticides and Toxic Substances.

Therefore, 40 CFR part 799 is

amended as follows:

PART 799—[AMENDED]

1. The authority citation for part 799

continues to read as follows:

Authority: 15 U.S.C. 2603, 2611, 2625.

2. By adding a new paragraph (d) to

§ 799.1 to read as follows:

§ 799.1 Scope and purpose.

* * * * *

(d) This part contains certain TSCA

test guidelines which are crossreferenced in the test rules contained in

this part.

3. By adding and reserving subparts E

through G.

4. By adding a new subpart H,

consisting of § § 799.9135–799.9780, to

read as follows:

Subpart H—Health Effects Test

Guidelines

799.9135 TSCA acute inhalation toxicity

with histopathology.

799.9346 TSCA subchronic inhalation

toxicity.

799.9370 TSCA prenatal developmental

toxicity.

799.9380 TSCA reproduction and fertility

effects.

799.9420 TSCA carcinogenicity.

799.9510 TSCA bacterial reverse mutation

test.

799.9530 TSCA in vitro mammalian cell

gene mutation test.

799.9538 TSCA mammalian bone marrow

chromosomal aberration test.

799.9539 TSCA mammalian erythrocyte

micronucleus test.

799.9620 TSCA neurotoxicity screening

battery.

799.9780 TSCA immunotoxicity.

Subpart H—Health Effects Test

Guidelines

§ 799.9135 TSCA acute inhalation toxicity

with histopathology.

(a) Scope. This section is intended to

meet the testing requirements under

section 4 of the Toxic Substances

Control Act (TSCA). In the assessment

and evaluation of the potential human

health effects of chemical substances, it

is appropriate to test for acute

inhalation toxic effects. The goals of this

test are to characterize the exposureresponse relationship for sensitive

endpoints following acute exposure and

to characterize toxicologic response

following acute high exposures. The

latter is of particular concern in relation

to spills and other accidental releases.

This testing is designed to determine the

gross pathology and histopathology

resulting from acute inhalation exposure

to a substance. Because toxic effects on

the respiratory tract are of particular

concern following inhalation exposure,

several indicators of respiratory toxicity

consisting of histopathology on fixed

tissue and evaluation of cellular and

biochemical parameters in

bronchoalveolar lavage fluid should be

employed. The respiratory

histopathology consists of specialized

techniques to preserve tissues of the

respiratory tract in order to allow

detailed microscopic examination to

identify adverse effects of chemical

substances on this organ system. The

bronchoalveolar lavage is designed to be

a rapid screening test to provide an

early indicator of pulmonary toxicity by

examining biochemical and cytologic

endpoints of material from the lungs of

animals exposed to potentially toxic

chemical substances. These acute tests

are designed to assess the relationship,

if any, between the animals’ exposure to

the test substance and to demonstrate

relationship between the animals’

exposure and the incidence and severity

of observed abnormalities, including

gross or histopathologic lesions, body

weight changes, effects on mortality,

and any other toxic effects. These acute

tests are not intended to provide a

complete evaluation of the toxicologic

effects of a substance, and additional

functional and morphological

evaluations may be necessary to assess

completely the potential effects

produced by a chemical substance.

Additional tests may include longerterm exposures, or more in-depth

evaluation of specific organ systems as

indicated by signs of toxicity following

acute exposure.

(b) Source. This a new section

developed by the United States

Environmental Protection Agency.

(c) Definitions. The following

definitions apply to this section.

Aerodynamic diameter (dae) refers to

the size of particles. It is the diameter

of a sphere of unit density that behaves

aerodynamically (has the same settling

velocity in air) as the particle of the test

substance. It is used to compare

particles of different size, shape, and

density, and to predict where in the

respiratory tract such particles may be

primarily deposited.

Exposure response is the relationship

between the exposure concentration and

the measured toxic response, whether

expressed as a group mean (± standard

deviation) in the case of a continuous

variable or as incidence in the case of

a quantal variable. This definiton

should not preclude the exploration of

other dose metrics in establishing this

relationship.

Geometric standard deviation (GSD)

is a dimensionless number equal to the

ratio between the mass median

aerodynamic diameter (MMAD) and

either 84% or 16% of the diameter size

distribution (e.g., MMAD = 2 μm; 84%

= 4 μm; GSD = 4/2 = 2.0.) The MMAD,

together with the GSD, describe the

particle size distribution of an aerosol.

Use of the GSD may not be valid for

non-lognormally distributed aerosols. (If

the size distribution deviates from the

lognormal, it shall be noted).

Inhalability is the ratio of the number

concentration of particles of a certain

aerodynamic diameter, dae, that are

inspired through the nose or mouth to

the number concentration of the same

dae present in the inspired volume of

ambient air. In humans, inhalability can

exceed 15 μm dae, whereas inhalability

dramatically decreases for particles

above 4 μm dae in small laboratory

animals.

Lower respiratory tract consists of

those structures of the respiratory tract

below the larynx.

Mass geometric mean aerodynamic

diameter or the mass median

aerodynamic diameter (MMAD) is the

calculated aerodynamic diameter that

divides the particles of an aerosol (a

gaseous suspension of fine liquid or

solid particles) in half, based on the

weight of the particles. By weight, 50%

of the particles will be larger than the

MMAD and 50% of the particles will be

smaller than the MMAD.

Particle regional deposition is the

fraction of inhaled particles that

deposits in the specific region of the

respiratory tract. The major mechanisms

of particle deposition in the respiratory

tract include impaction, sedimentation,

diffusion, interception, and electrostatic

precipitation. The deposition

mechanism that is dominant for a given

region depends on the respiratory tract

architecture and ventilation rate of the

species and the aerosol particle size and

distribution. The respiratory tract in

both humans and various experimental

mammals can be divided into three

regions on the basis of structure, size,

and function:

(1) The extrathoracic region or upper

respiratory tract that includes the nose,

mouth, nasopharynx, oropharynx,

laryngopharynx, and larynx.

(2) The tracheobronchial region that

includes the trachea, bronchi, and
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43825

bronchioles (including the terminal

bronchioles).

(3) The alveolar region that includes

the respiratory bronchioles (if present in

the species), alveolar ducts, alveolar

sacs, and alveoli.

Respiratory effects are any adverse

effects on the structure or functions of

the respiratory system related to

exposure to a chemical substance.

Target organ is any organ found to be

a target of toxicity in the 4-hour (hr)

high concentration group as a result of:

(1) The initial histopathologic

examination (respiratory tract, liver,

kidney, gross lesions); or

(2) The retrospective histopathologic

examination of archived organs

triggered by their identification as

targets of toxicity in a 90-day study.

Toxic effects are any adverse changes

(a change that is statistically and

biologically significant) in the structure

or function of an experimental animal as

a result of exposure to a chemical

substance.

Upper respiratory tract consists of

those structures of the respiratory tract

above and including the larynx.

(d) Principle of the test method. The

test substance shall be administered to

several groups of experimental animals;

one concentration level and duration

being used per group. Bronchoalveolar

lavage shall be used to evaluate early

effects on the respiratory system by

examining changes in the content of the

lavage fluid of the lung. At 24 hrs

following exposure, the animals shall be

sacrificed and necropsied, and tissue

samples from the respiratory tract and

other major organs will be prepared for

microscopic examination. The exposure

levels at which significant toxic effects

on the respiratory organ system are

produced are compared to those levels

that produce other toxic effects. As

triggered by the results of the 4-hr test,

additional exposure periods of 1 hr and

8 hrs will be required to determine the

effect of exposure time on the toxicity

observed. A 1-hr exposure study can be

elected as an option to provide data

suitable for risk assessment for very

short duration exposures as may occur

from chemical releases. In the absence

of adequate toxicological data for 1-hr

exposure, the Agency will extrapolate to

shorter-term exposures from the 4-hr

data on the basis of concentration alone.

This is a conservative method of

extrapolation, consistent with general

Agency methods for deriving criteria for

short-term exposure from longer-term

studies (a concentration x time

extrapolation would result in higher

concentration for a shorter duration).

(e) Test procedures—(1) Animal

selection—(i) Species. In general, the

laboratory rat and mouse should be

used. Under some circumstances, other

species, such as the hamster or guinea

pig, may be more appropriate, and if

these or other species are used,

justification should be provided.

(ii) Strain. If rats and mice are used,

the use of the F344 rat and the B6C3F1

mouse is preferred to facilitate

comparison with existing data.

(iii) Age. Young adults shall be used.

The weight variation of animals used in

a test should not exceed ± 20% of the

mean weight for each species.

(iv) Sex. Equal numbers of animals of

each sex shall be used for each

concentration level. The females shall

be nulliparous and nonpregnant.

(v) Health status. Body weight and

feed consumption are not sufficient

indicators of the health status of animals

prior to initiating an inhalation toxicity

study. Prior to initiating the study,

animals shall be monitored for known

viral and bacterial respiratory pathogens

determined by conventional

microbiological assays (e.g., serology).

The animals shall be free from

pathogens at the start of exposure.

(2) Number of animals. At least five

males and five females shall be used in

each concentration/duration and control

group. Animals shall be randomly

assigned to treatment and control

groups.

(3) Control groups. The control group

shall be a sham-treated group. Except

for treatment with the test substance,

animals in the control group shall be

handled in a manner identical to the

test-group animals. Where a vehicle is

used to help generate an appropriate

concentration of the substance in the

atmosphere, a vehicle control group

shall be used. If the 4- and 8-hr

exposure studies are conducted

concurrently, a concurrent 8-hr shamexposed control group may serve as the

control group for both the 4-hr and the

8-hr exposure studies, provided there is

adequate historical control data showing

no changes in histopathology or

bronchoalveolar lavage of controls

exposed for 4 and 8 hrs. Similarly, if the

optional 1-hr exposure study is

conducted concurrently with the 4- and/

or 8-hr study, the concurrent control

group for those studies may also be used

for the 1-hr study, provided adequate

historical control data show no changes

in histopathology or bronchoalveolar

lavage between controls exposed for

these time periods.

(4) Concentration level and

concentration selection. For the 4-hr

study, at least three concentrations shall

be used in addition to the control group.

Ideally, the data generated from the test

should be sufficient to produce an

exposure-response curve. The

concentrations can either be linearly or

logarithmically spaced depending on

the anticipated steepness of the

concentration-response curve. A

rationale for concentration selection

should be provided to indicate that the

selected concentrations will maximally

support detection of concentrationresponse relationship. The high

concentration should be clearly toxic or

a limit concentration, but should not

result in an incidence of fatalities that

would preclude a meaningful evaluation

of the data. The lowest concentration

should define a no-observed-adverseeffects level (NOAEL).

(i) Limit concentration. For aerosols

and particles, the high concentrations

need not be greater than 2 mg/L, or

concentrations that cannot maintain a

particle size distribution having an

MMAD between 1 and 4 μm (i.e., a

particle size that permits inhalability

and deposition throughout the

respiratory tract). For fibers, the

bivariate distribution of length and

diameter must ensure inhalability. For

gases and vapors, the concentrations

need not be greater than 50,000 ppm or

50% of the lower explosive limit,

whichever is lower. If a test at an

aerosol or particulate exposure of 2 mg/

L (actual concentration of respirable

substance) for 4 hrs or, where this is not

feasible, the maximum attainable

concentration, using the procedures

described for this study, produces no

observable toxic effects, then a full

study using three concentrations will

not be necessary. Similarly, if a test at

a gas or vapor exposure of 50,000 ppm

or 50% of the lower explosive limit,

whichever is lower, produces no

observable toxic effects, then a full

study using three concentrations will

not be necessary.

(ii) 8-Hr study and optional 1-hr

study. If the 8-hr study is triggered,

three concentrations shall be tested.

These concentrations should allow for

the determination of an effect level and

a NOAEL. If the option to perform a 1-

hr study is elected, three concentrations

shall be selected and tested in a similar

manner.

(5) Inhalation exposure. Animals can

be exposed to the substance by either a

nose-only procedure or in a whole-body

exposure chamber.

(i) Inhalation chambers. The animals

shall be tested in inhalation equipment

designed to sustain a dynamic airflow

for nose-only exposures of at least 300

ml/minute/animal or an airflow for

whole-body exposures of at least 12 to

15 air changes per hr and ensure an

adequate oxygen content of at least 19%

and an evenly distributed exposure
43826 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

atmosphere. Where a whole-body

chamber is used, its design shall

minimize crowding by providing

individual caging. As a general rule, to

ensure stability of a chamber

atmosphere, the total ‘‘volume’’ of the

test animals should not exceed 5% of

the volume of the test chamber.

(ii) Environmental conditions. The

temperature at which the test is

performed shall be maintained at 22 °C

( ±2 °C). Ideally, the relative humidity

should be maintained between 40% and

60%, but in certain instances (e.g., tests

using water as a vehicle), this may not

be practical.

(iii) Exposure periodicity. For acute

testing, the exposure design shall enable

4 hrs of exposure to the target

concentrations, as defined by an average

of ± 5% for gases and vapors and ± 15%

for particles and aerosols. If triggered by

the results of the 4-hr exposure,

additional testing shall be conducted in

a comparable manner using an 8-hr

exposure period.

(6) Physical measurements.

Measurements or monitoring shall be

made of the following:

(i) Chemical purity of the test material

shall be analyzed.

(ii) The rate of airflow shall be

monitored continuously, but shall be

recorded at least every 30 minutes.

(iii) The actual concentrations of the

test substance shall be measured in the

breathing zone. During the exposure

period, the actual concentrations of the

test substance shall be held as constant

as practical, monitored continuously or

intermittently depending on the method

of analysis, and recorded at least at the

beginning, at an intermediate time, and

at the end of the exposure period. Wellestablished and published monitoring

methods should be used where

available. If no standard methods are

available, then accuracy and precision

information must be supplied.

(iv) During the development of the

generating system, appropriate particle

size analysis shall be performed to

establish the stability of the aerosol.

During exposure, analysis should be

conducted as often as necessary to

determine the consistency of particle

size distribution. The particle size

distribution shall have an MMAD

between 1 and 4 μm. The particle size

of hygroscopic materials shall be small

enough when dry to assure that the size

of the particle at saturation will still

have an MMAD between 1 and 4 μm.

Characterization for fibers shall include

the bivariate distribution of length and

diameter; this distribution must ensure

inhalability.

(v) If the test substance is present in

a mixture, the mass and composition of

the entire mixture, as well as the

principal compound, shall be measured.

(vi) Temperature and humidity shall

be monitored continuously, but shall be

recorded at least every 30 minutes.

(7) Food and water during exposure

period. Food shall be withheld during

exposure. Water may also be withheld

in certain cases.

(8) Observation period. The

bronchoalveolar lavage and respiratory

pathology shall be conducted 24 hrs

following exposure to allow expression

of signs of toxicity. There is concern

that some latency time will be required

to allow migration of cells and

macromolecules into the lungs

following exposure, and that some

pathology may require macromolecular

synthesis or degradation before cell

damage develops.

(9) Gross pathology. (i) All animals

shall be subjected to a full gross

necropsy which includes examination

of orifices and the cranial, thoracic, and

abdominal cavities and their contents.

(ii) At least the lungs, liver, kidneys,

adrenals, brain, and gonads shall be

weighed wet, as soon as possible after

dissection to avoid drying.

(iii) The following organs and tissues,

or representative samples thereof, shall

be preserved in a suitable medium for

possible future histopathological

examination: All gross lesions; brainincluding sections of medulla/pons;

cerebellar cortex and cerebral cortex;

pituitary; thyroid/parathyroid; thymus;

heart; sternum with bone marrow;

salivary glands; liver; spleen; kidneys;

adrenals; pancreas; gonads; accessory

genital organs (epididymis, prostrate,

and, if present, seminal vesicles); aorta;

skin; gall bladder (if present);

esophagus; stomach; duodenum;

jejunum; ileum; cecum; colon; rectum;

urinary bladder; representative lymph

nodes; thigh musculature; peripheral

nerve; spinal cord at three levels

cervical, midthoracic, and lumbar; and

eyes. Respiratory tract tissues shall also

be preserved in a suitable medium.

(10) Histopathology. The following

histopathology shall be performed:

(i) Full histopathology shall be

performed on the respiratory tract, liver

and kidney of all animals in the control

and high concentration groups. The

histopathology of the respiratory tract is

described under paragraph (e)(11) of

this section.

(ii) All gross lesions which differ from

controls in frequency, distribution, type,

or severity in all concentration groups.

(iii) Target organs in all animals, as

indicated by the observations in the

high concentration group in this study.

Histopathologic examination of target

organs in animals at all concentration

levels (rather than only to the extent

necessary to define the NOAEL) can

support the application of exposureresponse analyses such as the

benchmark concentration approach.

(iv) Archived organs identified as

targets of toxicity from results of the 90-

day study (if a 90-day study is required

for this substance) should be elevated in

high concentration animals of the 4-hr

acute study to determine if they are also

targets of acute toxicity.

(11) Respiratory tract histopathology.

(i) Representative sections of the

respiratory tract shall be examined

histologically. These shall include the

trachea, major conducting airways,

alveolar region, terminal and respiratory

bronchioles (if present), alveolar ducts

and sacs, and interstitial tissues.

(ii) Care shall be taken that the

method used to kill the animal does not

result in damage to the tissues of the

upper or lower respiratory tract. The

lungs shall be infused with a fixative

while in an inflated state of fixed

pressure.

(iii) The upper respiratory tract shall

be examined for histopathologic lesions.

This examination shall use a minimum

of four sections located as specified

under paragraphs (e)(11)(iii)(A) through

(e)(11)(iii)(D) of this section. An

evaluation of the nasal vestibule shall be

conducted. The method described by

the reference under paragraph (h)(11) of

this section should be given

consideration. The use of additional

sections shall be left to the discretion of

the study pathologist, but consideration

should be given to additional sections as

recommended in the reference under

paragraph (h)(8) of this section to ensure

adequate evaluation of the entire upper

respiratory tract, particularly the

nasopharyngeal meatus. The following

transverse sections shall be examined:

(A) Immediately posterior to the

upper incisor teeth.

(B) At the incisor papilla.

(C) At the second palatal ridge.

(D) At the level of the first upper

molar teeth.

(iv) The laryngeal mucosa shall be

examined for histopathologic changes.

Sections of the larynx to be examined

include the epithelium covering the

base of the epiglottis, the ventral pouch,

and the medial surfaces of the vocal

processes of the arytenoid cartilages.

(12) Bronchoalveolar lavage. (i)

Animals can be exposed to the

substance by either a nose-only

procedure or in a whole-body exposure

chamber.

(ii) Care should be taken that the

method used to kill the animal results

in minimum changes in the fluid of the

lungs of the test animals.
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43827

(iii) At the appropriate time, the test

animals shall be killed and the heartlung including trachea removed in bloc.

Alternatively, lungs can be lavaged in

situ. If the study will not be

compromised, one lobe of the lungs may

be used for lung lavage while the other

is fixed for histologic evaluation. The

lungs should be lavaged using

physiological saline. The lavages shall

consist of two washes, each of which

consists of approximately 80% (e.g., 5

ml in rats and 1 ml in mice) of the total

lung volume. Additional washes merely

tend to reduce the concentrations of the

material collected. The lung lavage fluid

shall be stored on ice at 5 °C until

assayed.

(iv) The following parameters shall be

determined in the lavage fluid as

indicators of cellular damage in the

lungs: total protein, cell count, and

percent leukocytes. In addition, a

phagocytosis assay shall be performed

to determine macrophage activity. Assay

methods described in the references

under paragraphs (h)(1) and (h)(3) of

this section may be used.

(13) Combined protocol. The tests

described may be combined with any

other toxicity study, as long as none of

the requirements of either are violated

by the combination.

(f) Triggered testing. If no adverse

effects are seen in the 4-hr study as

compared with controls, no further

testing is necessary. If the 4-hr study

shows positive effects in histopathology

or the bronchoalveolar lavage, an 8-hr

study shall be conducted. Only those

tissues showing positive results in the 4-

hr study must be pursued in the followup 8-hr study. Similarly, if the option to

perform a 1-hr study is exercised, only

those tissues showing positive results in

the 4-hr study shall be pursued.

(g) Data reporting and evaluation. The

final test report shall include the

following information:

(1) Description of equipment and test

methods. A description of the general

design of the experiment and any

equipment used shall be provided.

(i) Description of exposure apparatus,

including design, type, dimensions,

source of air, system for generating

particles, aerosols, gasses, and vapors,

method of conditioning air, treatment of

exhaust air, and the method of housing

animals in a test chamber.

(ii) Description of the equipment for

measuring temperature, humidity, and

particulate aerosol concentration and

size.

(iii) Exposure data shall be tabulated

and presented with mean values and

measure of variability (e.g., standard

deviation) and should include:

(A) Chemical purity of the test

material.

(B) Airflow rates through the

inhalation equipment.

(C) Temperature and humidity of air.

(D) Nominal concentration (total

amount of test substance fed into the

inhalation equipment divided by the

volume of air).

(E) Actual concentration in test

breathing zone.

(F) Particle size distribution (e.g.,

MMAD with GSD) and the bivariate

distribution of fiber length and

diameter, where appropriate.

(2) Results—(i) General group animal

data. The following information shall be

arranged by test group exposure level.

(A) Number of animals exposed.

(B) Number of animals dying.

(C) Number of animals showing overt

signs of toxicity.

(D) Pre- and post-exposure body

weight change in animals, and weight

change during the observation period.

(ii) Counts and incidence of gross

alterations observed at necropsy in the

test and control groups. Data shall be

tabulated to show:

(A) The number of animals used in

each group and the number of animals

in which any gross lesions were found.

(B) The number of animals affected by

each different type of lesion, and the

locations and frequency of each type of

lesion.

(iii) Counts and incidence of general

histologic alterations in the test group.

Data shall be tabulated to show:

(A) The number of animals used in

each group and the number of animals

in which any histopathologic lesions

were found.

(B) The number of animals affected by

each different type of lesion, and the

locations, frequency, and average grade

of each type of lesion.

(iv) Counts and incidence of

respiratory histopathologic alterations

by the test group. Data shall be tabulated

to show:

(A) The number of animals used in

each group and the number of animals

in which any histopathologic lesions

were found.

(B) The number of animals affected by

each different type of lesion, and the

locations, frequency, and average grade

of each type of lesion.

(v) Results of the bronchoalveolar

lavage study. Data shall be tabulated to

show:

(A) The amount of administered

lavage fluid and recovered lavage fluid

for each test animal.

(B) The magnitude of change of

biochemical and cytologic indices in

lavage fluids at each test concentration

for each animal.

(C) Results shall be quantified as

amount of constituent/mL of lavage

fluid. This assumes that the amount of

lavage fluid recovered is a

representative sample of the total lavage

fluid.

(3) Evaluation of data. The findings

from this acute study should be

evaluated in the context of preceding

and/or concurrent toxicity studies and

any correlated functional findings. The

evaluation shall include the relationship

between the concentrations of the test

substance and the presence or absence,

incidence, and severity of any effects.

The evaluation should include

appropriate statistical analyses, for

example, parametric tests for

continuous data and non-parametric

tests for the remainder. Choice of

analyses should consider tests

appropriate to the experimental design,

including repeated measures. The report

must include concentration-response

curves for the bronchoalveolar lavage

and tables reporting observations at

each concentration level for necropsy

findings and gross, general, and

respiratory system histopathology.

(h) Reference. For additional

background information on this test

guideline, the following references

should be consulted. These references

are available for inspection at the TSCA

Nonconfidential Information Center,

Rm. NE–B607, Environmental

Protection Agency, 401 M St., SW.,

Washington, DC, 12 noon to 4 p.m.,

Monday through Friday, except legal

holidays.

(1) Burleson, G.R., Fuller, L.B.,

Me¬nache, M.G., and Graham, J.A. Poly

(I): poly (C)-enhanced alveolar

peritoneal macrophage phagocytosis:

Quantification by a new method

utilizing fluorescent beads. Proceedings

of the Society of Experimental Biology

and Medicine. 184:468–476 (1987).

(2) Gardner, D.E., Crapo, J.D., and

McClellan, R.O. (Eds.) Toxicology of the

Lung. (Raven Press, New York, 1993)

pp. i–xii, 1–30.

(3) Gilmour, G.I., and Selgrade, M.K.

A comparison of the pulmonary

defenses against streptococcal infection

in rats and mice following O3 exposure:

Differences in disease susceptibility and

neutrophil recruitment. Toxicology and

Applied Pharmacology. 123:211–218

(1993).

(4) Henderson, R.F., Benson, J.M.,

Hahn, F.F., Hobbs, C.H., Jones, R.K.,

Mauderly, J.L., McClellan, R.O., and

Pickrell, J.A. New approaches for the

evaluation of pulmonary toxicity:

Bronchoalveolar lavage fluid analysis.

Fundamental and Applied Toxicology.

5:451–458 (1985).
43828 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(5) Henderson, R.F. Use of

bronchoalveolar lavage to detect lung

damage. Environmental Health

Perspectives. 56:115–129 (1984).

(6) Henderson, R.F., Rebar, A.H.,

Pickrell, J.A., and Newton, G.J. Early

damage indicators in the lung. III.

Biochemical and cytological response of

the lung to inhaled metal salts.

Toxicology and Applied Pharmacology.

50:123–136 (1979).

(7) McClellan, R.O. and Henderson,

R.F. (Eds.) Second edition. Concepts in

Inhalation Toxicology. (Taylor and

Francis, Washington, DC, 1995) pp.i–

xxiv, 1–24, 441–470.

(8) Mery, S., Gross, E.A., Joyner, D.R.,

Godo, M., and Morgan, K.T. Nasal

Diagrams: A Tool for Recording the

Distribution of Nasal Lesions in Rats

and Mice. Toxicologic Pathology.

22:353–372 (1994).

(9) Phalen, R.F. (Ed) Methods in

Inhalation Toxicology. (CRC Press, Boca

Raton, FL, 1997) pp. i–xii, 1–12.

(10) Renne, R.A., Gideon, K.M.,

Miller, R.A., Mellick, P.W., and

Grumbein, S.L. Histologic methods and

interspecies variations in the laryngeal

histology of F344/N rats and B6C3F1

mice. Toxicology and Pathology. 20:44–

51 (1992).

(11) Young, J.T. Histopathologic

examination of the rat nasal cavity.

Fundamental and Applied Toxicology.

1:309–312 (1981).

§ 799.9346 TSCA subchronic inhalation

toxicity.

(a) Scope This section is intended to

meet the testing requirements under

section 4 of TSCA. In the assessment

and evaluation of the toxic

characteristics of a gas, volatile

substance, or aerosol/particulate,

determination of subchronic inhalation

toxicity may be carried out after initial

information on toxicity has been

obtained by acute testing. The

subchronic inhalation study has been

designed to permit the determination of

the no-observed-effect-level (NOEL) and

toxic effects associated with continuous

or repeated exposure to a test substance

for a period of 90 days. This study is not

capable of determining those effects that

have a long latency period for

development (e.g., carcinogenicity and

life shortening). Extrapolation from the

results of this study to humans is valid

only to a limited degree. It can,

however, provide useful 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 target

organs and the possibilities of

accumulation, and can be of use in

selecting concentration levels for

chronic studies and establishing safety

criteria for human exposure. Hazards of

inhaled substances are influenced by

the inherent toxicity and by physical

factors such as volatility and particle

size.

(b) Source. The source material used

in developing this TSCA test guideline

is the OPPTS harmonized test guideline

870.3465 (June 1996 Public Draft). This

source is available at the address in

paragraph (h) of this section.

(c) Definitions. The following

definitions apply to this section.

Aerodynamic equivalent diameter is

defined as the diameter of a unit density

sphere having the same terminal settling

velocity as the particle in question,

whatever its size, shape, and density. It

is used to predict where in the

respiratory tract such particles may be

deposited.

Concentration in a subchronic

inhalation study is the amount of test

substance administered via inhalation

for a period of 90–days. Concentration

is expressed as weight of the test

substance per unit volume of air

(milligrams per liter or parts per

million).

Cumulative toxicity is the adverse

effects of repeated exposures occurring

as a result of prolonged action on, or

increased concentration of the

administered test substance or its

metabolites in susceptible tissues.

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

Mass median aerodynamic diameter

(MMAD) is the median aerodynamic

diameter and along with the geometric

standard deviation (GSD) is used to

describe the particle size distribution of

any aerosol statistically based on the

weight and size of the particles. Fifty

percent of the particles by weight will

be smaller than the median diameter

and 50% of the particles will be larger.

No-observed-effect-level (NOEL) is the

maximum concentration used in a study

which produces no adverse effects.

Subchronic 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 (approximately 10%)

of a life span.

(d) Limit test. The exposure is at a

concentration of 1 mg/L or greater

(expected human exposure may indicate

the need for a higher concentration),

where such concentration is not

possible due to physical or chemical

properties of the test substance, or

where the maximum attainable

concentration produces no observable

toxic effects. A full study using three

concentrations may not be necessary.

(e) Test procedures—(1) Animal

selection—(i) Species and strain. A

mammalian species shall be used for

testing. A variety of rodent species may

be used, although the rat is the preferred

species. Commonly used laboratory

strains should be employed. If another

mammalian species is used, the tester

shall provide justification/reasoning for

its selection.

(ii) Age/weight. (A) Testing should be

started with young healthy animals as

soon as possible after weaning and

acclimatization.

(B) Exposure shall commence no later

than 8 weeks of age.

(C) At the commencement of the

study the weight variation of animals

used shall not exceed ± 20% of the mean

weight for each sex.

(iii) Sex. (A) Equal numbers of

animals of each sex shall be used at

each concentration.

(B) Females shall be nulliparous and

nonpregnant.

(iv) Numbers. (A) At least 20 rodents

(10 females and 10 males) should be

used for each test group. If another

mammalian species is selected (e.g. dog,

rabbit, or nonhuman primate), at least

eight animals per group (four males and

four females) shall be used.

(B) If interim sacrifices are planned,

the number of animals shall be

increased by the number of animals

scheduled to be sacrificed before the

completion of the study.

(C) To avoid bias, the use of adequate

randomization procedures for the

proper allocation of animals to test and

control groups is required.

(D) Each animal shall be assigned a

unique identification number. Dead

animals, their preserved organs and

tissues, and microscopic slides shall be

identified by reference to the animal’s

unique number.

(v) Husbandry. (A) Animals may be

group-caged by sex, but the number of

animals per cage must not interfere with

clear observation of each animal. The

biological properties of the test

substance or toxic effects (e.g.,

morbidity, excitability) may indicate a

need for individual caging. Animals

must be housed individually in

inhalation chambers during exposure to

aerosols.

(B) The temperature of the

experimental animal rooms should be at

22 ±3 °C.

(C) The relative humidity of the

experimental animal rooms should be

30–70%.
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43829

(D) Where lighting is artificial, the

sequence should be 12 h light/12 h dark.

(E) Control and test animals should be

fed from the same batch and lot. The

feed should be analyzed to assure

adequacy of nutritional requirements of

the species tested and for impurities

that might influence the outcome of the

test. Animals should be fed and watered

ad libitum with food replaced at least

weekly. For nonrodents feeding should

be at least daily and water ad libitum.

(F) The study should not be initiated

until animals have been allowed a

period of acclimatization/quarantine.

(2) Control and test substances. (i)

Whenever it is necessary to formulate

the test substance with a vehicle for

aerosol generation, the vehicle ideally

should not elicit toxic effects or

substantially alter the chemical or

toxicological properties of the test

substance.

(ii) One lot of the test substance

should be used, if possible throughout

the duration of the study, and the

research sample should be stored under

conditions that maintain its purity and

stability. Prior to the initiation of the

study, there should be a characterization

of the test substance, including the

purity of the test substance and, if

technically feasible, the name and

quantities of unknown contaminants

and impurities.

(3) Control groups. A concurrent

control group is required. This group

shall be an untreated or sham-treated

control group. Except for treatment with

the test substance, animals in the

control group shall be handled in a

manner identical to the test group

animals. Where a vehicle other than

water is used to generate a substance, a

vehicle control group should be used. If

the toxic properties of the vehicle are

not known or cannot be made available,

both untreated and vehicle control

groups are required.

(4) Satellite group. A satellite group of

20 animals (10 animals per sex) may be

treated with the high concentration

level for 90 days and observed for

reversibility, persistence, or delayed

occurrence of toxic effects for a posttreatment period of appropriate length,

normally not less than 28 days. In

addition, a control group of 20 animals

(10 animals of each sex) should be

added to the satellite study.

(5) Concentration levels and

concentration selection. (i) In

subchronic toxicity tests, it is desirable

to have a concentration-response

relationship as well as a NOEL.

Therefore, at least three concentration

levels plus a control and, where

appropriate, a vehicle control

(corresponding to the concentration of

vehicle at the highest exposure level)

shall be used. Concentrations should be

spaced appropriately to produce test

groups with a range of toxic effects. The

data should be sufficient to produce a

concentration-response curve.

(ii) The highest concentration should

result in toxic effects but not produce an

incidence of fatalities which would

prevent a meaningful evaluation.

(iii) The intermediate concentrations

should be spaced to produce a gradation

of toxic effects.

(iv) The lowest concentration should

produce no evidence of toxicity.

(v) In the case of potentially explosive

test substances, care should be taken to

avoid generating explosive

concentrations.

(6) Administration of the test

substance. Animals should be exposed

to the test substance for 6 h per day on

a 7–day per week basis for a period of

at least 90 days. Based primarily on

practical considerations, exposure for 6

h per day on a 5–day per week basis is

acceptable.

(7) Observation period. The animals

should be observed for a period of 90

days. Animals in the satellite group (if

used) scheduled for follow-up

observations should be kept for at least

28 days further without treatment to

assess reversibility.

(8) Exposure specifications. (i) The

animals shall be tested in dynamic

inhalation equipment designed to

sustain a minimum airflow of 10 air

changes per hr, an adequate oxygen

content of at least 19%, and uniform

conditions throughout the exposure

chamber. Maintenance of slight negative

pressure inside the chamber will

prevent leakage of the test substance

into the surrounding areas. It is not

normally necessary to measure chamber

oxygen concentration if airflow is

adequate.

(ii) The selection of a dynamic

inhalation chamber should be

appropriate for the test substance and

test system. Where a whole body

chamber is used to expose animals to an

aerosol, individual housing must be

used to minimize crowding of the test

animals and maximize their exposure to

the test substance. To ensure stability of

a chamber atmosphere, the total volume

occupied by the test animals shall not

exceed 5% of the volume of the test

chamber. It is recommended, but not

required, that nose-only or head-only

exposure be used for aerosol studies in

order to minimize oral exposures due to

animals licking compound off their fur.

Heat stress should be minimized.

(iii) The temperature at which the test

is performed should be maintained at

22 ± 2 °C. The relative humidity should

be maintained between 40 and 60%, but

in certain instances (e.g., use of water

vehicle) this may not be practicable.

(9) Physical measurements.

Measurements or monitoring shall be

made of the following:

(i) The rate of airflow shall be

monitored continuously but recorded at

least three times during the exposure.

(ii) The actual concentrations of the

test substance shall be measured in the

animal’s breathing zone. During the

exposure period, the actual

concentrations of the test substance

shall be held as constant as practicable

and monitored continuously or

intermittently depending on the method

of analysis. Chamber concentration may

be measured using gravimetric or

analytical methods as appropriate. If

trial run measurements are reasonably

consistent (± 10% for liquid, aerosol,

gas, or vapor; ± 20% for dry aerosol),

then two measurements should be

sufficient. If measurements are not

consistent, three to four measurements

should be taken. Whenever the test

substance is a formulation, or it is

necessary to formulate the test

substance with a vehicle for aerosol

generation, the analytical concentration

must be reported for the total

formulation, and not just for the active

ingredient (AI). If, for example, a

formulation contains 10% AI and 90%

inerts, a chamber analytical limit

concentration of 2 mg/L would consist

of 0.2 mg/L of the AI. It is not necessary

to analyze inert ingredients provided

the mixture at the animal’s breathing

zone is analogous to the formulation;

the grounds for this conclusion must be

provided in the study report. If there is

some difficulty in measuring chamber

analytical concentration due to

precipitation, nonhomogeneous

mixtures, volatile components, or other

factors, additional analyses of inert

components may be necessary.

(iii) During the development of the

generating system, particle size analysis

shall be performed to establish the

stability of aerosol concentrations with

respect to particle size. The MMAD

particle size range should be between 1–

3 μm. The particle size of hygroscopic

materials should be small enough when

dry to assure that the size of the swollen

particle will still be within the 1–3 μm

range. Measurements of aerodynamic

particle size in the animal’s breathing

zone should be measured during a trial

run. If MMAD valves for each exposure

level are within 10% of each other, then

two measurements during the exposures

should be sufficient. If pretest

measurements are not within 10% of

each other, three to four measurements

should be taken.
43830 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(iv) Temperature and humidity shall

be monitored continuously and

recorded at least three times during an

exposure.

(10) Feed and water during exposure

period. Feed shall be withheld during

exposure. Water may also be withheld

during exposure.

(11) Observation of animals. (i)

During and following exposure,

observations are made and recorded

systematically; individual records

should be maintained for each animal.

It is not always possible to observe

animals during exposure in a wholebody chamber.

(ii) Observations shall be made at

least once each day for morbidity and

mortality. Appropriate actions should

be taken to minimize loss of animals to

the study (e.g., Necropsy or refrigeration

of those animals found dead and

isolation or sacrifice of weak or

moribund animals).

(iii) A careful clinical examination

shall be made at least once weekly.

Observations should be detailed and

carefully recorded, preferably using

explicitly defined scales. Observations

should include, but not be limited to,

evaluation of skin and fur, eyes and

mucous membranes, respiratory and

circulatory effects, autonomic effects

such as salivation, central nervous

system effects, including tremors and

convulsions, changes in the level of

activity, gait and posture, reactivity to

handling or sensory stimuli, altered

strength, and stereotypes or bizarre

behavior (e.g., self-mutilation, walking

backwards).

(iv) Signs of toxicity should be

recorded as they are observed including

the time of onset, degree and duration.

(v) Individual weights of animals

shall be determined shortly before the

test substance is administered, and

weekly thereafter.

(vi) Food consumption shall also be

determined weekly if abnormal body

weight changes are observed.

(vii) Moribund animals should be

removed and sacrificed when noticed

and the time of death should be

recorded as precisely as possible.

(viii) At termination, all survivors in

the treatment groups shall be sacrificed.

(12) Clinical pathology. Hematology

and clinical chemistry examinations

shall be made on all animals, including

controls, of each sex in each group for

rodents and all animals when

nonrodents are used as test animals. For

rodents, the hematology and clinical

chemistry parameters should be

examined once prior to initiation of

exposure and at terminal sacrifice. For

nonrodents, the hematology and clinical

chemistry parameters should be

examined once prior to initiation of

exposure, at monthly intervals or

midway through the test period and at

termination.

(i) The recommended hematology

parameters are: Hemoglobin and

hematocrit concentrations, red blood

cell count, white blood cell count,

differential leukocyte count, platelet

count, and a measure of clotting

potential such as prothrombin time or

thromboplastin time.

(ii) Clinical chemistry parameters

which are considered appropriate to all

studies are electrolyte balance,

carbohydrate metabolism, and liver and

kidney function. Other determinations

which may be necessary for an adequate

toxicological evaluation include

analyses of lipids, hormones, acid/base

balance, methemoglobin and

cholinesterase activity. Additional

clinical biochemistry may be employed

where necessary to extend the

investigation of observed effects.The

selection of specific tests will be

influenced by observations on the mode

of action of the substance and signs of

clinical toxicity. Suggested blood

clinical chemistry determinations:

(A) Electrolytes.

(1) Calcium.

(2) Chloride.

(3) Magnesium.

(4) Inorganic phosphorus.

(5) Potassium.

(6) Sodium.

(B) Enzymes.

(1) Alkaline phosphatase.

(2) Alanine aminotransferase.

(3) Aspartate aminotransferase.

(4) Gamma glutamyl transferase.

(C) Other.

(1) Albumin.

(2) Blood creatinine.

(3) Blood urea nitrogen.

(4) Globulins.

(5) Glucose (fasting).

(6) Total bilirubin.

(7) Total cholesterol.

(8) Total serum protein.

(iii) Urinalysis is not recommended

on a routine basis, but only when there

is an indication based on expected or

observed toxicity.

(13) Ophthalmological examination.

Ophthalmological examinations shall be

made on all animals prior to the

administration of the test substance and

on all high concentration and control

groups at termination. If changes in the

eyes are detected, all animals in the

other concentration groups shall be

examined.

(14) Gross pathology. (i) All animals

shall be subjected to a full gross

necropsy which includes examination

of the external surface of the body, all

orifices and the cranial, thoracic, and

abdominal cavities and their contents.

(ii) At least the liver, kidneys, brain,

and gonads shall be trimmed and

weighed wet, as soon as possible after

dissection to avoid drying.

(iii) The following organs and tissues,

or representative samples thereof, shall

be preserved in a suitable medium for

possible future histopathological

examination:

(A) Digestive system.

(1) Salivary glands.

(2) Esophagus.

(3) Stomach.

(4) Duodenum.

(5) Jejunum.

(6) Ileum.

(7) Cecum.

(8) Colon.

(9) Rectum.

(10) Liver.

(11) Pancreas.

(12) Gallbladder (dogs).

(B) Nervous system.

(1) Brain (multiple sections).

(2) Pituitary.

(3) Peripheral nerve(s).

(4) Spinal cord (three levels).

(5) Eyes (retina, optic nerve).

(C) Glandular system.

(1) Adrenals.

(2) Parathyroids.

(3) Thyroids.

(D) Respiratory system.

(1) Trachea.

(2) Lung.

(3) Pharynx.

(4) Larynx.

(5) Nose.

(E) Cardiovascular/hematopoietic

system.

(1) Aorta (thoracic).

(2) Heart.

(3) Bone marrow.

(4) Lymph nodes.

(5) Spleen.

(6) Thymus.

(F) Urogenital system.

(1) Kidneys.

(2) Urinary bladder.

(3) Prostate.

(4) Testes.

(5) Epididymides.

(6) Seminal vesicle(s).

(7) Uterus.

(8) Ovaries.

(G) Other.

(1) Lacrimal gland.

(2) Mammary gland.

(3) Skin.

(4) Skeletal muscle.

(5) All gross lesions and masses.

(6) Sternum and/or femur.

(15) Histopathology. (i) The following

histopathology shall be performed:

(A) Full histopathology on the

respiratory tract and other organs and

tissues, listed under paragraph

(e)(15)(iii) of this section, of all animals

in the control and high exposure groups
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43831

and all animals that died or were killed

during the study.

(B) All gross lesions in all animals.

(C) Target organs in all animals.

(D) Lungs, liver and kidneys of all

animals. Special attention to

examination of the respiratory tract

should be made for evidence of

infection as this provides a convenient

assessment of the state of health of the

animals.

(E) When a satellite group is used,

histopathology shall be performed on

tissues and organs identified as showing

effects in the treated groups.

(ii) If excessive early deaths or other

problems occur in the high exposure

group compromising the significance of

the data, the next concentration should

be examined for complete

histopathology.

(iii) An attempt should be made to

correlate gross observations with

microscopic findings.

(iv) Tissues and organs designated for

microscopic examination should be

fixed in 10% buffered formalin or a

recognized suitable fixative as soon as

necropsy is performed and no less than

48 hrs prior to trimming. Tissues should

be trimmed to a maximum thickness of

0.4 cm for processing.

(f) Data and reporting—(1) Treatment

of results. (i) Data shall be summarized

in tabular form, showing for each test

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.

(ii) All observed results (quantitative

and qualitative) should be evaluated by

an appropriate statistical method. Any

generally accepted statistical method

may be used; the statistical methods

including significance criteria should be

selected during the design of the study.

(2) Evaluation of study results. The

findings of the subchronic inhalation

toxicity study should be evaluated in

conjunction with the findings of

preceding studies and considered in

terms of the observed toxic effects and

the necropsy and histopathological

findings. The evaluation will include

the relationship between the

concentration of the test substance and

duration of exposure, and the presence

or absence, the incidence and severity,

of abnormalities, including behavioral

and clinical abnormalities, gross lesions,

identified target organs, body weight

changes, effects on mortality and any

other general or specific toxic effects. A

properly conducted subchronic test

should provide a satisfactory estimation

of a no-effect level. It also can indicate

the need for an additional longer-term

study and provide information on the

selection of concentrations.

(3) Test report. In addition to

reporting requirements specified under

40 CFR part 792, subpart J, the following

specific information shall be reported.

Both individual and summary data

should be presented.

(i) Test substance characterization

shall include:

(A) Chemical identification.

(B) Lot or batch number.

(C) Physical properties.

(D) Purity/impurities.

(E) Identification and composition of

any vehicle used.

(ii) Test system information shall

include:

(A) Species and strain of animals used

and rationale for selection if other than

that recommended.

(B) Age, sex, and body weight.

(C) Test environment including cage

conditions, ambient temperature,

humidity, and light/dark periods.

(iii) Test procedure information shall

include:

(A) Method of randomization used.

(B) Full description of experimental

design and procedure.

(C) Exposure regimen including

concentration levels, methods, and

volume.

(D) Description of test conditions; the

following exposure conditions shall be

reported:

(1) Description of exposure apparatus

including design, type, volume, source

of air, system for generating aerosols,

method of conditioning air, treatment of

exhaust air and the method of housing

the animals in a test chamber.

(2) The equipment for measuring

temperature, humidity, and particulate

aerosol concentrations and size should

be described.

(E) Exposure data shall be tabulated

and presented with mean values and a

measure of variability (e.g., standard

deviation) and include:

(1) Airflow rates through the

inhalation equipment.

(2) Temperature and humidity of air.

(3) Actual (analytical or gravimetric)

concentration in the breathing zone.

(4) Nominal concentration (total

amount of test substance fed into the

inhalation equipment divided by

volume of air).

(5) Particle size distribution,

calculated mass median aerodynamic

diameter (MMAD) and geometric

standard deviation (GSD).

(6) Explanation as to why the desired

chamber concentration and/or particle

size could not be achieved (if

applicable) and the efforts taken to

comply with this aspect of the section.

(iv) Test results information shall

include:

(A) Group animal data. Tabulation of

toxic response data by species, strain,

sex and exposure level for:

(1) Number of animals exposed.

(2) Number of animals showing signs

of toxicity.

(3) Number of animals dying.

(B) Individual animal data. Data

should be presented as summary (group

mean) as well as for individual animals.

(1) Time of death during the study or

whether animals survived to

termination.

(2) Time of observation of each

abnormal sign and its subsequent

course.

(3) Body weight data.

(4) Feed consumption data, when

collected.

(5) Results of ophthalmological

examination, when performed.

(6) Results of hematological tests

performed. .

(7) Results of clinical chemistry tests

performed.

(8) Results of urinalysis tests

performed.

(9) Necropsy findings, including

absolute and relative organ weight data.

(10) Detailed description of all

histopathological findings.

(11) Statistical treatment of results,

where appropriate.

(g) Quality control. A system shall be

developed and maintained to assure and

document adequate performance of

laboratory staff and equipment. The

study shall be conducted in compliance

with 40 CFR Part 792—Good Laboratory

Practice Standards.

(h) References. For additional

background information on this test

guideline, the following references

should be consulted. These references

are available for inspection at the TSCA

Nonconfidential Information Center,

Rm. NE–B607, Environmental

Protection Agency, 401 M St., SW.,

Washington, DC, 12 noon to 4 p.m.,

Monday through Friday, except legal

holidays.

(1) Cage, J.C. Ed. Paget, G.E.

Experimental Inhalation Toxicology,

Methods in Toxicology. (F.A. Davis Co.,

Philadelphia, PA, 1970) pp. 258–277.

(2) Casarett, L.J. and Doull. Chapter 9.

Toxicology: The Basic Science of

Poisons (New York: Macmillan

Publishing Co., Inc., 1975).

(3) U.S. Environmental Protection

Agency, Office of Pesticide Programs,

Health Effects Division. Interim policy

for particle size and limit concentration

issues in inhalation toxicity studies

(February 1, 1994).

(4) MacFarland, H.N. Ed. Hayes, W.J.

Vol. 7. Respiratory Toxicology, Essays in

Toxicology. (Academic Press, New York,

NY, 1976) pp. 121–154.
43832 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(5) Organisation for Economic Cooperation and Development. Guidelines

for testing of chemicals, section 4-health

effects, part 413. Subchronic Inhalation

Toxicity Studies (Paris, 1981).

§ 799.9370 TSCA prenatal developmental

toxicity.

(a) Scope This section is intended to

meet the testing requirements under

section 4 of TSCA. This guideline for

developmental toxicity testing is

designed to provide general information

concerning the effects of exposure on

the pregnant test animal and on the

developing organism; this may include

death, structural abnormalities, or

altered growth and an assessment of

maternal effects. For information on

testing for functional deficiencies and

other postnatal effects, the guidelines

for the two-generation reproductive

toxicity study and the developmental

neurotoxicity study should be

consulted.

(b) Source. The source material used

in developing this TSCA test guideline

is the OPPTS harmonized test guideline

870.3700 (February 1996 Public Draft).

This source is available at the address

in paragraph (h) of this section.

(c) Good laboratory practice

standards. The study shall be conducted

in compliance with 40 CFR Part 792—

Good Laboratory Practice Standards.

(d) Principle of the test method. The

test substance is administered to

pregnant animals at least from

implantation to one day prior to the

expected day of parturition. Shortly

before the expected date of delivery, the

pregnant females are terminated, the

uterine contents are examined, and the

fetuses are processed for visceral and

skeletal evaluation.

(e) Test procedures—(1) Animal

selection—(i) Species and strain. It is

recommended that testing be performed

in the most relevant species, and that

laboratory species and strains which are

commonly used in prenatal

developmental toxicity testing be

employed. The preferred rodent species

is the rat and the preferred non-rodent

species is the rabbit.

(ii) Age. Young adult animals shall be

used.

(iii) Sex. Nulliparous female animals

shall be used at each dose level.

Animals should be mated with males of

the same species and strain, avoiding

the mating of siblings, if parentage is

known. Day 0 in the test is the day on

which a vaginal plug and/or sperm are

observed in the rodent or that

insemination is performed or observed

in the rabbit.

(iv) Number of animals. Each test and

control group shall contain a sufficient

number of animals to yield

approximately 20 animals with

implantation sites at necropsy.

(2) Administration of test and control

substances—(i) Dose levels and dose

selection. (A) At least three-dose levels

and a concurrent control shall be used.

Healthy animals shall be randomly

assigned to the control and treatment

groups, in a manner which results in

comparable mean body weight values

among all groups. The dose levels

should be spaced to produce a gradation

of toxic effects. Unless limited by the

physical/chemical nature or biological

properties of the test substance, the

highest dose shall be chosen with the

aim to induce some developmental and/

or maternal toxicity but not death or

severe suffering. In the case of maternal

mortality, this should not be more than

approximately 10%. The intermediate

dose levels should produce minimal

observable toxic effects. The lowest dose

level should not produce any evidence

of either maternal or developmental

toxicity (i.e., the no-observed-adverseeffect level, NOAEL) or should be at or

near the limit of detection for the most

sensitive endpoint. Two- or four-fold

intervals are frequently optimal for

spacing the dose levels, and the

addition of a fourth test group is often

preferable to using very large intervals

(e.g., more than a factor of 10) between

dosages.

(B) It is desirable that additional

information on metabolism and

pharmacokinetics of the test substance

be available to demonstrate the

adequacy of the dosing regimen. This

information should be available prior to

testing.

(C) The highest dose tested need not

exceed 1,000 mg/kg/day by oral or

dermal administration, or 2 mg/L (or the

maximum attainable concentration) by

inhalation, unless potential human

exposure data indicate the need for

higher doses. If a test performed at the

limit dose level, using the procedures

described for this study, produces no

observable toxicity and if an effect

would not be expected based upon data

from structurally related compounds,

then a full study using three-dose levels

may not be considered necessary.

(ii) Control group. (A) A concurrent

control group shall be used. This group

shall be a sham-treated control group or

a vehicle-control group if a vehicle is

used in administering the test

substance.

(B) The vehicle control group should

receive the vehicle in the highest

volume used.

(C) If a vehicle or other additive is

used to facilitate dosing, consideration

should be given to the following

characteristics: Effects on the

absorption, distribution, metabolism, or

retention of the test substance; effects on

the chemical properties of the test

substance which may alter its toxic

characteristics; and effects on the food

or water consumption or the nutritional

status of the animals.

(iii) Route of administration. (A) The

test substance or vehicle is usually

administered orally by intubation.

(B) If another route of administration

is used, for example, when the route of

administration is based upon the

principal route of potential human

exposure, the tester shall provide

justification and reasoning for its

selection, and appropriate modifications

may be necessary. Care should be taken

to minimize stress on the maternal

animals. For materials administered by

inhalation, whole-body exposure is

preferable to nose-only exposure due to

the stress of restraint required for noseonly exposure.

(C) The test substance shall be

administered at approximately the same

time each day.

(D) When administered by gavage or

dermal application, the dose to each

animal shall be based on the most recent

individual body weight determination.

(iv) Dosing schedule. At minimum,

the test substance shall be administered

daily from implantation to the day

before cesarean section on the day prior

to the expected day of parturition.

Alternatively, if preliminary studies do

not indicate a high potential for

preimplantation loss, treatment may be

extended to include the entire period of

gestation, from fertilization to

approximately 1 day prior to the

expected day of termination.

(f) Observation of animals—(1)

Maternal. (i) Each animal shall be

observed at least once daily, considering

the peak period of anticipated effects

after dosing. Mortality, moribundity,

pertinent behavioral changes, and all

signs of overt toxicity shall be recorded

at this cageside observation. In addition,

thorough physical examinations shall be

conducted at the same time maternal

body weights are recorded.

(ii) Animals shall be weighed on day

0, at termination, and at least at 3–day

intervals during the dosing period.

(iii) Food consumption shall be

recorded on at least 3-day intervals,

preferably on days when body weights

are recorded.

(iv) (A) Females shall be terminated

immediately prior to the expected day

of delivery.

(B) Females showing signs of abortion

or premature delivery prior to

scheduled termination shall be killed
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43833

and subjected to a thorough

macroscopic examination.

(v) At the time of termination or death

during the study, the dam shall be

examined macroscopically for any

structural abnormalities or pathological

changes which may have influenced the

pregnancy. Evaluation of the dams

during cesarean section and subsequent

fetal analyses should be conducted

without knowledge of treatment group

in order to minimize bias.

(vi) (A) Immediately after termination

or as soon as possible after death, the

uteri shall be removed and the

pregnancy status of the animals

ascertained. Uteri that appear nongravid

shall be further examined (e.g. by

ammonium sulfide staining) to confirm

the nonpregnant status.

(B) Each gravid uterus (with cervix)

shall be weighed. Gravid uterine

weights should not be obtained from

dead animals if autolysis or

decomposition has occurred.

(C) The number of corpora lutea shall

be determined for pregnant animals.

(D) The uterine contents shall be

examined for embryonic or fetal deaths

and the number of viable fetuses. The

degree of resorption shall be described

in order to help estimate the relative

time of death of the conceptus.

(2) Fetal. (i) The sex and body weight

of each fetus shall be determined.

(ii) Each fetus shall be examined for

external anomalies.

(iii) Fetuses shall be examined for

skeletal and soft tissue anomalies (e.g.

variations and malformations or other

categories of anomalies as defined by

the performing laboratory).

(A) For rodents, approximately onehalf of each litter shall be prepared by

standard techniques and examined for

skeletal alterations, preferably bone and

cartilage. The remainder shall be

prepared and examined for soft tissue

anomalies, using appropriate serial

sectioning or gross dissection

techniques. It is also acceptable to

examine all fetuses by careful dissection

for soft tissue anomalies followed by an

examination for skeletal anomalies.

(B) For rabbits, all fetuses shall be

examined for both soft tissue and

skeletal alterations. The bodies of these

fetuses should be evaluated by careful

dissection for soft-tissue anomalies,

followed by preparation and

examination for skeletal anomalies. An

adequate evaluation of the internal

structures of the head, including the

eyes, brain, nasal passages, and tongue,

should be conducted for at least half of

the fetuses.

(g) Data and reporting—(1) Treatment

of results. Data shall be reported

individually and summarized in tabular

form, showing for each test group the

types of change and the number of

dams, fetuses, and litters displaying

each type of change.

(2) Evaluation of study results. The

following shall be provided:

(i) Maternal and fetal test results,

including an evaluation of the

relationship, or lack thereof, between

the exposure of the animals to the test

substance and the incidence and

severity of all findings.

(ii) Criteria used for categorizing fetal

external, soft tissue, and skeletal

anomalies.

(iii) When appropriate, historical

control data to enhance interpretation of

study results. Historical data (on litter

incidence and fetal incidence within

litter), when used, should be compiled,

presented, and analyzed in an

appropriate and relevant manner. In

order to justify its use as an analytical

tool, information such as the dates of

study conduct, the strain and source of

the animals, and the vehicle and route

of administration should be included.

(iv) Statistical analysis of the study

findings should include sufficient

information on the method of analysis,

so that an independent reviewer/

statistician can reevaluate and

reconstruct the analysis. In the

evaluation of study data, the litter

should be considered the basic unit of

analysis.

(v) 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) Test report. In addition to the

reporting requirements as specified

under 40 CFR part 792, subpart J, the

following specific information shall be

reported. Both individual and summary

data should be presented.

(i) Species and strain.

(ii) Maternal toxic response data by

dose, including but not limited to:

(A) The number of animals at the start

of the test, the number of animals

surviving, the number pregnant, and the

number aborting.

(B) Day of death during the study or

whether animals survived to

termination.

(C) Day of observation of each

abnormal clinical sign and its

subsequent course.

(D) Body weight and body weight

change data, including body weight

change adjusted for gravid uterine

weight.

(E) Food consumption and, if

applicable, water consumption data.

(F) Necropsy findings, including

gravid uterine weight.

(iii) Developmental endpoints by dose

for litters with implants, including:

(A) Corpora lutea counts.

(B) Implantation data, number and

percent of live and dead fetuses, and

resorptions (early and late).

(C) Pre- and postimplantation loss

calculations.

(iv) Developmental endpoints by dose

for litters with live fetuses, including:

(A) Number and percent of live

offspring.

(B) Sex ratio.

(C) Fetal body weight data, preferably

by sex and with sexes combined.

(D) External, soft tissue, and skeletal

malformation and variation data. The

total number and percent of fetuses and

litters with any external, soft tissue, or

skeletal alteration, as well as the types

and incidences of individual anomalies,

should be reported.

(v) The numbers used in calculating

all percentages or indices.

(vi) Adequate statistical treatment of

results.

(vii) A copy of the study protocol and

any amendments should be included.

(h) References. For additional

background information on this test

guideline, the following references

should be consulted. These references

are available for inspection at the TSCA

Nonconfidential Information Center,

Rm. NE–B607, Environmental

Protection Agency, 401 M St., SW.,

Washington, DC, 12 noon to 4 p.m.,

Monday through Friday, except legal

holidays.

(1) Aliverti, V.L. et al. The extent of

fetal ossification as an index of delayed

development in teratogenicity studies in

the rat. Teratology. 20:237–242 (1979).

(2) Barrow, M.V. and W.J. Taylor. A

rapid method for detecting

malformations in rat fetuses. Journal of

Morphology 127:291–306 (1969).

(3) Burdi, A.R. Toluidine blue-alizarin

red S staining of cartilage and bone in

whole-mount skeltons in vitro. Stain

Technolology. 40:45–48 (1965).

(4) Edwards, J.A. Ed. Woolam,D.H.M.

The external development of the rabbit

and rat embryo. Vol. 3. Advances in

Teratology (Academic, NY, 1968).

(5) Fritz, H. Prenatal ossification in

rabbits as indicative of fetal maturity.

Teratology. 11:313–320 (1974).

(6) Fritz, H. and Hess, R. Ossification

of the rat and mouse skeleton in the

perinatal period. Teratology. 3:331–338

(1970).

(7) Gibson, J.P. et al. Use of the rabbit

in teratogenicity studies. Toxicology

and Applied Pharmacology. 9:398–408

(1966).

(8) Inouye, M. Differential staining of

cartilage and bone in fetal mouse

skeleton by alcian blue and alizarin red

S. Congenital Anomalies. 16(3):171–173

(1976).
43834 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(9) Igarashi, E. et al. Frequence of

spontaneous axial skeletal variations

detected by the double staining

technique for ossified and cartilaginous

skeleton in rat fetuses. Congenital

Anomalies. 32:381–391 (1992).

(10) Kimmel, C.A. et al. Skeletal

development following heat exposure in

the rat. Teratology. 47:229–242 (1993).

(11) Kimmel, C.A. and Francis, E.Z.

Proceedings of the workshop on the

acceptability and interpretation of

dermal developmental toxicity studies.

Fundamental and Applied Toxicology.

14:386–398 (1990).

(12) Kimmel, C.A. and C. Trammell. A

rapid procedure for routine double

staining of cartilage and bone in fetal

and adult animals. Stain Technology.

56:271–273 (1981).

(13) Kimmel, C.A. and Wilson, J.G.

Skeletal deviation in rats: malformations

or variations? Teratology. 8:309–316

(1973).

(14) Marr, M.C. et al. Comparison of

single and double staining for

evaluation of skeletal development: the

effects of ethylene glycol (EG) in CD

rats. Teratology. 37:476 (1988).

(15) Marr, M.C. et al. Developmental

stages of the CD (Sprague-Dawley) rat

skeleton after maternal exposure to

ethylene glycol. Teratology. 46:169–181

(1992).

(16) McLeod, M.J. Differential staining

of cartilage and bone in whole mouse

fetuses by Alcian blue and alizarin red

S. Teratology. 22:299–301 (1980).

(17) Monie, I.W. et al. Dissection

procedures for rat fetuses permitting

alizarin red staining of skeleton and

histological study of viscera.

Supplement to Teratology Workshop

Manual. pp. 163–173 (1965).

(18) Organisation for Economic Cooperation and Development, No. 414:

Teratogenicity, Guideline for Testing of

Chemicals. [C(83)44 (Final)] (1983).

(19) Salewski (Koeln), V.E.

Faerbermethode zum makroskopischen

nachweis von implantations stellen am

uterus der ratte. Naunyn-Schmeidebergs

Archiv fu ̈r Pharmakologie und

Experimentelle Pathologie. 247:367

(1964).

(20) Spark, C. and Dawson,A.B. The

order and time of appearance of centers

of ossification in the fore and hind

limbs of the albino rat, with special

reference to the possible influence of the

sex factor. American Journal of

Anatomy. 41:411–445 (1928).

(21) Staples, R.E. Detection of visceral

alterations in mammalian fetuses.

Teratology. 9(3):A37–A38 (1974).

(22) Staples, R.E. and Schnell, V.L.

Refinements in rapid clearing technique

in the KOH—alizarin red S method for

fetal bone. Stain Technology. 39:61–63

(1964).

(23) Strong, R.M. The order time and

rate of ossification of the albino rat (mus

norvegicus albinus) skeleton. American

Journal of Anatomy. 36: 313–355 (1928).

(24) Stuckhardt, J.L. and Poppe, S.M.

Fresh visceral examination of rat and

rabbit fetuses used in teratogenicity

testing. Teratogenesis, Carcinogenesis,

and Mutagenesis. 4:181–188 (1984).

(25) Van Julsingha, E.B. and

Bennett,C.G. Eds. Neubert, D., Merker,

H.J., and Kwasigroch, T.E. A dissecting

procedure for the detection of anomalies

in the rabbit foetal head. Methods in

Prenatal Toxicology (University of

Chicago, Chicago, IL, 1977) pp. 126–144

.

(26) Whitaker, J. and Dix, D.M.

Double-staining for rat foetus skeletons

in teratological studies. Laboratory

Animals. 13:309–310 (1979).

(27) Wilson, J.G. Eds. Wilson, J.G. and

Warkany, J. Embryological

considerations in teratology. Teratology:

Principles and Techniques (University

of Chicago, Chicago, IL, 1965) pp. 251–

277.

§ 799.9380 TSCA reproduction and fertility

effects.

(a) Scope. This section is intended to

meet the testing requirements under

section 4 of the TSCA. This section is

for two-generation reproduction testing

and is designed to provide general

information concerning the effects of a

test substance on the integrity and

performance of the male and female

reproductive systems, including gonadal

function, the estrous cycle, mating

behavior, conception, gestation,

parturition, lactation, and weaning, and

on the growth and development of the

offspring. The study may also provide

information about the effects of the test

substance on neonatal morbidity,

mortality, target organs in the offspring,

and preliminary data on prenatal and

postnatal developmental toxicity and

serve as a guide for subsequent tests.

Additionally, since the study design

includes in utero as well as postnatal

exposure, this study provides the

opportunity to examine the

susceptibility of the immature/neonatal

animal.

(b) Source. The source material used

in developing this TSCA test guideline

is the OPPTS harmonized test guideline

870.3800 (February 1996 Public Draft).

This source is available at the address

in paragraph (g) of this section.

(c) Good laboratory practice

standards. The study shall be conducted

in compliance with 40 CFR Part 792—

Good Laboratory Practice Standards.

(d) Principle of the test method. The

test substance is administered to

parental (P) animals prior to and during

their mating, during the resultant

pregnancies, and through the weaning

of their F1 offspring. The substance is

then administered to selected F1

offspring during their growth into

adulthood, mating, and production of an

F2 generation, until the F2 generation is

weaned.

(e) Test procedures—(1) Animal

selection—(i) Species and strain. The rat

is the most commonly used species for

testing. If another mammalian species is

used, the tester shall provide

justification/reasoning for its selection,

and appropriate modifications will be

necessary. Healthy parental animals,

which have been acclimated to

laboratory conditions for at least 5 days

and have not been subjected to previous

experimental procedures, should be

used. Strains of low fecundity shall not

be used.

(ii) Age. Parental (P) animals shall be

5 to 9 weeks old at the start of dosing.

The animals of all test groups should be

of uniform weight, age, and parity as

nearly as practicable, and should be

representative of the species and strain

under study.

(iii) Sex. (A) For an adequate

assessment of fertility, both males and

females shall be studied.

(B) The females shall be nulliparous

and nonpregnant.

(iv) Number of animals. Each control

group shall contain a sufficient number

of mating pairs to yield approximately

20 pregnant females. Each test group

shall contain a similar number of mating

pairs.

(v) Identification of animals. Each

animal shall be assigned a unique

identification number. For the P

generation, this should be done before

dosing starts. For the F1 generation, this

should be done for animals selected for

mating; in addition, records indicating

the litter of origin shall be maintained

for all selected F1 animals.

(2) Administration of test and control

substances—(i) Dose levels and dose

selection. (A) At least three-dose levels

and a concurrent control shall be used.

Healthy animals should be randomly

assigned to the control and treatment

groups, in a manner which results in

comparable mean body weight values

among all groups. The dose levels

should be spaced to produce a gradation

of toxic effects. Unless limited by the

physical/chemical nature or biological

properties of the test substance, the

highest dose should be chosen with the

aim to induce some reproductive and/or

systemic toxicity but not death or severe

suffering. In the case of parental
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43835

mortality, this should not be more than

approximately 10%. The intermediate

dose levels should produce minimal

observable toxic effects. The lowest dose

level should not produce any evidence

of either systemic or reproductive

toxicity (i.e., the no-observed-adverseeffect level, NOAEL) or should be at or

near the limit of detection for the most

sensitive endpoint. Two- or four-fold

intervals are frequently optimal for

spacing the dose levels, and the

addition of a fourth test group is often

preferable to using very large intervals

(e.g., more than a factor of 10) between

dosages.

(B) It is desirable that additional

information on metabolism and

pharmacokinetics of the test substance

be available to demonstrate the

adequacy of the dosing regimen. This

information should be available prior to

testing.

(C) The highest dose tested should not

exceed 1,000 mg/kg/day (or 20,000 ppm

in the diet), unless potential human

exposure data indicate the need for

higher doses. If a test performed at the

limit dose level, using the procedures

described for this study, produces no

observable toxicity and if an effect

would not be expected based upon data

from structurally related compounds,

then a full study using three dose levels

may not be considered necessary.

(ii) Control group. (A) A concurrent

control group shall be used. This group

shall be an untreated or sham treated

group or a vehicle-control group if a

vehicle is used in administering the test

substance.

(B) If a vehicle is used in

administering the test substance, the

control group shall receive the vehicle

in the highest volume used.

(C) If a vehicle or other additive is

used to facilitate dosing, consideration

should be given to the following

characteristics: Effects on the

absorption, distribution, metabolism, or

retention of the test substance; effects on

the chemical properties of the test

substance which may alter its toxic

characteristics; and effects on the food

or water consumption or the nutritional

status of the animals.

(D) If a test substance is administered

in the diet and causes reduced dietary

intake or utilization, the use of a pairfed control group may be considered

necessary.

(iii) Route of administration. (A) The

test substance is usually administered

by the oral route (diet, drinking water,

or gavage).

(B) If administered by gavage or

dermal application, the dosage

administered to each animal prior to

mating and during gestation and

lactation shall be based on the

individual animal body weight and

adjusted weekly at a minimum.

(C) If another route of administration

is used, for example, when the route of

administration is based upon the

principal route of potential human

exposure, the tester should provide

justification and reasoning for its

selection, and appropriate modifications

may be necessary. Care should be taken

to minimize stress on the maternal

animals and their litters during

gestation and lactation.

(D) All animals should be dosed by

the same method during the appropriate

experimental period.

(iv) Dosing schedule. (A) The animals

should be dosed with the test substance

on a 7–days–a–week basis.

(B) Daily dosing of the parental (P)

males and females shall begin when

they are 5 to 9 weeks old. Daily dosing

of the F1 males and females shall begin

at weaning. For both sexes (P and F1),

dosing shall be continued for at least 10

weeks before the mating period.

(C) Daily dosing of the P and F1 males

and females shall continue until

termination.

(3) Mating procedure—(i) Parental.

(A) For each mating, each female shall

be placed with a single randomly

selected male from the same dose level

(1:1 mating) until evidence of

copulation is observed or either 3

estrous periods or 2 weeks has elapsed.

Animals should be separated as soon as

possible after evidence of copulation is

observed. If mating has not occurred

after 2 weeks or 3 estrous periods, the

animals should be separated without

further opportunity for mating. Mating

pairs should be clearly identified in the

data.

(B) Vaginal smears shall be collected

daily and examined for all females

during mating, until evidence of

copulation is observed.

(C) Each day, the females shall be

examined for presence of sperm or

vaginal plugs. Day 0 of pregnancy is

defined as the day a vaginal plug or

sperm are found.

(ii) F1 mating. For mating the F1

offspring, at least one male and one

female should be randomly selected

from each litter for mating with another

pup of the same dose level but different

litter, to produce the F2 generation.

(iii) Second mating. In certain

instances, such as poor reproductive

performance in the controls, or in the

event of treatment-related alterations in

litter size, the adults may be remated to

produce an F1b or F2b litter. If

production of a second litter is deemed

necessary in either generation, the dams

should be remated approximately 1–2

weeks following weaning of the last F1a

or F2a litter.

(iv) Special housing. After evidence of

copulation, animals that are presumed

to be pregnant shall be caged separately

in delivery or maternity cages. Pregnant

animals shall be provided with nesting

materials when parturition is near.

(v) Standardization of litter sizes. (A)

Animals should be allowed to litter

normally and rear their offspring to

weaning. Standardization of litter sizes

is optional.

(B) If standardization is performed,

the following procedure should be used.

On day 4 after birth, the size of each

litter may be adjusted by eliminating

extra pups by random selection to yield,

as nearly as possible, four males and

four females per litter or five males and

five females per litter. Selective

elimination of pups, i.e. based upon

body weight, is not appropriate.

Whenever the number of male or female

pups prevents having four (or five) of

each sex per litter, partial adjustment

(for example, five males and three

females, or four males and six females)

is acceptable. Adjustments are not

appropriate for litters of eight pups or

less.

(4) Observation of animals—(i)

Parental. (A) Throughout the test

period, each animal shall be observed at

least once daily, considering the peak

period of anticipated effects after

dosing. Mortality, moribundity,

pertinent behavioral changes, signs of

difficult or prolonged parturition, and

all signs of overt toxicity shall be

recorded at this cageside examination.

In addition, thorough physical

examinations should be conducted

weekly on each animal.

(B) Parental animals (P and F1) shall

be weighed on the first day of dosing

and weekly thereafter. Parental females

(P and F1) should be weighed at a

minimum on approximately gestation

days 0, 7, 14, and 21, and during

lactation on the same days as the

weighing of litters.

(C) During the premating and

gestation periods, food consumption

shall be measured weekly at a

minimum. Water consumption should

be measured weekly at a minimum if

the test substance is administered in the

water.

(D) Estrous cycle length and normality

should be evaluated by vaginal smears

for all P and F1 females during a

minimum of 3 weeks prior to mating

and throughout cohabitation; care

should be taken to prevent the

induction of pseudopregnancy.

(E) For all P and F1 males at

termination, sperm from one testis and

one epididymis shall be collected for
43836 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

enumeration of homogenizationresistant spermatids and cauda

epididymal sperm reserves,

respectively. In addition, sperm from

the cauda epididymis (or vas deferens)

should be collected for evaluation of

sperm motility and sperm morphology.

(1) The total number of

homogenization-resistant testicular

sperm and cauda epididymal sperm

should be enumerated. The method

described in the reference under

paragraph (g)(8) of this section may be

used. Cauda sperm reserves can be

derived from the concentration and

volume of sperm in the suspension used

to complete the qualitative evaluations,

and the number of sperm recovered by

subsequent mincing and/or

homogenizing of the remaining cauda

tissue. Enumeration in only control and

high-dose P and F1 males may be

performed unless treatment-related

effects are observed; in that case, the

lower dose groups should also be

evaluated.

(2) An evaluation of epididymal (or

vas deferens) sperm motility should be

performed. Sperm should be recovered

while minimizing damage (the

evaluation techniques as described in

the reference under paragraph (g)(8) of

this section may be used), and the

percentage of progressively motile

sperm should be determined either

subjectively or objectively. For objective

evaluations, an acceptable counting

chamber of sufficient depth can be used

to effectively combine the assessment of

motility with sperm count and sperm

morphology. When computer-assisted

motion analysis is performed, the

derivation of progressive motility relies

on user-defined thresholds for average

path velocity and straightness or linear

index. If samples are videotaped, or

images otherwise recorded, at the time

of necropsy, subsequent analysis of only

control and high-dose P and F1 males

may be performed unless treatmentrelated effects are observed; in that case,

the lower dose groups should also be

evaluated. In the absence of a video or

digital image, all samples in all

treatment groups should be analyzed at

necropsy.

(3) A morphological evaluation of an

epididymal (or vas deferens) sperm

sample shall be performed. Sperm (at

least 200 per sample) should be

examined as fixed, wet preparations (the

techniques for such examinations is

described in the references under

paragraphs (g)(4) and (g)(8) of this

section may be used) and classified as

either normal (both head and midpiece/

tail appear normal) or abnormal.

Examples of morphologic sperm

abnormalities would include fusion,

isolated heads, and misshapen heads

and/or tails. Evaluation of only control

and high-dose P and F1 males may be

performed unless treatment-related

effects are observed; in that case, the

lower dose groups should also be

evaluated.

(ii) Offspring. (A) Each litter should be

examined as soon as possible after

delivery (lactation day 0) to establish

the number and sex of pups, stillbirths,

live births, and the presence of gross

anomalies. Pups found dead on day 0

should be examined for possible defects

and cause of death.

(B) Live pups should be counted,

sexed, and weighed individually at

birth, or soon thereafter, at least on days

4, 7, 14, and 21 of lactation, at the time

of vaginal patency or balanopreputial

separation, and at termination.

(C) The age of vaginal opening and

preputial separation should be

determined for F1 weanlings selected

for mating. If there is a treatment-related

effect in F1 sex ratio or sexual

maturation, anogenital distance should

be measured on day 0 for all F2 pups.

(5) Termination schedule. (i) All P

and F1 adult males and females should

be terminated when they are no longer

needed for assessment of reproductive

effects.

(ii) F1 offspring not selected for

mating and all F2 offspring should be

terminated at comparable ages after

weaning.

(6) Gross necropsy. (i) At the time of

termination or death during the study,

all parental animals (P and F1) and

when litter size permits at least three

pups per sex per litter from the

unselected F1 weanlings and the F2

weanlings shall be examined

macroscopically for any structural

abnormalities or pathological changes.

Special attention shall be paid to the

organs of the reproductive system.

(ii) Dead pups or pups that are

terminated in a moribund condition

should be examined for possible defects

and/or cause of death.

(iii) At the time of necropsy, a vaginal

smear should be examined to determine

the stage of the estrous cycle. The uteri

of all cohabited females should be

examined, in a manner which does not

compromise histopathological

evaluation, for the presence and number

of implantation sites.

(7) Organ weights. (i) At the time of

termination, the following organs of all

P and F1 parental animals shall be

weighed:

(A) Uterus (with oviducts and cervix),

ovaries.

(B) Testes, epididymides (total

weights for both and cauda weight for

either one or both), seminal vesicles

(with coagulating glands and their

fluids), and prostate.

(C) Brain, pituitary, liver, kidneys,

adrenal glands, spleen, and known

target organs.

(ii) For F1 and F2 weanlings that are

examined macroscopically, the

following organs shall be weighed for

one randomly selected pup per sex per

litter.

(A) Brain.

(B) Spleen and thymus.

(8) Tissue preservation. The following

organs and tissues, or representative

samples thereof, shall be fixed and

stored in a suitable medium for

histopathological examination.

(i) For the parental (P and F1)

animals:

(A) Vagina, uterus with oviducts,

cervix, and ovaries.

(B) One testis (preserved in Bouins

fixative or comparable preservative),

one epididymis, seminal vesicles,

prostate, and coagulating gland.

(C) Pituitary and adrenal glands.

(D) Target organs, when previously

identified, from all P and F1 animals

selected for mating.

(E) Grossly abnormal tissue.

(ii) For F1 and F2 weanlings selected

for macroscopic examination: Grossly

abnormal tissue and target organs, when

known.

(9) Histopathology—(i) Parental

animals. Full histopathology of the

organs listed under paragraph (e)(8)(i) of

this section shall be performed for ten

randomly chosen high dose and control

P and F1 animals per sex, for those

animals that were selected for mating.

Organs demonstrating treatment-related

changes shall also be examined for the

remainder of the high-dose and control

animals and for all parental animals in

the low- and mid-dose groups.

Additionally, reproductive organs of the

low- and mid-dose animals suspected of

reduced fertility, e.g., those that failed to

mate, conceive, sire, or deliver healthy

offspring, or for which estrous cyclicity

or sperm number, motility, or

morphology were affected, shall be

subjected to histopathological

evaluation. Besides gross lesions such as

atrophy or tumors, testicular

histopathological examination should

be conducted in order to to identify

treatment-related effects such as

retained spermatids, missing germ cell

layers or types, multinucleated giant

cells, or sloughing of spermatogenic

cells into the lumen. Examination of the

intact epididymis should include the

caput, corpus, and cauda, which can be

accomplished by evaluation of a

longitudinal section, and should be

conducted in order to identify such

lesions as sperm granulomas, leukocytic
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43837

infiltration (inflammation), aberrant cell

types within the lumen, or the absence

of clear cells in the cauda epididymal

epithelium. The postlactational ovary

should contain primordial and growing

follicles as well as the large corpora

lutea of lactation. Histopathological

examination should detect qualitative

depletion of the primordial follicle

population. A quantitative evaluation of

primordial follicles should be

conducted for all F1 females if any of

the following treatment-related findings

were observed:

(A) Reductions in ovarian weight and

abnormal ovarian histopathology

findings, e.g., follicular cysts or

qualitative evidence of a reduced

population of primordial follicles.

(B) Abnormal estrous cyclicity and

female infertility.

(C) Depletion of testicular spermatid

counts in F1 males and evidence of

germ cell depletion in testicular

histopathology evaluations.

(ii) Examination of ovarian sections. If

a quantitative evaluation is performed,

ten ovarian sections shall be taken at

least 100 μm apart from the inner third

of each ovary. Examination should

include enumeration of the total number

of primordial and antral follicles from

these 20 sections (the technique for this

histological assessment as described in

the reference under paragraph (g)(2) of

this section may be used) for

comparison with control ovaries.

(iii) Weanlings. For F1 and F2

weanlings, histopathological

examination of treatment-related

abnormalities noted at macroscopic

examination should be considered, if

such evaluation were deemed

appropriate and would contribute to the

interpretation of the study data.

(f) Data and reporting—(1) Treatment

of results. Data shall be reported

individually and summarized in tabular

form, showing for each test group the

types of change and the number of

animals displaying each type of change.

(2) Evaluation of study results. (i) An

evaluation of test results, including the

statistical analysis, shall be provided.

This should include an evaluation of the

relationship, or lack thereof, between

the exposure of the animals to the test

substance and the incidence and

severity of all abnormalities.

(ii) When appropriate, historical

control data should be used to enhance

interpretation of study results.

Historical data, when used, should be

compiled, presented, and analyzed in an

appropriate and relevant manner. In

order to justify its use as an analytical

tool, information such as the dates of

study conduct, the strain and source of

the animals, and the vehicle and route

of administration should be included.

(iii) Statistical analysis of the study

findings should include sufficient

information on the method of analysis,

so that an independent reviewer/

statistician can reevaluate and

reconstruct the analysis.

(iv) 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) Test report. In addition to the

reporting requirements as specified

under 40 CFR part 792, subpart J, the

following specific information shall be

reported. Both individual and summary

data should be presented.

(i) Species and strain.

(ii) Toxic response data by sex and

dose, including indices of mating,

fertility, gestation, birth, viability, and

lactation; offspring sex ratio; precoital

interval, including the number of days

until mating and the number of estrous

periods until mating; and duration of

gestation calculated from day 0 of

pregnancy. The report should provide

the numbers used in calculating all

indices.

(iii) Day (week) of death during the

study or whether animals survived to

termination; date (age) of litter

termination.

(iv) Toxic or other effects on

reproduction, offspring, or postnatal

growth.

(v) Developmental milestone data

(mean age of vaginal opening and

preputial separation, and mean

anogenital distance, when measured).

(vi) Number of P and F1 females

cycling normally and mean estrous

cycle length.

(vii) Day (week) of observation of each

abnormal sign and its subsequent

course.

(viii) Body weight and body weight

change data by sex for P, F1, and F2

animals.

(ix) Food (and water, if applicable)

consumption, food efficiency (body

weight gain per gram of food

consumed), and test material

consumption for P and F1 animals,

except for the period of cohabitation.

(x) Total cauda epididymal sperm

number, homogenization-resistant testis

spermatid number, number and percent

of progressively motile sperm, number

and percent of morphologically normal

sperm, and number and percent of

sperm with each identified anomaly.

(xi) Stage of the estrous cycle at the

time of termination for P and F1

parental females.

(xii) Necropsy findings.

(xiii) Implantation data and

postimplantation loss calculations for P

and F1 parental females.

(xiv) Absolute and adjusted organ

weight data.

(xv) Detailed description of all

histopathological findings.

(xvi) Adequate statistical treatment of

results.

(xvii) A copy of the study protocol

and any amendments should be

included.

(g) References. For additional

backgound information on this test

guideline, the following references

should be consulted. These references

are available for inspection at the TSCA

Nonconfidential Information Center,

Rm. NE–B607, Environmental

Protection Agency, 401 M St., SW.,

Washington, DC, 12 noon to 4 p.m.,

Monday through Friday, except legal

holidays.

(1) Gray, L.E. et al. A dose-response

analysis of methoxychlor-induced

alterations of reproductive development

and function in the rat. Fundamental

and Applied Toxicology. 12:92–108

(1989).

(2) Heindel, J.J. et al. Ed. Hirshfield,

A.N. Histological assessment of ovarian

follicle number in mice as a screen of

ovarian toxicity. Growth Factors and the

Ovary (Plenum, NY, 1989) pp. 421–426.

(3) Korenbrot, C.C. et al. Preputial

separation as an external sign of

pubertal development in the male rat.

Biology of Reproduction. 17:298–303

(1977).

(4) Linder, R.E. et al. Endpoints of

spermatoxicity in the rat after short

duration exposures to fourteen

reproductive toxicants. Reproductive

Toxicology. 6:491–505 (1992).

(5) Manson, J.M. and Kang, Y.J. Ed.

Hayes, A.W. Test methods for assessing

female reproductive and developmental

toxicology. Principles and Methods of

Toxicology (Raven, NY, 1989).

(6) Organisation for Economic Cooperation and Development, No. 416:

Two Generation Reproduction Toxicity

Study, Guidelines for Testing of

Chemicals. [C(83)44 (Final)] (1983).

(7) Pederson, T. and Peters, H.

Proposal for classification of oocytes

and follicles in the mouse ovary. Journal

of Reproduction and Fertility. 17:555–

557 (1988).

(8) Seed, J., Chapin, R.E. E.D. Clegg,

L.A. Dostal, R.H. Foote, M.E. Hurtt, G.R.

Klinefelter, S.L. Makris, S.D. Perreault,

S. Schrader, D. Seyler, R. Sprando, K.A.

Treinen, D.N.R. Veeramachaneni, and

Wise, L.D. Methods for assessing sperm

motility, morphology, and counts in the

rat, rabbit, and dog: a consensus report.

Reproductive Toxicology. 10(3):237–244

(1996).
43838 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(9) Smith, B.J. et al. Comparison of

random and serial sections in

assessment of ovarian toxicity.

Reproductive Toxicology. 5:379–383

(1991).

(10) Thomas, J.A. Eds. M.O. Amdur, J.

Doull, and C.D. Klaassen. Toxic

responses of the reproductive system.

Casarett and Doull’s Toxicology

(Pergamon, NY, 1991).

(11) Working, P.K. and Hurtt, M.

Computerized videomicrographic

analysis of rat sperm motility. Journal of

Andrology. 8:330–337 (1987).

(12) Zenick, H. et al. Ed. Hayes, A.W.

Assessment of male reproductive

toxicity: a risk assessment approach.

Principles and Methods of Toxicology

(Raven, NY, 1994).

§ 799.9420 TSCA carcinogenicity.

(a) Scope. This section is intended to

meet the testing requirements under

section 4 of TSCA. The objective of a

long-term carcinogenicity study is to

observe test animals for a major portion

of their life span for development of

neoplastic lesions during or after

exposure to various doses of a test

substance by an appropriate route of

administration.

(b) Source. The source material used

in developing this TSCA test guideline

is the OPPTS harmonized test guideline

870.4200 (June 1996 Public Draft). This

source is available at the address in

paragraph (g) of this section.

(c) Definitions. The following

definitions apply to this section.

Carcinogenicity is the development of

neoplastic lesions as a result of the

repeated daily exposure of experimental

animals to a chemical by the oral,

dermal, or inhalation routes of

exposure.

Cumulative toxicity is the adverse

effects of repeated dose occurring as a

result of prolonged action on, or

increased concentration of, the

administered test substance or its

metabolites in susceptible tissues.

Dose in a carcinogenicity study is the

amount of test substance administered

via the oral, dermal or inhalation routes

for a period of up to 24 months. Dose

is expressed as weight of the test

substance (grams, milligrams) per unit

body weight of test animal (milligram

per kilogram), or as weight of the test

substance in parts per million (ppm) in

food or drinking water. When exposed

via inhalation, dose is expressed as

weight of the test substance per unit

volume of air (milligrams per liter) or as

parts per million.

Target organ is any organ of a test

animal showing evidence of an effect

induced by a test substance.

(d) Test procedures—(1) Animal

selection—(i) Species and strain.

Testing shall be performed on two

mammalian species. Rats and mice are

the species of choice because of their

relatively short life spans, limited cost

of maintenance, widespread use in

pharmacological and toxicological

studies, susceptibility to tumor

induction, and the availability of inbred

or sufficiently characterized strains.

Commonly used laboratory strains shall

be used. If other mammalian species are

used, the tester shall provide

justification/reasoning for their

selection.

(ii) Age/weight. (A) Testing shall be

started with young healthy animals as

soon as possible after weaning and

acclimatization.

(B) Dosing should generally begin no

later than 8 weeks of age.

(C) At commencement of the study,

the weight variation of animals used

shall not exceed ± 20% of the mean

weight for each sex.

(D) Studies using prenatal or neonatal

animals may be recommended under

special conditions.

(iii) Sex. (A) Equal numbers of

animals of each sex shall be used at

each dose level.

(B) Females shall be nulliparous and

nonpregnant.

(iv) Numbers. (A) At least 100 rodents

(50 males and 50 females) shall be used

at each dose level and concurrent

control group.

(B) If interim sacrifices are planned,

the number shall be increased by the

number of animals scheduled to be

sacrificed during the course of the

study.

(C) For a meaningful and valid

statistical evaluation of long term

exposure and for a valid interpretation

of negative results, the number of

animals in any group should not fall

below 50% at 15 months in mice and 18

months in rats. Survival in any group

should not fall below 25% at 18 months

in mice and 24 months in rats.

(D) The use of adequate

randomization procedures for the

proper allocation of animals to test and

control groups is required to avoid bias.

(E) Each animal shall be assigned a

unique identification number. Dead

animals, their preserved organs and

tissues, and microscopic slides shall be

identified by reference to the unique

numbers assigned.

(v) Husbandry. (A) Animals may be

group-caged by sex, but the number of

animals per cage must not interfere with

clear observation of each animal. The

biological properties of the test

substance or toxic effects (e.g.,

morbidity, excitability) may indicate a

need for individual caging. Animals

should be housed individually in

dermal studies and during exposure in

inhalation studies.

(B) The temperature of the

experimental animal rooms should be at

22 ± 3 °C.

(C) The relative humidity of the

experimental animal rooms should be

30 to 70%.

(D) Where lighting is artificial, the

sequence should be 12 h light/12 h dark.

(E) Control and test animals should be

fed from the same batch and lot. The

feed should be analyzed to assure

uniform distribution and adequacy of

nutritional requirements of the species

tested and for impurities that might

influence the outcome of the test.

Animals should be fed and watered ad

libitum with food replaced at least

weekly.

(F) The study should not be initiated

until animals have been allowed a

period of acclimatization/quarantine to

environmental conditions, nor should

animals from outside sources be placed

on test without an adequate period of

quarantine.

(2) Control and test substances. (i)

Where necessary, the test substance is

dissolved or suspended in a suitable

vehicle. If a vehicle or diluent is

needed, it should not elicit toxic effects

itself. It is recommended that wherever

possible the use of an aqueous solution

be considered first, followed by

consideration of solution in oil, and

finally solution in other vehicles.

(ii) One lot of the test substance

should be used, if possible, throughout

the duration of the study, and the

research sample should be stored under

conditions that maintain its purity and

stability. Prior to the initiation of the

study, there should be a characterization

of the test substance, including the

purity of the test compound, and, if

possible, the name and quantities of

contaminants and impurities.

(iii) If the test or control substance is

to be incorporated into feed or another

vehicle, the period during which the

test substance is stable in such a

mixture should be determined prior to

the initiation of the study. Its

homogeneity and concentration should

be determined prior to the initiation of

the study and periodically during the

study. Statistically randomized samples

of the mixture should be analyzed to

ensure that proper mixing, formulation,

and storage procedures are being

followed, and that the appropriate

concentration of the test or control

substance is contained in the mixture.

(3) Control groups. A concurrent

control group (50 males and 50 females)

is required. This group shall be
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43839

untreated or if a vehicle is used in

administering the test substance, a

vehicle control group. If the toxic

properties of the vehicle are not known,

both untreated and vehicle control

groups are required.

(4) Dose levels and dose selection. (i)

For risk assessment purposes, at least

three dose levels shall be used, in

addition to the concurrent control

group. Dose levels should be spaced to

produce a gradation of effects. A

rationale for the doses selected must be

provided.

(ii) The highest dose level should

elicit signs of toxicity without

substantially altering the normal life

span due to effects other than tumors.

The highest dose should be determined

based on the findings from a 90–day

study to ensure that the dose used is

adequate to asses the carcinogenic

potential of the test substance. Thus, the

selection of the highest dose to be tested

is dependent upon changes observed in

several toxicological parameters in

subchronic studies. The highest dose

tested need not exceed 1,000 mg/kg/day.

(iii) The intermediate-dose level

should be spaced to produce a gradation

of toxic effects.

(iv) The lowest dose level should

produce no evidence of toxicity.

(v) For skin carcinogenicity studies,

when toxicity to the skin is a

determining factor, the highest dose

selected should not destroy the

functional integrity of the skin, the

intermediate dose should be a

minimally irritating dose, and the low

dose should be the highest nonirritating

dose.

(vi) The criteria for selecting the dose

levels for skin carcinogenicity studies,

based on gross and histopathologic

dermal lesions, are as follows:

(A) Gross criteria for reaching the high

dose:

(1) Erythema (moderate).

(2) Scaling.

(3) Edema (mild).

(4) Alopecia.

(5) Thickening.

(B) Histologic criteria for reaching the

high dose:

(1) Epidermal hyperplasia.

(2) Epidermal hyperkeratosis.

(3) Epidermal parakeratosis.

(4) Adnexal atrophy/hyperplasia.

(5) Fibrosis.

(6) Spongiosis (minimal-mild).

(7) Epidermal edema (minimal-mild).

(8) Dermal edema (minimalmoderate).

(9) Inflammation (moderate).

(C) Gross criteria for exceeding the

high dose:

(1) Ulcers, fissures.

(2) Exudate/crust (eschar).

(3) nonviable (dead) tissues.

(4) Anything leading to destruction of

the functional integrity of the epidermis

(e.g., caking, fissuring, open sores,

eschar).

(D) Histologic criteria for exceeding

the high dose:

(1) Crust (interfollicular and

follicular).

(2) Microulcer.

(3) Degeneration/necrosis (mild to

moderate).

(4) Epidermal edema (moderate to

marked).

(5) Dermal edema (marked).

(6) Inflammation (marked).

(5) Administration of the test

substance. The three main routes of

administration are oral, dermal, and

inhalation. The choice of the route of

administration depends upon the

physical and chemical characteristics of

the test substance and the form

typifying exposure in humans.

(i) Oral studies. If the test substance

is administered by gavage, the animals

are dosed with the test substance on a

7–day per week basis for a period of at

least 18 months for mice and hamsters

and 24 months for rats. However, based

primarily on practical considerations,

dosing by gavage or via a capsule on a

5–day per week basis is acceptable. If

the test substance is administered in the

drinking water or mixed in the diet,

then exposure should be on a 7–day per

week basis.

(ii) Dermal studies. (A) The animals

should be treated with the test

substance for at least 6 h/day on a 7–day

per week basis for a period of at least

18 months for mice and hamsters and 24

months for rats. However, based

primarily on practical considerations,

application on a 5–day per week basis

is acceptable. Dosing should be

conducted at approximately the same

time each day.

(B) Fur should be clipped weekly

from the dorsal area of the trunk of the

test animals. Care should be taken to

avoid abrading the skin which could

alter its permeability. A minimum of 24

hrs should be allowed for the skin to

recover before the next dosing of the

animal.

(C) The test substance shall be applied

uniformly over a shaved area which is

approximately 10% of the total body

surface area. In order to dose

approximately 10% of the body surface,

the area starting at the scapulae

(shoulders) to the wing of the ileum

(hipbone) and half way down the flank

on each side of the animal should be

shaved. The volume of application

should be kept constant and should not

exceed 100 μL for the mouse and 300 μL

for the rat; different concentrations of

the test solution should be prepared for

different dose levels. With highly toxic

substances, the surface area covered

may be less, but as much of the area as

possible should be covered with as thin

and uniform a film as practical. The test

material is not removed after

application.

(D) During the exposure period, the

application site should not be covered

when mice or hamsters are the species

of choice. For rats, the test substance

may be held in contact with the skin

with a porous gauze dressing and

nonirritating tape if necessary. The test

site should be further covered in a

suitable manner to retain the gauze

dressing and test substance and ensure

that the animals cannot ingest the test

substance.

(iii) Inhalation studies. (A) The

animals should be exposed to the test

substance for 6 h/day on a 7–day per

week basis, for a period of at least 18

months in mice and 24 months in rats.

However, based primarily on practical

considerations, exposure for 6 h/day on

a 5–day per week basis is acceptable.

(B) The animals shall be tested in

dynamic inhalation equipment designed

to sustain a minimum air flow of 10 air

changes per hr, an adequate oxygen

content of at least 19%, and uniform

conditions throughout the exposure

chamber. Maintenance of slight negative

pressure inside the chamber will

prevent leakage of the test substance

into surrounding areas.

(C) The selection of a dynamic

inhalation chamber should be

appropriate for the test substance and

test system. Where a whole body

chamber is used to expose animals to an

aerosol, individual housing must be

used to minimize crowding of the test

animals and maximize their exposure to

the test substance. To ensure stability of

a chamber atmosphere, the total volume

occupied by the test animals shall not

exceed 5% of the volume of the test

chamber. It is recommended, but not

required, that nose-only or head-only

exposure be used for aerosol studies in

order to minimize oral exposures due to

animals licking compound off their fur.

Heat stress to the animals should be

minimized.

(D) The temperature at which the test

is performed should be maintained at

22 ± 2 °C. The relative humidity should

be maintained between 40 to 60%, but

in certain instances (e.g., tests of

aerosols, use of water vehicle) this may

not be practicable.

(E) The rate of air flow shall be

monitored continuously but recorded at

least three times during exposure.
43840 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(F) Temperature and humidity shall

be monitored continuously but should

be recorded at least every 30 minutes.

(G) The actual concentrations of the

test substance shall be measured in the

breathing zone. During the exposure

period, the actual concentrations of the

test substance should be held as

constant as practicable, monitored

continuously or intermittently

depending on the method of analysis.

Chamber concentrations may be

measured using gravimetric or

analytical methods as appropriate. If

trial run measurements are reasonably

consistent (± 10% for liquid aerosol, gas,

or dry aerosol), the two measurements

should be sufficient. If measurements

are not consistent, then three to four

measurements should be taken.

(H) During the development of the

generating system, particle size analysis

shall be performed to establish the

stability of aerosol concentrations with

respect to particle size. Measurement of

aerodynamic particle size in the

animals’s breathing zone should be

measured during a trial run. If median

aerodynamic diameter (MMAD) values

for each exposure level are within 10%

of each other, then two measurements

during the exposures should be

sufficient. If pretest measurements are

not within 10% of each other, three to

four measurements should be taken. The

MMAD particle size range should be

between 1–3 μm. The particle size of

hygroscopic materials should be small

enough to allow pulmonary deposition

once the particles swell in the moist

environment of the respiratory tract.

(I) Feed shall be withheld during

exposure. Water may also be withheld

during exposure.

(6) Observation period. It is necessary

that the duration of the carcinogenicity

study comprise the majority of the

normal life span of the strain of animals

used. This time period shall not be less

than 24 months for rats and 18 months

for mice, and ordinarily not longer than

30 months for rats and 24 months for

mice. For longer time periods, and

where any other species are used,

consultation with the Agency in regard

to the duration of the study is advised.

(7) Observation of animals. (i)

Observations shall be made at least once

each day for morbidity and mortality.

Appropriate actions should be taken to

minimize loss of animals from the study

(e.g., necropsy or refrigeration of those

animals found dead and isolation or

sacrifice of weak or moribund animals).

(ii) A careful clinical examination

shall be made at least once weekly.

Observations should be detailed and

carefully recorded, preferably using

explicitly defined scales. Observations

should include, but not be limited to,

evaluation of skin and fur, eyes and

mucous membranes, respiratory and

circulatory effects, autonomic effects

such as salivation, central nervous

system effects, including tremors and

convulsions, changes in the level of

activity, gait and posture, reactivity to

handling or sensory stimuli, altered

strength and stereotypes or bizarre

behavior (e.g., self-mutilation, walking

backwards).

(iii) Body weights shall be recorded

individually for all animals; once a

week during the first 13 weeks of the

study and at least once every 4 weeks,

thereafter, unless signs of clinical

toxicity suggest more frequent weighing

to facilitate monitoring of health status.

(iv) When the test substance is

administered in the feed or drinking

water, measurements of feed or water

consumption, respectively, should be

determined weekly during the first 13

weeks of the study and then at

approximately monthly intervals unless

health status or body weight changes

dictate otherwise.

(v) Moribund animals shall be

removed and sacrificed when noticed

and the time of death should be

recorded as precisely as possible. At the

end of the study period, all survivors

shall be sacrificed.

(8) Clinical pathology. At 12 months,

18 months, and at terminal sacrifice, a

blood smear shall be obtained from all

animals. A differential blood count

should be performed on blood smears

from those animals in the highest

dosage group and the controls from the

terminal sacrifice. If these data, or data

from the pathological examination

indicate a need, then the 12– and 18–

month blood smears should also be

examined. Differential blood counts

should be performed for the next lower

groups if there is a major discrepancy

between the highest group and the

controls. If clinical observations suggest

a deterioration in health of the animals

during the study, a differential blood

count of the affected animals shall be

performed.

(9) Gross necropsy. (i) A complete

gross examination shall be performed on

all animals, including those that died

during the experiment or were killed in

a moribund condition.

(ii) The liver, lungs, kidneys, brain,

and gonads should be trimmed and

weighed wet as soon as possible after

dissection to avoid drying. The organs

should be weighed from interim

sacrifice animals as well as from at least

10 animals per sex per group at terminal

sacrifice.

(iii) The following organs and tissues,

or representative samples thereof, shall

be preserved in a suitable medium for

possible future histopathological

examination.

(A) Digestive system.

(1) Salivary glands.

(2) Esophagus.

(3) Stomach.

(4) Duodenum.

(5) Jejunum.

(6) Ileum.

(7) Cecum.

(8) Colon.

(9) Rectum.

(10) Liver.

(11) Pancreas.

(12) Gallbladder (mice).

(13) Bile duct (rat).

(B) Nervous system.

(1) Brain (multiple sections).

(2) Pituitary.

(3) Peripheral nerves.

(4) Spinal cord (three levels).

(5) Eyes (retina, optic nerve).

(C) Glandular system.

(1) Adrenals.

(2) Parathyroids.

(3) Thyroids.

(D) Respiratory system.

(1) Trachea.

(2) Lung.

(3) Pharynx.

(4) Larynx.

(5) Nose (inhalation studies only).

(E) Cardiovascular/hematopoietic

system.

(1) Aorta (thoracic).

(2) Heart.

(3) Bone marrow.

(4) Lymph nodes.

(5) Spleen.

(6) Thymus.

(F) Urogenital system.

(1) Kidneys.

(2) Urinary bladder.

(3) Prostate.

(4) Testes/epididymides.

(5) Seminal vesicles.

(6) Uterus.

(7) Ovaries.

(G) Other.

(1) Lacrimal gland.

(2) Mammary gland.

(3) Skin.

(4) Skeletal muscle.

(5) All gross lesions and masses.

(6) Sternum and/or femur.

(iv) In inhalation studies, the entire

respiratory tract, including nose,

pharynx, larynx, and paranasal sinuses

should be examined and preserved. In

dermal studies, skin from treated and

adjacent control skin sites should be

examined and preserved.

(v) Inflation of lungs and urinary

bladder with a fixative is the optimal

method for preservation of these tissues.

The proper inflation and fixation of the

lungs in inhalation studies is essential

for appropriate and valid

histopathological examination.
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43841

(vi) Information from clinical

pathology, and other in-life data should

be considered before microscopic

examination, since they may provide

significant guidance to the pathologist.

(10) Histopathology. (i) The following

histopathology shall be performed:

(A) Full histopathology on the organs

and tissues under paragraph (d)(9) (iii)

of this section of all animals in the

control and high dose groups and all

animals that died or were killed during

the study.

(B) All gross lesions in all animals.

(C) Target organs in all animals.

(D) Lungs, liver, and kidneys of all

animals. Special attention to

examination of the lungs of rodents

should be made for evidence of

infection since this provides an

assessment of the state of health of the

animals.

(ii) If the results show substantial

alteration of the animal’s normal life

span, the induction of effects that might

affect a neoplastic response, or other

effects that might compromise the

significance of the data, the next lower

dose levels shall be examined as

described under paragraph (d)(11)(i) of

this section.

(iii) An attempt should be made to

correlate gross observations with

microscopic findings.

(iv) Tissues and organs designated for

microscopic examination should be

fixed in 10% buffered formalin or a

recognized suitable fixative as soon as

necropsy is performed and no less than

48 hrs prior to trimming. Tissues should

be trimmed to a maximum thickness of

0.4 cm for processing.

(e) Data and reporting—(1) Treatment

of results. (i) Data shall be summarized

in tabular form, showing for each test

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.

(ii) All observed results (quantitative

and qualitative) shall be evaluated by an

appropriate statistical method. Any

generally accepted statistical methods

may be used; the statistical methods

including significance criteria shall be

selected during the design of the study.

(2) Evaluation of study results. (i) The

findings of a carcinogenicity study

should be evaluated in conjunction with

the findings of previous studies and

considered in terms of the toxic effects,

the necropsy and histopathological

findings. The evaluation shall include

the relationship between the dose of the

test substance and the presence,

incidence, and severity of abnormalities

(including behavioral and clinical

abnormalities), gross lesions, identified

target organs, body weight changes,

effects on mortality, and any other

general or specific toxic effects.

(ii) In any study which demonstrates

an absence of toxic effects, further

investigation to establish absorption and

bioavailablity of the test substance

should be considered.

(iii) In order for a negative test to be

acceptable, it must meet the following

criteria: No more than 10% of any group

is lost due to autolysis, cannibalism, or

management problems; and survival in

each group is no less than 50% at 15

months for mice and 18 months for rats.

Survival should not fall below 25% at

18 months for mice and 24 months for

rats.

(iv) The use of historical control data

from an appropriate time period from

the same testing laboratory (i.e., the

incidence of tumors and other suspect

lesions normally occurring under the

same laboratory conditions and in the

same strain of animals employed in the

test) is helpful for assessing the

significance of changes observed in the

current study.

(3) Test report. (i) In addition to the

reporting requirements as specified

under 40 CFR part 792, subpart J, the

following specific information shall be

reported. Both individual and summary

data should be presented.

(A) Test substance characterization

should include:

(1) Chemical identification.

(2) Lot or batch number.

(3) Physical properties.

(4) Purity/impurities.

(5) Identification and composition of

any vehicle used.

(B) Test system should contain data

on:

(1) Species and strain of animals used

and rationale for selection if other than

that recommended.

(2) Age including body weight data

and sex

(3) Test environment including cage

conditions, ambient temperature,

humidity, and light/dark periods.

(C) Test procedure should include the

following data:

(1) Method of randomization used.

(2) Full description of experimental

design and procedure.

(3) Dose regimen including levels,

methods, and volume.

(4) Test results—(i) Group animal

data. Tabulation of toxic response data

by species, strain, sex, and exposure

level for:

(A) Number of animals exposed.

(B) Number of animals showing signs

of toxicity.

(C) Number of animals dying.

(ii) Individual animal data. Data

should be presented as summary (group

mean) as well as for individual animals.

(A) Time of death during the study or

whether animals survived to

termination.

(B) Time of observation of each

abnormal sign and its subsequent

course.

(C) Body weight data.

(D) Feed and water consumption data,

when collected.

(E) Results of clinical pathology and

immunotoxicity screen when

performed.

(F) Necropsy findings including

absolute/relative organ weight data.

(G) Detailed description of all

histopathological findings.

(H) Statistical treatment of results

where appropriate.

(I) Historical control data.

(iii) Inhalation studies. In addition,

for inhalation studies the following

shall be reported:

(A) Test conditions. The following

exposure conditions shall be reported.

(1) Description of exposure apparatus

including design, type, dimensions,

source of air, system for generating

particulate and aerosols, method of

conditioning air, treatment of exhaust

air and the method of housing the

animals in a test chamber.

(2) The equipment for measuring

temperature, humidity, and particulate

aerosol concentrations and size should

be described.

(B) Exposure data. These shall be

tabulated and presented with mean

values and a measure of variability (e.g.

standard deviation) and should include:

(1) Airflow rates through the

inhalation equipment.

(2) Temperature and humidity of air.

(3) Actual (analytical or gravimetric)

concentration in the breathing zone.

(4) Nominal concentration (total

amount of test substance fed into the

inhalation equipment divided by

volume of air).

(5) Particle size distribution,

calculated MMAD and geometric

standard deviation (GSD).

(6) Explanation as to why the desired

chamber concentration and/or particle

size could not be achieved (if

applicable) and the efforts taken to

comply with this aspect of the sections.

(f) Quality assurance. A system shall

be developed and maintained to assure

and document adequate performance of

laboratory staff and equipment. The

study shall be conducted in compliance

with 40 CFR Part 792—Good Laboratory

Practice Standards.

(g) References. For additional

background information on this test

guideline, the following references

should be consulted. These references

are available for inspection at the TSCA

Nonconfidential Information Center,
43842 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

Rm. NE–B607, Environmental

Protection Agency, 401 M St., SW.,

Washington, DC, 12 noon to 4 p.m.,

Monday through Friday, except legal

holidays.

(1) Benitz, K.F. Ed. Paget, G.E.

Measurement of Chronic Toxicity.

Methods of Toxicology (Blackwell,

Oxford, 1970) pp. 82–131.

(2) Fitzhugh, O.G. Chronic Oral

Toxicity, Appraisal of the Safety of

Chemicals in Foods, Drugs and

Cosmetics. The Association of Food and

Drug Officials of the United States. pp.

36–45 (1959, 3rd Printing 1975).

(3) Goldenthal, E.I. and D’Aguanno,

W. Evaluation of Drugs, Appraisal of the

Safety of Chemicals in Foods, Drugs,

and Cosmetics. The Association of Food

and Drug Officials of the United States.

pp. 60–67 (1959, 3rd Printing 1975).

(4) Organisation for Economic Cooperation and Development. Guidelines

for Testing of Chemicals, Section 4–

Health Effects, Part 451 Carcinogenicity

Studies (Paris, 1981).

(5) Page, N.P. Chronic Toxicity and

Carcinogenicity Guidelines. Journal of

Environmental Pathology and

Toxicology. 11:161–182 (1977).

(6) Page, N.P. Eds. Kraybill and

Mehlman. Concepts of a Bioassay

Program in Environmental

Carcinogenesis. Vol.3. Advances in

Modern Toxicology (Hemisphere,

Washington, DC., 1977) pp. 87–171.

(7) Sontag, J.M. et al. Guidelines for

Carcinogen Bioassay in Small Rodents.

NCI-CS-TR-1 United States Cancer

Institute, Division of Cancer Control and

Prevention, Carcinogenesis Bioassay

Program (Bethesda, MD).

§ 799.9510 TSCA bacterial reverse

mutation test.

(a) Scope. This section is intended to

meet the testing requirements under

section 4 of TSCA.

(1) The bacterial reverse mutation test

uses amino-acid requiring strains of

Salmonella typhimurium and

Escherichia coli to detect point

mutations, which involve substitution,

addition or deletion of one or a few

DNA base pairs. The principle of this

bacterial reverse mutation test is that it

detects mutations which revert

mutations present in the test strains and

restore the functional capability of the

bacteria to synthesize an essential

amino acid. The revertant bacteria are

detected by their ability to grow in the

absence of the amino acid required by

the parent test strain.

(2) Point mutations are the cause of

many human genetic diseases and there

is substantial evidence that point

mutations in oncogenes and tumor

suppressor genes of somatic cells are

involved in tumor formation in humans

and experimental animals. The bacterial

reverse mutation test is rapid,

inexpensive and relatively easy to

perform. Many of the test strains have

several features that make them more

sensitive for the detection of mutations,

including responsive DNA sequences at

the reversion sites, increased cell

permeability to large molecules and

elimination of DNA repair systems or

enhancement of error-prone DNA repair

processes. The specificity of the test

strains can provide some useful

information on the types of mutations

that are induced by genotoxic agents. A

very large data base of results for a wide

variety of structures is available for

bacterial reverse mutation tests and

well-established methodologies have

been developed for testing chemicals

with different physico-chemical

properties, including volatile

compounds.

(b) Source. The source material used

in developing this TSCA test guideline

are the OECD replacement guidelines

for 471 and 472 (February 1997). This

source is available at the address in

paragraph (g) of this section.

(c) Definitions. The following

definitions apply to this section:

A reverse mutation test in either

Salmonella typhimurium or Escherichia

coli detects mutation in an amino-acid

requiring strain (histidine or

tryptophan, respectively) to produce a

strain independent of an outside supply

of amino-acid.

Base pair substitution mutagens are

agents that cause a base change in DNA.

In a reversion test this change may

occur at the site of the original

mutation, or at a second site in the

bacterial genome.

Frameshift mutagens are agents that

cause the addition or deletion of one or

more base pairs in the DNA, thus

changing the reading frame in the RNA

(d) Initial considerations. (1) The

bacterial reverse mutation test utilizes

prokaryotic cells, which differ from

mammalian cells in such factors as

uptake, metabolism, chromosome

structure and DNA repair processes.

Tests conducted in vitro generally

require the use of an exogenous source

of metabolic activation. In vitro

metabolic activation systems cannot

mimic entirely the mammalian in vivo

conditions. The test therefore does not

provide direct information on the

mutagenic and carcinogenic potency of

a substance in mammals.

(2) The bacterial reverse mutation test

is commonly employed as an initial

screen for genotoxic activity and, in

particular, for point mutation-inducing

activity. An extensive data base has

demonstrated that many chemicals that

are positive in this test also exhibit

mutagenic activity in other tests. There

are examples of mutagenic agents which

are not detected by this test; reasons for

these shortcomings can be ascribed to

the specific nature of the endpoint

detected, differences in metabolic

activation, or differences in

bioavailability. On the other hand,

factors which enhance the sensitivity of

the bacterial reverse mutation test can

lead to an overestimation of mutagenic

activity.

(3) The bacterial reverse mutation test

may not be appropriate for the

evaluation of certain classes of

chemicals, for example highly

bactericidal compounds (e.g. certain

antibiotics) and those which are thought

(or known) to interfere specifically with

the mammalian cell replication system

(e.g. some topoisomerase inhibitors and

some nucleoside analogues). In such

cases, mammalian mutation tests may

be more appropriate.

(4) Although many compounds that

are positive in this test are mammalian

carcinogens, the correlation is not

absolute. It is dependent on chemical

class and there are carcinogens that are

not detected by this test because they

act through other, non-genotoxic

mechanisms or mechanisms absent in

bacterial cells.

(e) Test method—(1) Principle. (i)

Suspensions of bacterial cells are

exposed to the test substance in the

presence and in the absence of an

exogenous metabolic activation system.

In the plate incorporation method, these

suspensions are mixed with an overlay

agar and plated immediately onto

minimal medium. In the preincubation

method, the treatment mixture is

incubated and then mixed with an

overlay agar before plating onto minimal

medium. For both techniques, after 2 or

3 days of incubation, revertant colonies

are counted and compared to the

number of spontaneous revertant

colonies on solvent control plates.

(ii) Several procedures for performing

the bacterial reverse mutation test have

been described. Among those commonly

used are the plate incorporation

method, the preincubation method, the

fluctuation method, and the suspension

method. Suggestions for modifications

for the testing of gases or vapors are

described in the reference in paragraph

(g)(12) of this section.

(iii) The procedures described in this

section pertain primarily to the plate

incorporation and preincubation

methods. Either of them is acceptable

for conducting experiments both with

and without metabolic activation. Some

compounds may be detected more
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43843

efficiently using the preincubation

method. These compounds belong to

chemical classes that include short

chain aliphatic nitrosamines, divalent

metals, aldehydes, azo-dyes and diazo

compounds, pyrollizidine alkaloids,

allyl compounds and nitro compounds.

It is also recognized that certain classes

of mutagens are not always detected

using standard procedures such as the

plate incorporation method or

preincubation method. These should be

regarded as ‘‘special cases’’ and it is

strongly recommended that alternative

procedures should be used for their

detection. The following ‘‘special cases’’

could be identified (together with

examples of procedures that could be

used for their detection): azo-dyes and

diazo compounds (alterative procedures

are described in the references in

paragraphs (g)(3), (g)(5), (g)(6), and

(g)(13) of this section), gases and volatile

chemicals (alterative procedures are

described in the references in

paragraphs (g)(12), (g)(14), (g)(15), and

(g)(16) of this section), and glycosides

(alterative procedures are described in

the references in paragraphs (g)(17) and

(g)(18) of this section). A deviation from

the standard procedure needs to be

scientifically justified.

(2) Description—(i) Preparations—(A)

Bacteria. (1) Fresh cultures of bacteria

should be grown up to the late

exponential or early stationary phase of

growth (approximately 109 cells per ml).

Cultures in late stationary phase should

not be used. The cultures used in the

experiment shall contain a high titre of

viable bacteria. The titre may be

demonstrated either from historical

control data on growth curves, or in

each assay through the determination of

viable cell numbers by a plating

experiment.

(2) The culture temperature shall be

37°C.

(3) At least five strains of bacteria

should be used. These should include

four strains of S. typhimurium (TA1535;

TA1537 or TA97a or TA97; TA98; and

TA100) that have been shown to be

reliable and reproducibly responsive

between laboratories. These four S.

typhimurium strains have GC base pairs

at the primary reversion site and it is

known that they may not detect certain

oxidizing mutagens, cross-linking

agents, and hydrazines. Such substances

may be detected by E.coli WP2 strains

or S. typhimurium TA102 (see

paragraph (g)(19) of this section) which

have an AT base pair at the primary

reversion site. Therefore the

recommended combination of strains is:

(i) S. typhimurium TA1535.

(ii) S. typhimurium TA1537 or TA97

or TA97a.

(iii) S. typhimurium TA98.

(iv) S. typhimurium TA100.

(v) E. coli WP2 uvrA, or E. coli WP2

uvrA (pKM101), or S. typhimurium

TA102. In order to detect cross-linking

mutagens it may be preferable to

include TA102 or to add a DNA repairproficient strain of E.coli [e.g. E.coli

WP2 or E.coli WP2 (pKM101).]

(4) Established procedures for stock

culture preparation, marker verification

and storage should be used. The aminoacid requirement for growth should be

demonstrated for each frozen stock

culture preparation (histidine for S.

typhimurium strains, and tryptophan for

E. coli strains). Other phenotypic

characteristics should be similarly

checked, namely: the presence or

absence of R-factor plasmids where

appropriate [i.e. ampicillin resistance in

strains TA98, TA100 and TA97a or

TA97, WP2 uvrA and WP2 uvrA

(pKM101), and ampicillin + tetracycline

resistance in strain TA102]; the

presence of characteristic mutations (i.e.

rfa mutation in S. typhimurium through

sensitivity to crystal violet, and uvrA

mutation in E. coli or uvrB mutation in

S. typhimurium, through sensitivity to

ultra-violet light). The strains should

also yield spontaneous revertant colony

plate counts within the frequency

ranges expected from the laboratory’s

historical control data and preferably

within the range reported in the

literature.

(B) Medium. An appropriate minimal

agar (e.g. containing Vogel-Bonner

minimal medium E and glucose) and an

overlay agar containing histidine and

biotin or tryptophan, to allow for a few

cell divisions, shall be used. The

procedures described in the references

under paragraphs (g)(1), (g)(2), and (g)(9)

of this section may be used for this

analysis.

(C) Metabolic activation. Bacteria

shall be exposed to the test substance

both in the presence and absence of an

appropriate metabolic activation system.

The most commonly used system is a

cofactor-supplemented postmitochondrial fraction (S9) prepared

from the livers of rodents treated with

enzyme-inducing agents such as Aroclor

1254 (the system described in the

references under paragraphs (g)(1) and

(g)(2) of this section may be used) or a

combination of phenobarbitone and βnaphthoflavone (the system described in

the references under paragraphs (g)(18),

(g)(20), and (g)(21) of this section may

be used). The post-mitochondrial

fraction is usually used at

concentrations in the range from 5 to

30% v/v in the S9-mix. The choice and

condition of a metabolic activation

system may depend upon the class of

chemical being tested. In some cases it

may be appropriate to utilize more than

one concentration of post-mitochondrial

fraction. For azo-dyes and diazocompounds, using a reductive metabolic

activation system may be more

appropriate (the system described in the

references under paragraphs (g)(6) and

(g)(13) of this section may be used).

(D) Test substance/preparation. Solid

test substances should be dissolved or

suspended in appropriate solvents or

vehicles and diluted if appropriate prior

to treatment of the bacteria. Liquid test

substances may be added directly to the

test systems and/or diluted prior to

treatment. Fresh preparations should be

employed unless stability data

demonstrate the acceptability of storage.

(ii) Test conditions—(A) Solvent/

vehicle. The solvent/vehicle shall not be

suspected of chemical reaction with the

test substance and shall be compatible

with the survival of the bacteria and the

S9 activity (see paragraph (g)(22) of this

section). If other than well-known

solvent/vehicles are used, their

inclusion should be supported by data

indicating their compatibility. It is

recommended that wherever possible,

the use of an aqueous solvent/vehicle be

considered first. When testing waterunstable substances, the organic

solvents used should be free of water.

(B) Exposure concentrations. (1)

Amongst the criteria to be taken into

consideration when determining the

highest amount of test substance to be

used are cytotoxicity and solubility in

the final treatment mixture. It may be

useful to determine toxicity and

insolubility in a preliminary

experiment. Cytotoxicity may be

detected by a reduction in the number

of revertant colonies, a clearing or

diminution of the background lawn, or

the degree of survival of treated

cultures. The cytotoxicity of a substance

may be altered in the presence of

metabolic activation systems.

Insolubility should be assessed as

precipitation in the final mixture under

the actual test conditions and evident to

the unaided eye. The recommended

maximum test concentration for soluble

non-cytotoxic substances is 5 mg/plate

or 5 μl/plate. For non-cytotoxic

substances that are not soluble at 5mg/

plate or 5μl/plate, one or more

concentrations tested should be

insoluble in the final treatment mixture.

Test substances that are cytotoxic

already below 5mg/plate or 5μl/plate

should be tested up to a cytotoxic

concentration. The precipitate should

not interfere with the scoring.

(2) At least five different analyzable

concentrations of the test substance

shall be used with approximately half
43844 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

log (i.e. √10) intervals between test

points for an initial experiment. Smaller

intervals may be appropriate when a

concentration-response is being

investigated.

(3) Testing above the concentration of

5 mg/plate or 5μl/plate may be

considered when evaluating substances

containing substantial amounts of

potentially mutagenic impurities.

(C) Controls. (1) Concurrent strainspecific positive and negative (solvent

or vehicle) controls, both with and

without metabolic activation, shall be

included in each assay. Positive control

concentrations that demonstrate the

effective performance of each assay

should be selected.

(2)(i) For assays employing a

metabolic activation system, the

positive control reference substance(s)

should be selected on the basis of the

type of bacteria strains used. The

following chemicals are examples of

suitable positive controls for assays with

metabolic activation:

Chemical CAS No.

9,10-Dimethylanthracene ....................................................................................................... [CAS no. 781–43–1]

7,12-Dimethylbenzanthracene ............................................................................................... [CAS no. 57–97–6]

Congo Red (for the reductive metabolic activation method) ................................................ [CAS no. 573–58–0]

Benzo(a)pyrene ..................................................................................................................... [CAS no. 50–32–8]

Cyclophosphamide (monohydrate) ........................................................................................ [CAS no. 50–18–0]

[CAS no. 6055–19–2]

2-Aminoanthracene ............................................................................................................... [CAS no. 613–13–8]

(ii) 2-Aminoanthracene should not be

used as the sole indicator of the efficacy

of the S9-mix. If 2-aminoanthracene is

used, each batch of S9 should also be

characterized with a mutagen that

requires metabolic activation by

microsomal enzymes, e.g.,

benzo(a)pyrene,

dimethylbenzanthracene.

(3) For assays performed without

metabolic activation system, examples

of strain-specific positive controls are:

Chemical CAS No. Strain

(a) Sodium azide .................................................... [CAS no. 26628–22–8] .................................. TA1535 and TA100

(b) 2-Nitrofluorene .................................................. [CAS no. 607–57–8] ...................................... TA 98

(c) 9-Aminoacridine or ICR 191 ............................. [CAS no. 90–45–9] or ....................................

[CAS no. 17070–45–0] ..................................

TA1537, TA97 and TA97a

(d) Cumene hydroperoxide .................................... [CAS no. 80–15–9] ........................................ TA102

(e) Mitomycin C ...................................................... [CAS no. 50–07–7] ........................................ WP2 uvrA and TA102

(f) N-Ethyl-N-nitro-N-nitrosoguanidine or

4-nitroquinoline 1-oxide ..........................................

[CAS no. 70–25–7] or ....................................

[CAS no. 56–57–5] ........................................

WP2, WP2 uvrA and WP2 uvrA (pKM101)

(g) Furylfuramide (AF-2) ......................................... [CAS no. 3688–53–7] .................................... Plasmid-containing strains

(4) Other appropriate positive control

reference substances may be used. The

use of chemical class-related positive

control chemicals may be considered,

when available.

(5) Negative controls, consisting of

solvent or vehicle alone, without test

substance, and otherwise treated in the

same way as the treatment groups, shall

be included. In addition, untreated

controls should also be used unless

there are historical control data

demonstrating that no deleterious or

mutagenic effects are induced by the

chosen solvent.

(3) Procedure—(i) Treatment with test

substance. (A) For the plate

incorporation method, without

metabolic activation, usually 0.05 ml or

0.1 ml of the test solutions, 0.1 ml of

fresh bacterial culture (containing

approximately 108 viable cells) and 0.5

ml of sterile buffer are mixed with 2.0

ml of overlay agar. For the assay with

metabolic activation, usually 0.5 ml of

metabolic activation mixture containing

an adequate amount of postmitochondrial fraction (in the range

from 5 to 30% v/v in the metabolic

activation mixture) are mixed with the

overlay agar (2.0 ml), together with the

bacteria and test substance/test solution.

The contents of each tube are mixed and

poured over the surface of a minimal

agar plate. The overlay agar is allowed

to solidify before incubation.

(B) For the preincubation method the

test substance/test solution is

preincubated with the test strain

(containing approximately 108 viable

cells) and sterile buffer or the metabolic

activation system (0.5 ml) usually for 20

min. or more at 30–37 °C prior to mixing

with the overlay agar and pouring onto

the surface of a minimal agar plate.

Usually, 0.05 or 0.1 ml of test substance/

test solution, 0.1 ml of bacteria, and 0.5

ml of S9-mix or sterile buffer, are mixed

with 2.0 ml of overlay agar. Tubes

should be aerated during pre-incubation

by using a shaker.

(C) For an adequate estimate of

variation, triplicate plating should be

used at each dose level. The use of

duplicate plating is acceptable when

scientifically justified. The occasional

loss of a plate does not necessarily

invalidate the assay.

(D) Gaseous or volatile substances

should be tested by appropriate

methods, such as in sealed vessels

(methods described in the references

under paragraphs (g)(12), (g)(14), (g)(15),

and (g)(16) of this section may be used).

(ii) Incubation. All plates in a given

assay shall be incubated at 37 °C for 48–

72 hrs. After the incubation period, the

number of revertant colonies per plate is

counted.

(f) Data and reporting—(1) Treatment

of results. (i) Data shall be presented as

the number of revertant colonies per

plate. The number of revertant colonies

on both negative (solvent control, and
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43845

untreated control if used) and positive

control plates shall also be given.

(ii) Individual plate counts, the mean

number of revertant colonies per plate

and the standard deviation shall be

presented for the test substance and

positive and negative (untreated and/or

solvent) controls.

(iii) There is no requirement for

verification of a clear positive response.

Equivocal results shall be clarified by

further testing preferably using a

modification of experimental

conditions. Negative results need to be

confirmed on a case-by-case basis. In

those cases where confirmation of

negative results is not considered

necessary, justification should be

provided. Modification of study

parameters to extend the range of

conditions assessed should be

considered in follow-up experiments.

Study parameters that might be

modified include the concentration

spacing, the method of treatment (plate

incorporation or liquid preincubation),

and metabolic activation conditions.

(2) Evaluation and interpretation of

results. (i) There are several criteria for

determining a positive result, such as a

concentration-related increase over the

range tested and/or a reproducible

increase at one or more concentrations

in the number of revertant colonies per

plate in at least one strain with or

without metabolic activation system.

Biological relevance of the results

should be considered first. Statistical

methods may be used as an aid in

evaluating the test results. However,

statistical significance should not be the

only determining factor for a positive

response.

(ii) A test substance for which the

results do not meet the criteria

described under paragraph (f)(2)(i) of

this section is considered nonmutagenic in this test

(iii) Although most experiments will

give clearly positive or negative results,

in rare cases the data set will preclude

making a definite judgement about the

activity of the test substance. Results

may remain equivocal or questionable

regardless of the number of times the

experiment is repeated.

(iv) Positive results from the bacterial

reverse mutation test indicate that a

substance induces point mutations by

base substitutions or frameshifts in the

genome of either Salmonella

typhimurium and/or Escherichia coli.

Negative results indicate that under the

test conditions, the test substance is not

mutagenic in the tested species.

(3) Test report. In addition to the

reporting requirements as specified

under 40 CFR part 792, subpart J, the

following specific information shall be

reported. Both individual and summary

data should be presented.

(i) Test substance:

(A) Identification data and CAS no., if

known.

(B) Physical nature and purity.

(C) Physicochemical properties

relevant to the conduct of the study.

(D) Stability of the test substance, if

known.

(ii) Solvent/vehicle:

(A) Justification for choice of solvent/

vehicle.

(B) Solubility and stability of the test

substance in solvent/vehicle, if known.

(iii) Strains:

(A) Strains used.

(B) Number of cells per culture.

(C) Strain characteristics.

(iv) Test conditions:

(A) Amount of test substance per plate

(mg/plate or ml/plate) with rationale for

selection of dose and number of plates

per concentration.

(B) Media used.

(C) Type and composition of

metabolic activation system, including

acceptability criteria.

(D) Treatment procedures.

(v) Results:

(A) Signs of toxicity.

(B) Signs of precipitation.

(C) Individual plate counts.

(D) The mean number of revertant

colonies per plate and standard

deviation.

(E) Dose-response relationship, where

possible.

(F) Statistical analyses, if any.

(G) Concurrent negative (solvent/

vehicle) and positive control data, with

ranges, means and standard deviations.

(H) Historical negative (solvent/

vehicle) and positive control data, with

e.g. ranges, means and standard

deviations.

(vi) Discussion of the results.

(vii) Conclusion.

(g) References. For additional

background information on this test

guideline, the following references

should be consulted. These references

are available for inspection at the TSCA

Nonconfidential Information Center,

Rm. NE–B607, Environmental

Protection Agency, 401 M St., SW.,

Washington, DC, 12 noon to 4 p.m.,

Monday through Friday, except legal

holidays.

(1) Ames, B.N., McCann, J., and

Yamasaki, E. Methods for Detecting

Carcinogens and Mutagens With the

Salmonella/Mammalian-Microsome

Mutagenicity Test. Mutation Research.

31, 347–364 (1975).

(2) Maron, D.M. and Ames, B.N.

Revised Methods for the Salmonella

Mutagenicity Test. Mutation Research.

113, 173–215 (1983).

(3) Gatehouse, D., Haworth, S.,

Cebula, T., Gocke, E., Kier, L.,

Matsushima, T., Melcion, C., Nohmi, T.,

Venitt, S., and Zeiger, E.

Recommendations for the Performance

of Bacterial Mutation Assays. Mutation

Research. 312, 217–233 (1994).

(4) Kier, L.D., Brusick, D.J., Auletta,

A.E., Von Halle, E.S., Brown, M.M.,

Simmon, V.F., Dunkel, V., McCann, J.,

Mortelmans, K., Prival, M., Rao, T.K.,

and Ray V. The Salmonella

Typhimurium/Mammalian Microsomal

Assay: A Report of the U.S.

Environmental Protection Agency GeneTox Program. Mutation Research. 168,

69–240 (1986).

(5) Yahagi, T., Degawa, M., Seino,

Y.Y., Matsushima, T., Nagao, M.,

Sugimura, T., and Hashimoto, Y.

Mutagenicity of Carcinogen Azo Dyes

and Their Derivatives. Cancer Letters, 1.

91–96 (1975).

(6) Matsushima, M., Sugimura, T.,

Nagao, M., Yahagi, T., Shirai, A., and

Sawamura, M. Factors Modulating

Mutagenicity Microbial Tests. Eds.

Norpoth, K.H. and Garner, R.C. ShortTerm Test Systems for Detecting

Carcinogens (Springer, BerlinHeidelberg-New York, 1980) pp. 273–

285.

(7) Gatehouse, D.G., Rowland, I.R.,

Wilcox, P., Callender, R.D., and Foster,

R. Bacterial Mutation Assays. Ed.

Kirkland, D.J. Basic Mutagenicity Tests.

UKEMS Part 1 Revised (Cambridge

University Press, 1990) pp. 13–61.

(8) Aeschbacher, H.U., Wolleb, U.,

and Porchet, L.J. Liquid Preincubation

Mutagenicity Test for Foods. Food

Safety. 8, 167–177 (1987).

(9) Green, M.H.L., Muriel, W.J., and

Bridges, B.A. Use of a Simplified

Fluctuation Test to Detect Low Levels of

Mutagens. Mutation Research. 38, 33–42

(1976).

(10) Hubbard, S.A., Green, M.H.L.,

Gatehouse, D., and J.W. Bridges. The

Fluctuation Test in Bacteria. 2nd

Edition. Ed. Kilbey, B.J., Legator, M.,

Nichols, W., and Ramel C. Handbook of

Mutagenicity Test Procedures (Elsevier,

Amsterdam-New York-Oxford, 1984)

pp. 141–161.

(11) Thompson, E.D. and Melampy,

P.J. An Examination of the Quantitative

Suspension Assay for Mutagenesis With

Strains of Salmonella Typhimurium.

Environmental Mutagenesis. 3, 453–465

(1981).

(12) Araki, A., Noguchi, T., Kato, F.,

and T. Matsushima. Improved Method

for Mutagenicity Testing of Gaseous

Compounds by Using a Gas Sampling

Bag. Mutation Research. 307, 335–344

(1994).

(13) Prival, M.J., Bell, S.J., Mitchell,

V.D., Reipert, M.D., and Vaughn, V.L.
43846 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

Mutagenicity of Benzidine and

Benzidine-Congener Dyes and Selected

Monoazo Dyes in a Modified Salmonella

Assay. Mutation Research. 136, 33–47

(1984).

(14) Zeiger, E., Anderson, B. E.,

Haworth, S, Lawlor, T., and

Mortelmans, K. Salmonella

Mutagenicity Tests. V. Results from the

Testing of 311 Chemicals. Environ. Mol.

Mutagen. 19, 2–141 (1992).

(15) Simmon, V., Kauhanen, K., and

Tardiff, R.G. Mutagenic Activity of

Chemicals Identified in Drinking Water.

Ed. Scott, D., Bridges, B., and Sobels, F.

Progress in Genetic Toxicology (Elsevier,

Amsterdam, 1977) pp. 249–258.

(16) Hughes, T.J., Simmons, D.M.,

Monteith, I.G., and Claxton, L.D.

Vaporization Technique to Measure

Mutagenic Activity of Volatile Organic

Chemicals in the Ames/Salmonella

Assay. Environmental Mutagenesis. 9,

421–441 (1987).

(17) Matsushima, T., Matsumoto, A.,

Shirai, M., Sawamura, M., and

Sugimura, T. Mutagenicity of the

Naturally Occurring Carcinogen Cycasin

and Synthetic Methylazoxy Methane

Conjugates in Salmonella

Typhimurium. Cancer Research. 39,

3780–3782 (1979).

(18) Tamura, G., Gold, C., Ferro-Luzzi,

A., and Ames. B.N. Fecalase: A Model

for Activation of Dietary Glycosides to

Mutagens by Intestinal Flora. Proc.

National Academy of Science. (USA,

1980) 77, 4961–4965.

(19) Wilcox, P., Naidoo, A., Wedd, D.

J., and Gatehouse, D. G. Comparison of

Salmonella Typhimurium TA 102 With

Escherichia Coli WP2 Tester Strains.

Mutagenesis. 5, 285–291 (1990).

(20) Matsushima, T., Sawamura, M.,

Hara, K., and Sugimura, T. A Safe

Substitute for Polychlorinated

Biphenyls as an Inducer of Metabolic

Activation Systems. Ed. F.J. de Serres et

al. In Vitro Metabolic Activation in

Mutagenesis Testing. (Elsevier, North

Holland, 1976) pp. 85–88.

(21) Elliott, B.M., Combes, R.D.,

Elcombe, C.R., Gatehouse, D.G., Gibson,

G.G., Mackay, J.M., and Wolf, R.C.

Alternatives to Aroclor 1254-Induced S9

in In Vitro Genotoxicity Assays.

Mutagenesis. 7, 175–177 (1992).

(22) Maron, D., Katzenellenbogen, J.,

and Ames, B.N. Compatibility of

Organic Solvents With the Salmonella/

Microsome Test. Mutation Research. 88,

343–350 (1981).

(23) Claxton, L.D., Allen, J., Auletta,

A., Mortelmans, K., Nestmann, E., and

Zeiger, E. Guide for the Salmonella

Typhimurium/Mammalian Microsome

Tests for Bacterial Mutagenicity.

Mutation Research. 189, 83–91 (1987).

(24) Mahon, G.A.T., Green, M.H.L.,

Middleton, B., Mitchell, I., Robinson,

W.D., and Tweats, D.J. Analysis of Data

from Microbial Colony Assays. UKEMS

Sub-Committee on Guidelines for

Mutagenicity Testing Part II. Ed.

Kirkland, D.J. Statistical Evaluation of

Mutagenicity Test Data (Cambridge

University Press, 1989) pp. 28–65.

§ 799.9530 TSCA in vitro mammalian cell

gene mutation test.

(a) Scope. This section is intended to

meet the testing requirements under

section 4 of TSCA. The in vitro

mammalian cell gene mutation test can

be used to detect gene mutations

induced by chemical substances.

Suitable cell lines include L5178Y

mouse lymphoma cells, the CHO, AS52

and V79 lines of Chinese hamster cells,

and TK6 human lymphoblastoid cells

under paragraph (g)(1) of this section. In

these cell lines the most commonlyused genetic endpoints measure

mutation at thymidine kinase (TK) and

hypoxanthine-guanine phosphoribosyl

transferase (HPRT), and a transgene of

xanthine-guanine phosphoribosyl

transferase (XPRT). The TK, HPRT and

XPRT mutation tests detect different

spectra of genetic events. The autosomal

location of TK and XPRT may allow the

detection of genetic events (e.g. large

deletions) not detected at the HPRT

locus on X-chromosomes (For a

discussion see the references in

paragraphs (g)(2), (g)(3), (g)(4),(g)(5), and

(g)(6) of this section).

(b) Source. The source material used

in developing this TSCA test guideline

is the OECD guideline 476 (February

1997). This source is available at the

address in paragraph (g) of this section.

(c) Definitions. The following

definitions apply to this section:

Base pair substitution mutagens are

substances which cause substitution of

one or several base pairs in the DNA.

Forward mutation is a gene mutation

from the parental type to the mutant

form which gives rise to an alteration or

a loss of the enzymatic activity or the

function of the encoded protein.

Frameshift mutagens are substances

which cause the addition or deletion of

single or multiple base pairs in the DNA

molecule.

Mutant frequency is the number of

mutant cells observed divided by the

number of viable cells.

Phenotypic expression time is a

period during which unaltered gene

products are depleted from newly

mutated cells.

Relative suspension growth is an

increase in cell number over the

expression period relative to the

negative control.

Relative total growth is an increase in

cell number over time compared to a

control population of cells; calculated as

the product of suspension growth

relative to the negative control times

cloning efficiency relative to negative

control.

Survival is the cloning efficiency of

the treated cells when plated at the end

of the treatment period; survival is

usually expressed in relation to the

survival of the control cell population.

Viability is the cloning efficiency of

the treated cells at the time of plating in

selective conditions after the expression

period.

(d) Initial considerations. (1) In the in

vitro mammalian cell gene mutation

test, cultures of established cell lines or

cell strains can be used. The cells used

are selected on the basis of growth

ability in culture and stability of the

spontaneous mutation frequency. Tests

conducted in vitro generally require the

use of an exogenous source of metabolic

activation. This metabolic activation

system cannot mimic entirely the

mammalian in vivo conditions. Care

should be taken to avoid conditions

which would lead to results not

reflecting intrinsic mutagenicity.

Positive results which do not reflect

intrinsic mutagenicity may arise from

changes in pH, osmolality or high levels

of cytotoxicity.

(2) This test is used to screen for

possible mammalian mutagens and

carcinogens. Many compounds that are

positive in this test are mammalian

carcinogens; however, there is not a

perfect correlation between this test and

carcinogenicity. Correlation is

dependent on chemical class and there

is increasing evidence that there are

carcinogens that are not detected by this

test because they appear to act through

other, non-genotoxic mechanisms or

mechanisms absent in bacterial cells.

(e) Test method—(1) Principle. (i)

Cells deficient in thymidine kinase (TK)

due to the mutation TK∂/- -≤ TK-

/- are

resistant to the cytotoxic effects of the

pyrimidine analogue trifluorothymidine

(TFT). Thymidine kinase proficient cells

are sensitive to TFT, which causes the

inhibition of cellular metabolism and

halts further cell division. Thus mutant

cells are able to proliferate in the

presence of TFT, whereas normal cells,

which contain thymidine kinase, are

not. Similarly, cells deficient in HPRT

or XPRT are selected by resistance to 6-

thioguanine (TG) or 8-azaguanine (AG).

The properties of the test substance

should be considered carefully if a base

analogue or a compound related to the

selective agent is tested in any of the

mammalian cell gene mutation tests. For

example, any suspected selective
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43847

toxicity by the test substance for mutant

and non-mutant cells should be

investigated. Thus, performance of the

selection system/agent shall be

confirmed when testing chemicals

structurally related to the selective

agent.

(ii) Cells in suspension or monolayer

culture shall be exposed to the test

substance, both with and without

metabolic activation, for a suitable

period of time and subcultured to

determine cytotoxicity and to allow

phenotypic expression prior to mutant

selection. Cytotoxicity is usually

determined by measuring the relative

cloning efficiency (survival) or relative

total growth of the cultures after the

treatment period. The treated cultures

shall be maintained in growth medium

for a sufficient period of time,

characteristic of each selected locus and

cell type, to allow near-optimal

phenotypic expression of induced

mutations. Mutant frequency is

determined by seeding known numbers

of cells in medium containing the

selective agent to detect mutant cells,

and in medium without selective agent

to determine the cloning efficiency

(viability). After a suitable incubation

time, colonies shall be counted. The

mutant frequency is derived from the

number of mutant colonies in selective

medium and the number of colonies in

non-selective medium.

(2) Description—(i) Preparations—(A)

Cells. (1) A variety of cell types are

available for use in this test including

subclones of L5178Y, CHO, CHO-AS52,

V79, or TK6 cells. Cell types used in

this test should have a demonstrated

sensitivity to chemical mutagens, a high

cloning efficiency and a stable

spontaneous mutant frequency. Cells

should be checked for mycoplasma

contamination and should not be used

if contaminated.

(2) The test should be designed to

have a predetermined sensitivity and

power. The number of cells, cultures,

and concentrations of test substance

used should reflect these defined

parameters. The parameters discussed

in the reference under paragraph (g)(13)

of this section may be used. The

minimal number of viable cells

surviving treatment and used at each

stage in the test should be based on the

spontaneous mutation frequency. A

general guide is to use a cell number

which is at least ten times the inverse

of the spontaneous mutation frequency.

However, it is recommended to utilize

at least 106 cells. Adequate historical

data on the cell system used should be

available to indicate consistent

performance of the test.

(B) Media and culture conditions.

Appropriate culture media and

incubation conditions (culture vessels,

temperature, CO2 concentration and

humidity) shall be used. Media should

be chosen according to the selective

systems and cell type used in the test.

It is particularly important that culture

conditions should be chosen that ensure

optimal growth of cells during the

expression period and colony forming

ability of both mutant and non-mutant

cells.

(C) Preparation of cultures. Cells are

propagated from stock cultures, seeded

in culture medium and incubated at

37°C. Prior to use in this test, cultures

may need to be cleansed of pre-existing

mutant cells.

(D) Metabolic activation. Cells shall

be exposed to the test substance both in

the presence and absence of an

appropriate metabolic activation system.

The most commonly used system is a

co-factor-supplemented postmitochondrial fraction (S9) prepared

from the livers of rodents treated with

enzyme-inducing agents such as Aroclor

1254 or a combination of

phenobarbitone and β-naphthoflavone.

The post-mitochondrial fraction is

usually used at concentrations in the

range from 1–10% v/v in the final test

medium. The choice and condition of a

metabolic activation system may

depend upon the class of chemical

being tested. In some cases it may be

appropriate to utilize more than one

concentration of post-mitochondrial

fraction. A number of developments,

including the construction of genetically

engineered cell lines expressing specific

activating enzymes, may provide the

potential for endogenous activation. The

choice of the cell lines used should be

scientifically justified (e.g. by the

relevance of the cytochrome P450

isoenzyme to the metabolism of the test

substance).

(E) Test substance/preparations. Solid

test substances should be dissolved or

suspended in appropriate solvents or

vehicles and diluted if appropriate prior

to treatment of the cells. Liquid test

substances may be added directly to the

test systems and/or diluted prior to

treatment. Fresh preparations should be

employed unless stability data

demonstrate the acceptability of storage.

(ii) Test conditions—(A) Solvent/

vehicle. The solvent/vehicle shall not be

suspected of chemical reaction with the

test substance and shall be compatible

with the survival of the cells and the S9

activity. If other than well-known

solvent/vehicles are used, their

inclusion should be supported by data

indicating their compatibility. It is

recommended that wherever possible,

the use of an aqueous solvent/vehicle be

considered first. When testing waterunstable substances, the organic

solvents used should be free of water.

Water can be removed by adding a

molecular sieve.

(B) Exposure concentrations. (1)

Among the criteria to be considered

when determining the highest

concentration are cytotoxicity and

solubility in the test system and changes

in pH or osmolality.

(2) Cytotoxicity should be determined

with and without metabolic activation

in the main experiment using an

appropriate indicator of cell integrity

and growth, such as relative cloning

efficiency (survival) or relative total

growth. It may be useful to determine

cytotoxicity and solubility in a

preliminary experiment.

(3) At least four analyzable

concentrations shall be used. Where

there is cytotoxicity, these

concentrations shall cover a range from

the maximum to little or no toxicity;

this will usually mean that the

concentration levels should be

separated by no more than a factor

between 2 and √10. If the maximum

concentration is based on cytotoxicity

then it shall result in approximately 10–

20% but not less than 10% relative

survival (relative cloning efficiency) or

relative total growth. For relatively noncytotoxic compounds the maximum

concentration should be 5 mg/ml, 5 μl/

ml, or 0.01 M, whichever is the lowest.

(4) Relatively insoluble substances

should be tested up to or beyond their

limit of solubility under culture

conditions. Evidence of insolubility

should be determined in the final

treatment medium to which cells are

exposed. It may be useful to assess

solubility at the beginning and end of

the treatment, as solubility can change

during the course of exposure in the test

system due to presence of cells, S9,

serum etc. Insolubility can be detected

by using the unaided eye. The

precipitate should not interfere with the

scoring.

(C) Controls. (1) Concurrent positive

and negative (solvent or vehicle)

controls both with and without

metabolic activation shall be included

in each experiment. When metabolic

activation is used the positive control

chemical shall be one that requires

activation to give a mutagenic response.

(2) Examples of positive control

substances include:
43848 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

Metabolic Activation condition Locus Chemical CAS No.

Absence of exogenous metabolic activationHPRT ................................... Ethylmethanesulfonate .............................................. [CAS no. 62–50–0]

Ethylnitrosourea ......................................................... [CAS no. 759–73–9]

TK (small and large colonies).Methylmethanesulfonate ............................................ [CAS no. 66–27–3]

XPRT ................................... Ethylmethanesulfonate .............................................. [CAS no. 62–50–0]

Ethylnitrosourea ......................................................... [CAS no. 759–73–9]

Presence of exogenous

metabolic activation.

HPRT ................................... 3-Methylcholanthrene ................................................ [CAS no. 56–49–5]

N-Nitrosodimethylamine ............................................. [CAS no. 62–75–9]

7,12-Dimethylbenzanthracene ................................... [CAS no. 57–97–6]

TK (small and large colonies).Cyclophosphamide (monohydrate) ............................ [CAS no. 50–18–0]

[CAS no. 6055–19–2]

Benzo(a)pyrene ......................................................... [CAS no. 50–32–8]

3-Methylcholanthrene ................................................ [CAS no. 56–49–5]

XPRT ................................... N-Nitrosodimethylamine (for high levels of S-9) ....... [CAS no. 62–75–9]

Benzo(a)pyrene ......................................................... [CAS no. 50–32–8]

(3) Other appropriate positive control

reference substances may be used, e.g.,

if a laboratory has a historical data base

on 5-Bromo 2′-deoxyuridine [CAS No.

59–14–3], this reference substance could

be used as well. The use of chemical

class-related positive control chemicals

may be considered, when available.

(4) Negative controls, consisting of

solvent or vehicle alone in the treatment

medium, and treated in the same way as

the treatment groups shall be included.

In addition, untreated controls should

also be used unless there are historical

control data demonstrating that no

deleterious or mutagenic effects are

induced by the chosen solvent.

(3) Procedure—(i) Treatment with test

substance. (A) Proliferating cells shall

be exposed to the test substance both

with and without metabolic activation.

Exposure shall be for a suitable period

of time (usually 3 to 6 hrs is effective).

Exposure time may be extended over

one or more cell cycles.

(B) Either duplicate or single treated

cultures may be used at each

concentration tested. When single

cultures are used, the number of

concentrations should be increased to

ensure an adequate number of cultures

for analysis (e.g. at least eight

analyzsable concentrations). Duplicate

negative (solvent) control cultures

should be used.

(C) Gaseous or volatile substances

should be tested by appropriate

methods, such as in sealed culture

vessels. Methods described in the

references under paragraphs (g)(20) and

(g)(21) of this section may be used.

(ii) Measurement of survival, viability,

and mutant frequency. (A) At the end of

the exposure period, cells shall be

washed and cultured to determine

survival and to allow for expression of

the mutant phenotype. Measurement of

cytotoxicity by determining the relative

cloning efficiency (survival) or relative

total growth of the cultures is usually

initiated after the treatment period.

(B) Each locus has a defined

minimum time requirement to allow

near optimal phenotypic expression of

newly induced mutants (HPRT and

XPRT require at least 6–8 days, and TK

at least 2 days). Cells are grown in

medium with and without selective

agent(s) for determination of numbers of

mutants and cloning efficiency,

respectively. The measurement of

viability (used to calculate mutant

frequency) is initiated at the end of the

expression time by plating in nonselective medium.

(C) If the test substance is positive in

the L5178Y TK∂/- test, colony sizing

should be performed on at least one of

the test cultures (the highest positive

concentration) and on the negative and

positive controls. If the test substance is

negative in the L5178Y TK∂/- test,

colony sizing should be performed on

the negative and positive controls. In

studies using TK6TK∂/-

, colony sizing

may also be performed.

(f) Data and reporting—(1) Treatment

of results. (i) Data shall include

cytotoxicity and viability determination,

colony counts and mutant frequencies

for the treated and control cultures. In

the case of a positive response in the

L5178Y TK∂/- test, colonies are scored

using the criteria of small and large

colonies on at least one concentration of

the test substance (highest positive

concentration) and on the negative and

positive control. The molecular and

cytogenetic nature of both large and

small colony mutants has been explored

in detail and is discussed in the

references under paragraphs (g)(22) and

(g)(23) of this section. In the TK∂/- test,

colonies are scored using the criteria of

normal growth (large) and slow growth

(small) colonies (a scoring system

similar to the one described in the

reference under paragraph (g)(24) of this

section may be used). Mutant cells that

have suffered the most extensive genetic

damage have prolonged doubling times

and thus form small colonies. This

damage typically ranges in scale from

the losses of the entire gene to

karyotypically visible chromosome

aberrations. The induction of small

colony mutants has been associated

with chemicals that induce gross

chromosome aberrations. Less seriously

affected mutant cells grow at rates

similar to the parental cells and form

large colonies.

(ii) Survival (relative cloning

efficiencies) or relative total growth

shall be given. Mutant frequency shall

be expressed as number of mutant cells

per number of surviving cells.

(iii) Individual culture data shall be

provided. Additionally, all data shall be

summarized in tabular form.

(iv) There is no requirement for

verification of a clear positive response.

Equivocal results shall be clarified by

further testing preferably using a

modification of experimental

conditions. Negative results need to be

confirmed on a case-by-case basis. In

those cases where confirmation of

negative results is not considered

necessary, justification should be

provided. Modification of study

parameters to extend the range of

conditions assessed should be

considered in follow-up experiments for

either equivocal or negative results.

Study parameters that might be

modified include the concentration
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43849

spacing, and the metabolic activation

conditions.

(2) Evaluation and interpretation of

results. (i) There are several criteria for

determining a positive result, such as a

concentration-related, or a reproducible

increase in mutant frequency. Biological

relevance of the results should be

considered first. Statistical methods

may be used as an aid in evaluating the

test results. Statistical significance

should not be the only determining

factor for a positive response.

(ii) A test substance, for which the

results do not meet the criteria

described in paragraph (f)(2)(i) of this

section is considered non-mutagenic in

this system.

(iii) Although most studies will give

clearly positive or negative results, in

rare cases the data set will preclude

making a definite judgement about the

activity of the test substance. Results

may remain equivocal or questionable

regardless of the number of times the

experiment is repeated.

(iv) Positive results for an in vitro

mammalian cell gene mutation test

indicate that the test substance induces

gene mutations in the cultured

mammalian cells used. A positive

concentration-response that is

reproducible is most meaningful.

Negative results indicate that, under the

test conditions, the test substance does

not induce gene mutations in the

cultured mammalian cells used.

(3) Test report. The test report shall

include the following information:

(i) Test substance:

(A) Identification data and CAS no., if

known.

(B) Physical nature and purity.

(C) Physicochemical properties

relevant to the conduct of the study.

(D) Stability of the test substance.

(ii) Solvent/vehicle:

(A) Justification for choice of vehicle/

solvent.

(B) Solubility and stability of the test

substance in solvent/vehicle, if known.

(iii) Cells:

(A) Type and source of cells.

(B) Number of cell cultures.

(C) Number of cell passages, if

applicable.

(D) Methods for maintenance of cell

cultures, if applicable.

(E) Absence of mycoplasma.

(iv) Test conditions:

(A) Rationale for selection of

concentrations and number of cell

cultures including e.g., cytotoxicity data

and solubility limitations, if available.

(B) Composition of media, CO2

concentration.

(C) Concentration of test substance.

(D) Volume of vehicle and test

substance added.

(E) Incubation temperature.

(F) Incubation time.

(G) Duration of treatment.

(H) Cell density during treatment.

(I) Type and composition of metabolic

activation system including

acceptability criteria.

(J) Positive and negative controls.

(K) Length of expression period

(including number of cells seeded, and

subcultures and feeding schedules, if

appropriate).

(L) Selective agent(s).

(M) Criteria for considering tests as

positive, negative or equivocal.

(N) Methods used to enumerate

numbers of viable and mutant cells.

(O) Definition of colonies of which

size and type are considered (including

criteria for ‘‘small’’ and ‘‘large’’

colonies, as appropriate).

(v) Results:

(A) Signs of toxicity.

(B) Signs of precipitation.

(C) Data on pH and osmolality during

the exposure to the test substance, if

determined.

(D) Colony size if scored for at least

negative and positive controls.

(E) Laboratory’s adequacy to detect

small colony mutants with the L5178Y

TK∂/- system, where appropriate.

(F) Dose-response relationship, where

possible.

(G) Statistical analyses, if any.

(H) Concurrent negative (solvent/

vehicle) and positive control data.

(I) Historical negative (solvent/

vehicle) and positive control data with

ranges, means, and standard deviations.

(J) Mutant frequency.

(vi) Discussion of the results.

(vii) Conclusion.

(g) References. For additional

background information on this test

guideline, the following references

should be consulted. These references

are available for inspection at the TSCA

Nonconfidential Information Center,

Rm. NE–B607, Environmental

Protection Agency, 401 M St., SW.,

Washington, DC, 12 noon to 4 p.m.,

Monday through Friday, except legal

holidays.

(1) Chu, E.H.Y. and Malling, H.V.

Mammalian Cell Genetics. II. Chemical

Induction of Specific Locus Mutations

in Chinese Hamster Cells In Vitro, Proc.

National Academy Science (USA, 1968)

61, 1306–1312.

(2) Liber, H.L. and Thilly, W.G.

Mutation Assay at the Thymidine

Kinase Locus in Diploid Human

Lymphoblasts. Mutation Research. 94,

467–485 (1982).

(3) Moore, M.M., Harrington-Brock,

K., Doerr, C.L., and Dearfield, K.L.

Differential Mutant Quantitation at the

Mouse Lymphoma TK and CHO HGPRT

Loci. Mutagenesis. 4, 394–403 (1989).

(4) Aaron, C.S. and Stankowski, Jr.,

L.F. Comparison of the AS52/XPRT and

the CHO/HPRT Assays: Evaluation of

Six Drug Candidates. Mutation

Research. 223, 121–128 (1989).

(5) Aaron, C.S., Bolcsfoldi, G., Glatt,

H.R., Moore, M., Nishi, Y., Stankowski,

L., Theiss, J., and Thompson, E.

Mammalian Cell Gene Mutation Assays

Working Group Report. Report of the

International Workshop on

Standardization of Genotoxicity Test

Procedures. Mutation Research. 312,

235–239 (1994).

(6) Scott, D., Galloway, S.M.,

Marshall, R.R., Ishidate, M., Brusick, D.,

Ashby, J., and Myhr, B.C. Genotoxicity

Under Extreme Culture Conditions. A

report from ICPEMC Task Group 9.

Mutation Research. 257, 147–204

(1991).

(7) Clive, D., McCuen, R., Spector,

J.F.S., Piper, C., and Mavournin, K.H.

Specific Gene Mutations in L5178Y

Cells in Culture. A Report of the U.S.

Environmental Protection Agency GeneTox Program. Mutation Research. 115,

225–251 (1983).

(8) Li, A.P., Gupta, R.S., Heflich, R.H.,

and Wasson, J. S. A Review and

Analysis of the Chinese Hamster Ovary/

Hypoxanthine Guanine Phosphoribosyl

Transferase System to Determine the

Mutagenicity of Chemical Agents: A

Report of Phase III of the U.S.

Environmental Protection Agency GeneTox Program. Mutation Research. 196,

17–36 (1988).

(9) Li, A.P., Carver, J.H., Choy, W.N.,

Hsie, A.W., Gupta, R.S., Loveday, K.S.,

O’Neill, J.P., Riddle, J.C., Stankowski,

Jr., L.F., and Yang, L.L. A Guide for the

Performance of the Chinese Hamster

Ovary Cell/Hypoxanthine-Guanine

Phosphoribosyl Transferase Gene

Mutation Assay. Mutation Research.

189, 135–141 (1987).

(10) Liber, H.L., Yandell, D.W., and

Little, J.B. A Comparison of Mutation

Induction at the tk and hprt Loci in

Human Lymphoblastoid Cells;

Quantitative Differences are Due to an

Additional Class of Mutations at the

Autosomal TK Locus. Mutation

Research. 216, 9–17 (1989).

(11) Stankowski, L.F. Jr., Tindall,

K.R., and Hsie, A.W. Quantitative and

Molecular Analyses of Ethyl

Methanesulfonate- and ICR 191-Induced

Molecular Analyses of Ethyl

Methanesulfonate- and ICR 191-Induced

Mutation in AS52 Cells. Mutation

Reseach. 160, 133–147 (1986).

(12) Turner, N.T., Batson, A.G., and

Clive, D. Eds. Kilbey, B.J. et al.

Procedures for the L5178Y/TK∂/- >

TK∂/- Mouse Lymphoma Cell

Mutagenicity Assay. Handbook of

Mutagenicity Test Procedures (Elsevier
43850 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

Science Publishers, New York, 1984)

pp. 239–268.

(13) Arlett, C.F., Smith, D.M., Clarke,

G.M., Green, M.H.L., Cole, J., McGregor,

D.B., and Asquith, J.C. Ed. Kirkland, D.J.

Mammalian Cell Gene Mutation Assays

Based Upon Colony Formation.

Statistical Evaluation of Mutagenicity

Test Data (Cambridge University Press,

1989) pp. 66–101.

(14) Abbondandolo, A., Bonatti, S.,

Corti, G., Fiorio, R., Loprieno, N., and

Mazzaccaro, A. Induction of 6-

Thioguanine-Resistant Mutants in V79

Chinese Hamster Cells by Mouse-Liver

Microsome-Activated

Dimethylnitrosamine. Mutation

Research. 46, 365–373 (1977).

(15) Ames, B.N., McCann, J., and

Yamasaki, E. Methods for Detecting

Carcinogens and Mutagens with the

Salmonella/Mammalian-Microsome

Mutagenicity Test. Mutation Reseach.

31, 347–364 (1975).

(16) Clive, D., Johnson, K.O., Spector,

J.F.S., Batson, A.G., and Brown M.M.M.

Validation and Characterization of the

L5178Y/TK∂/- Mouse Lymphoma

Mutagen Assay System. Mutation

Reseach. 59, 61–108 (1979).

(17) Maron, D.M. and Ames, B.N.

Revised Methods for the Salmonella

Mutagenicity Test. Mutation Reseach.

113, 173, 215 (1983).

(18) Elliott, B.M., Combes, R.D.,

Elcombe, C.R., Gatehouse, D.G., Gibson,

G.G., Mackay, J.M., and Wolf, R.C.

Alternatives to Aroclor 1254-Induced S9

in In Vitro Genotoxicity Assays.

Mutagenesis. 7, 175–177 (1992).

(19) Matsushima, T., Sawamura, M.,

Hara, K., and Sugimura, T. A Safe

Substitute for Polychlorinated

Biphenyls as an Inducer of Metabolic

Activation Systems. (Eds.) de Serres,

F.J., Fouts, J.R., Bend, J.R., and Philpot,

R.M. In Vitro Metabolic Activation in

Mutagenesis Testing (Elsevier, NorthHolland, 1976) pp. 85–88.

(20) Krahn, D.F., Barsky, F.C., and

McCooey, K.T. Eds. Tice, R.R., Costa,

D.L., and Schaich, K.M. CHO/HGPRT

Mutation Assay: Evaluation of Gases

and Volatile Liquids. Genotoxic Effects

of Airborne Agents (New York, Plenum,

1982) pp. 91–103.

(21) Zamora, P.O., Benson, J.M., Li,

A.P., and Brooks, A.L. Evaluation of an

Exposure System Using Cells Grown on

Collagen Gels for Detecting Highly

Volatile Mutagens in the CHO/HGPRT

Mutation Assay. Environmental

Mutagenesis. 5, 795–801 (1983).

(22) Applegate, M.L., Moore, M.M.,

Broder, C.B., Burrell, A., and Hozier, J.C.

Molecular Dissection of Mutations at the

Heterozygous Thymidine Kinase Locus

in Mouse Lymphoma Cells. Proc.

National Academy Science (USA, 1990)

87, 51–55.

(23) Moore, M.M., Clive, D., Hozier,

J.C., Howard, B.E., Batson, A.G., Turner,

N.T., and Sawyer, J. Analysis of

Trifluorothymidine-Resistant (TFTr

)

Mutants of L5178Y/TK∂/- Mouse

Lymphoma Cells. Mutation Research.

151, 161–174 (1985).

(24) Yandell, D.W., Dryja, T.P., and

Little J.B. Molecular Genetic Analysis of

Recessive Mutations at a Heterozygous

Autosomal Locus in Human Cells.

Mutation Research. 229, 89–102 (1990).

(25) Moore, M.M. and Doerr, C.L.

Comparison of Chromosome Aberration

Frequency and Small-Colony TKDeficient Mutant Frequency in L5178Y/

TK∂/- 3.7.2C Mouse Lymphoma Cells.

Mutagenesis. 5, 609–614 (1990).

§ 799.9538 TSCA mammalian bone marrow

chromosomal aberration test.

(a) Scope. This section is intended to

meet the testing requirements under

section 4 of TSCA. The mammalian

bone marrow chromosomal aberration

test is used for the detection of

structural chromosome aberrations

induced by test compounds in bone

marrow cells of animals, usually

rodents. Structural chromosome

aberrations may be of two types,

chromosome or chromatid. An increase

in polyploidy may indicate that a

chemical has the potential to induce

numerical aberrations. With the

majority of chemical mutagens, induced

aberrations are of the chromatid-type,

but chromosome-type aberrations also

occur. Chromosome mutations and

related events are the cause of many

human genetic diseases and there is

substantial evidence that chromosome

mutations and related events causing

alterations in oncogenes and tumor

suppressor genes are involved in cancer

in humans and experimental systems.

(b) Source. The source material used

in developing this TSCA test guideline

is the OECD guideline 475 (February

1997). This source is available at the

address in paragraph (g) of this section.

(c) Definitions. The following

definitions apply to this section:

Chromatid-type aberration is

structural chromosome damage

expressed as breakage of single

chromatids or breakage and reunion

between chromatids.

Chromosome-type aberration is

structural chromosome damage

expressed as breakage, or breakage and

reunion, of both chromatids at an

identical site.

Endoreduplication is a process in

which after an S period of DNA

replication, the nucleus does not go into

mitosis but starts another S period. The

result is chromosomes with

2,4,8,...chromatids.

Gap is an achromatic lesion smaller

than the width of one chromatid, and

with minimum misalignment of the

chromatids.

Numerical aberration is a change in

the number of chromosomes from the

normal number characteristic of the

animals utilized.

Polyploidy is a multiple of the

haploid chromosome number (n) other

than the diploid number (i.e., 3n, 4n

and so on).

Structural aberration is a change in

chromosome structure detectable by

microscopic examination of the

metaphase stage of cell division,

observed as deletions and fragments,

intrachanges or interchanges.

(d) Initial considerations. (1) Rodents

are routinely used in this test. Bone

marrow is the target tissue in this test,

since it is a highly vascularised tissue,

and it contains a population of rapidly

cycling cells that can be readily isolated

and processed. Other species and target

tissues are not the subject of this

section.

(2) This chromosome aberration test is

especially relevant to assessing

mutagenic hazard in that it allows

consideration of factors of in vivo

metabolism, pharmacokinetics and

DNA-repair processes although these

may vary among species and among

tissues. An in vivo test is also useful for

further investigation of a mutagenic

effect detected by an in vitro test.

(3) If there is evidence that the test

substance, or a reactive metabolite, will

not reach the target tissue, it is not

appropriate to use this test.

(e) Test method—(1) Principle.

Animals are exposed to the test

substance by an appropriate route of

exposure and are sacrificed at

appropriate times after treatment. Prior

to sacrifice, animals are treated with a

metaphase-arresting agent (e.g.,

colchicine or Colcemid). Chromosome

preparations are then made from the

bone marrow cells and stained, and

metaphase cells are analyzed for

chromosome aberrations.

(2) Description—(i) Preparations—(A)

Selection of animal species. Rats, mice

and Chinese hamsters are commonly

used, although any appropriate

mammalian species may be used.

Commonly used laboratory strains of

young healthy adult animals should be

employed. At the commencement of the

study, the weight variation of animals

should be minimal and not exceed ±

20% of the mean weight of each sex.

(B) Housing and feeding conditions.

The temperature in the experimental

animal room should be 22°C (± 3°C).
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43851

Although the relative humidity should

be at least 30% and preferably not

exceed 70% other than during room

cleaning, the aim should be 50–60%.

Lighting should be artificial, the

sequence being 12 hrs light, 12 hrs dark.

For feeding, conventional laboratory

diets may be used with an unlimited

supply of drinking water. The choice of

diet may be influenced by the need to

ensure a suitable admixture of a test

substance when administered by this

method. Animals may be housed

individually, or be caged in small

groups of the same sex.

(C) Preparation of the animals.

Healthy young adult animals shall be

randomly assigned to the control and

treatment groups. Cages should be

arranged in such a way that possible

effects due to cage placement are

minimized. The animals are identified

uniquely. The animals are acclimated to

the laboratory conditions for at least 5

days.

(D) Preparation of doses. Solid test

substances shall be dissolved or

suspended in appropriate solvents or

vehicles and diluted, as appropriate,

prior to dosing of the animals. Liquid

test substances may be dosed directly or

diluted prior to dosing. Fresh

preparations of the test substance

should be employed unless stability

data demonstrate the acceptability of

storage.

(ii) Test conditions—(A) Solvent/

vehicle. The solvent/vehicle shall not

produce toxic effects at the dose levels

used, and shall not be suspected of

chemical reaction with the test

substance. If other than well-known

solvents/vehicles are used, their

inclusion should be supported with data

indicating their compatibility. It is

recommended that wherever possible,

the use of an aqueous solvent/vehicle

should be considered first.

(B) Controls. (1) Concurrent positive

and negative (solvent/vehicle) controls

shall be included for each sex in each

test. Except for treatment with the test

substance, animals in the control groups

should be handled in an identical

manner to the animals in the treated

groups.

(2) Positive controls shall produce

structural chromosome aberrations in

vivo at exposure levels expected to give

a detectable increase over background.

Positive control doses should be chosen

so that the effects are clear but do not

immediately reveal the identity of the

coded slides to the reader. It is

acceptable that the positive control be

administered by a route different from

the test substance and sampled at only

a single time. The use of chemical class

related positive control chemicals may

be considered, when available.

Examples of positive control substances

include:

Chemical CAS No.

Triethylenemelamine ............................................................................................................. [CAS no. 51–18–3]

Ethyl methanesulphonate ...................................................................................................... [CAS no. 62–50–0]

Ethyl nitrosourea .................................................................................................................... [CAS no. 759–73–9]

Mitomycin C ........................................................................................................................... [CAS no. 50–07–7]

Cyclophosphamide (monohydrate) ........................................................................................ [CAS no. 50–18–0]

[CAS no. 6055–19–2]

(3) Negative controls, treated with

solvent or vehicle alone, and otherwise

treated in the same way as the treatment

groups, shall be included for every

sampling time, unless acceptable interanimal variability and frequencies of

cells with chromosome aberrations are

available from historical control data. If

single sampling is applied for negative

controls, the most appropriate time is

the first sampling time. In the absence

of historical or published control data

demonstrating that no deleterious or

mutagenic effects are induced by the

chosen solvent/vehicle, untreated

controls shall be used .

(3) Procedure—(i) Number and sex of

animals. Each treated and control group

shall include at least 5 analyzable

animals per sex. If at the time of the

study there are data available from

studies in the same species and using

the same route of exposure that

demonstrate that there are no

substantial differences in toxicity

between sexes, then testing in a single

sex will be sufficient. Where human

exposure to chemicals may be sexspecific, as for example with some

pharmaceutical agents, the test should

be performed with animals of the

appropriate sex.

(ii) Treatment schedule. (A) Test

substances are preferably administered

as a single treatment. Test substances

may also be administered as a split

dose, i.e. two treatments on the same

day separated by no more than a few

hrs, to facilitate administering a large

volume of material. Other dose regimens

should be scientifically justified.

(B) Samples shall be taken at two

separate times following treatment on

one day. For rodents, the first sampling

interval is 1.5 normal cell cycle length

(the latter being normally 12–18 hr)

following treatment. Since the time

required for uptake and metabolism of

the test substance as well as its effect on

cell cycle kinetics can affect the

optimum time for chromosome

aberration detection, a later sample

collection 24 hr after the first sample

time is recommended. If dose regimens

of more than one day are used, one

sampling time at 1.5 normal cell cycle

lengths after the final treatment should

be used.

(C) Prior to sacrifice, animals shall be

injected intraperitoneally with an

appropriate dose of a metaphase

arresting agent (e.g. Colcemid or

colchicine). Animals are sampled at an

appropriate interval thereafter. For mice

this interval is approximately 3–5 hrs;

for Chinese hamsters this interval is

approximately 4–5 hrs. Cells shall be

harvested from the bone marrow and

analyzed from chromosome aberrations.

(iii) Dose levels. If a range finding

study is performed because there are no

suitable data available, it shall be

performed in the same laboratory, using

the same species, strain, sex, and

treatment regimen to be used in the

main study (an approach to dose

selection is presented in the reference

under paragraph (g)(5) of this section).

If there is toxicity, three dose levels

shall be used for the first sampling time.

These dose levels shall cover a range

from the maximum to little or no

toxicity. At the later sampling time only

the highest dose needs to be used. The

highest dose is defined as the dose

producing signs of toxicity such that

higher dose levels, based on the same

dosing regimen, would be expected to

produce lethality. Substances with

specific biological activities at low nontoxic doses (such as hormones and

mitogens) may be exceptions to the
43852 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

dose-setting criteria and should be

evaluated on a case-by-case basis. The

highest dose may also be defined as a

dose that produces some indication of

toxicity in the bone marrow (e.g. greater

than 50% reduction in mitotic index).

(iv) Limit test. If a test at one dose

level of at least 2,000 mg/kg body

weight using a single treatment, or as

two treatments on the same day,

produces no observable toxic effects,

and if genotoxicity would not be

expected based on data from

structurally related compounds, then a

full study using three dose levels may

not be considered necessary. For studies

of a longer duration, the limit dose is

2,000 mg/kg/body weight/day for

treatment up to 14 days, and 1,000 mg/

kg/body weight/day for treatment longer

than 14 days. Expected human exposure

may indicate the need for a higher dose

level to be used in the limit test.

(v) Administration of doses. The test

substance is usually administered by

gavage using a stomach tube or a

suitable intubation cannula, or by

intraperitoneal injection. Other routes of

exposure may be acceptable where they

can be justified. The maximum volume

of liquid that can be administered by

gavage or injection at one time depends

on the size of the test animal. The

volume should not exceed 2 ml/100g

body weight. The use of volumes higher

than these must be justified. Except for

irritating or corrosive substances which

will normally reveal exacerbated effects

with higher concentrations, variability

in test volume should be minimized by

adjusting the concentration to ensure a

constant volume at all dose levels.

(vi) Chromosome preparation.

Immediately after sacrifice, bone

marrow shall be obtained, exposed to

hypotonic solution and fixed. The cells

shall be then spread on slides and

stained.

(vii) Analysis. (A) The mitotic index

should be determined as a measure of

cytotoxicity in at least 1,000 cells per

animal for all treated animals (including

positive controls) and untreated

negative control animals.

(B) At least 100 cells should be

analyzed for each animal. This number

could be reduced when high numbers of

aberrations are observed. All slides,

including those of positive and negative

controls, shall be independently coded

before microscopic analysis. Since slide

preparation procedures often result in

the breakage of a proportion of

metaphases with loss of chromosomes,

the cells scored should therefore contain

a number of centromeres equal to the

number 2n ± 2.

(f) Data and reporting—(1) Treatment

of results. Individual animal data shall

be presented in tabular form. The

experimental unit is the animal. For

each animal the number of cells scored,

the number of aberrations per cell and

the percentage of cells with structural

chromosome aberration(s) shall be

evaluated. Different types of structural

chromosome aberrations shall be listed

with their numbers and frequencies for

treated and control groups. Gaps shall

be recorded separately and reported but

generally not included in the total

aberration frequency. If there is no

evidence for a difference in response

between the sexes, the data may be

combined for statistical analysis.

(2) Evaluation and interpretation of

results. (i) There are several criteria for

determining a positive result, such as a

dose-related increase in the relative

number of cells with chromosome

aberrations or a clear increase in the

number of cells with aberrations in a

single dose group at a single sampling

time. Biological relevance of the results

should be considered first. Statistical

methods may be used as an aid in

evaluating the test results (some

statistical methods are described in the

reference under paragraph (g)(6) of this

section). Statistical significance should

not be the only determining factor for a

positive response. Equivocal results

should be clarified by further testing

preferably using a modification of

experimental conditions.

(ii) An increase in polyploidy may

indicate that the test substance has the

potential to induce numerical

chromosome aberrations. An increase in

endoreduplication may indicate that the

test substance has the potential to

inhibit cell cycle progression. This

phenomenon is described in the

references under paragraphs (g)(7) and

(g)(8) of this section.

(iii) A test substance for which the

results do not meet the criteria

described in paragraph (f)(2)(i) of this

section is considered non-mutagenic in

this test.

(iv) Although most experiments will

give clearly positive or negative results,

in rare cases the data set will preclude

making a definite judgment about the

activity of the test substance. Results

may remain equivocal or questionable

regardless of the number of experiments

performed.

(v) Positive results from the in vivo

chromosome aberration test indicate

that a substance induces chromosome

aberrations in the bone marrow of the

species tested. Negative results indicate

that, under the test conditions, the test

substance does not induce chromosome

aberrations in the bone marrow of the

species tested.

(vi) The likelihood that the test

substance or its metabolites reach the

general circulation or specifically the

target tissue (e.g., systemic toxicity)

should be discussed.

(3) Test report. The test report shall

include the following information:

(i) Test substance:

(A) Identification data and CAS No.,

if known.

(B) Physical nature and purity.

(C) Physicochemical properties

relevant to the conduct of the study.

(D) Stability of the test substance, if

known.

(ii) Solvent/vehicle:

(A) Justification for choice of vehicle.

(B) Solubility and stability of the test

substance in solvent/vehicle, if known.

(iii) Test animals:

(A) Species/strain used.

(B) Number, age and sex of animals.

(C) Source, housing conditions, diet,

etc.

(D) Individual weight of the animals

at the start of the test, including body

weight range, mean and standard

deviation for each group.

(iv) Test conditions:

(A) Positive and negative (vehicle/

solvent) controls.

(B) Data from range-finding study, if

conducted.

(C) Rationale for dose level selection.

(D) Details of test substance

preparation.

(E) Details of the administration of the

test substance.

(F) Rationale for route of

administration.

(G) Methods for verifying that the test

substance reached the general

circulation or target tissue, if applicable.

(H) Conversion from diet/drinking

water test substance concentration parts

per million (ppm) to the actual dose

(mg/kg body weight/day), if applicable.

(I) Details of food and water quality.

(J) Detailed description of treatment

and sampling schedules.

(K) Methods for measurement of

toxicity.

(L) Identity of metaphase arresting

substance, its concentration and

duration of treatment.

(M) Methods of slide preparation.

(N) Criteria for scoring aberrations.

(O) Number of cells analyzed per

animal.

(P) Criteria for considering studies as

positive, negative or equivocal.

(v) Results:

(A) Signs of toxicity.

(B) Mitotic index.

(C) Type and number of aberrations,

given separately for each animal.

(D) Total number of aberrations per

group with means and standard

deviations.
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43853

(E) Number of cells with aberrations

per group with means and standard

deviations.

(F) Changes in ploidy, if seen.

(G) Dose-response relationship, where

possible.

(H) Statistical analyses, if any.

(I) Concurrent negative control data.

(J) Historical negative control data

with ranges, means and standard

deviations.

(K) Concurrent positive control data.

(vi) Discussion of the results.

(vii) Conclusion.

(g) References. For additional

background information on this test

guideline, the following references

should be consulted. These references

are available for inspection at the TSCA

Nonconfidential Information Center,

Rm. NE–B607, Environmental

Protection Agency, 401 M St., SW.,

Washington, DC, 12 noon to 4 p.m.,

Monday through Friday, except legal

holidays.

(1) Adler, I.D. Eds. S. Venitt and J.M.

Parry. Cytogenetic Tests in Mammals.

Mutagenicity Testing: A Practical

Approach. (IRL Press, Oxford,

Washington DC, 1984) pp. 275–306.

(2) Preston, R.J., Dean, B.J., Galloway,

S., Holden, H., McFee, A.F., and Shelby,

M. Mammalian In Vivo Cytogenetic

Assays: Analysis of Chromosome

Aberrations in Bone Marrow Cells.

Mutation Research. 189, 157–165

(1987).

(3) Richold, M., Chandley, A., Ashby,

J., Gatehouse, D.G., Bootman, J., and

Henderson, L. Ed. D.J. Kirkland. In Vivo

Cytogenetic Assays. Basic Mutagenicity

Tests, UKEMS Recommended

Procedures. UKEMS Subcommittee on

Guidelines for Mutagenicity Testing.

Report. Part I revised. (Cambridge

University Press, Cambridge, NY, Port

Chester, Melbourne, Sydney, 1990) pp.

115–141.

(4) Tice, R.R., Hayashi, M.,

MacGregor, J.T., Anderson, D., Blakey,

D.H., Holden, H.E., Kirsch-Volders, M.,

Oleson Jr., F.B., Pacchierotti, F., Preston,

R.J., Romagna, F., Shimada, H., Sutou,

S., and Vannier, B. Report from the

Working Group on the In Vivo

Mammalian Bone Marrow Chromosomal

Aberration Test. Mutation Research.

312, 305–312 (1994).

(5) Fielder, R.J., Allen, J.A., Boobis,

A.R., Botham, P.A., Doe, J., Esdaile, D.J.,

Gatehouse, D.G., Hodson-Walker, G.,

Morton, D.B., Kirkland, D. J., and

Richold, M. Report of British Toxicology

Society/UK Environmental Mutagen

Society Working Group: Dose Setting in

In Vivo Mutagenicity Assays.

Mutagenesis. 7, 313–319 (1992).

(6) Lovell, D.P., Anderson, D.,

Albanese, R., Amphlett, G.E., Clare, G.,

Ferguson, R., Richold, M., Papworth,

D.G., and Savage, J.R.K. Ed. Kirkland,D.

J. Statistical Analysis of In Vivo

Cytogenetic Assays. UKEMS SubCommittee on Guidelines for

Mutagenicity Testing. Report Part III.

Statistical Evaluation of Mutagenicity

Test Data (Cambridge University Press,

Cambridge, 1989) pp. 184–232.

(7) Locke-Huhle, C.

Endoreduplication in Chinese Hamster

Cells During Alpha-Radiation Induced

G2 Arrest. Mutation Research. 119, 403–

413 (1983).

(8) Huang, Y., Change, C., and Trosko,

J. E. Aphidicolin-Induced

Endoreduplication in Chinese Hamster

Cells. Cancer Research. 43, 1362–1364

(1983).

§ 799.9539 TSCA mammalian erythrocyte

micronucleus test.

(a) Scope. This section is intended to

meet the testing requirements under

section 4 of TSCA.

(1) The mammalian erythrocyte

micronucleus test is used for the

detection of damage induced by the test

substance to the chromosomes or the

mitotic apparatus of erythroblasts by

analysis of erythrocytes as sampled in

bone marrow and/or peripheral blood

cells of animals, usually rodents.

(2) The purpose of the micronucleus

test is to identify substances that cause

cytogenetic damage which results in the

formation of micronuclei containing

lagging chromosome fragments or whole

chromosomes.

(3) When a bone marrow erythroblast

develops into a polychromatic

erythrocyte, the main nucleus is

extruded; any micronucleus that has

been formed may remain behind in the

otherwise anucleated cytoplasm.

Visualization of micronuclei is

facilitated in these cells because they

lack a main nucleus. An increase in the

frequency of micronucleated

polychromatic erythrocytes in treated

animals is an indication of induced

chromosome damage.

(b) Source. The source material used

in developing this TSCA test guideline

is the OECD guideline 474 (February

1997). This source is available at the

address in paragraph (g) of this section.

(c) Definitions. The following

definitions apply to this section:

Centromere (kinetochore) is a region

of a chromosome with which spindle

fibers are associated during cell

division, allowing orderly movement of

daughter chromosomes to the poles of

the daughter cells.

Micronuclei are small nuclei, separate

from and additional to the main nuclei

of cells, produced during telophase of

mitosis (meiosis) by lagging

chromosome fragments or whole

chromosomes.

Normochromatic erythrocyte is a

mature erythrocyte that lacks ribosomes

and can be distinguished from

immature, polychromatic erythrocytes

by stains selective for ribosomes.

Polychromatic erythrocyte is a

immature erythrocyte, in an

intermediate stage of development, that

still contains ribosomes and therefore

can be distinguished from mature,

normochromatic erythrocytes by stains

selective for ribosomes.

(d) Initial considerations. (1) The bone

marrow of rodents is routinely used in

this test since polychromatic

erythrocytes are produced in that tissue.

The measurement of micronucleated

immature (polychromatic) erythrocytes

in peripheral blood is equally

acceptable in any species in which the

inability of the spleen to remove

micronucleated erythrocytes has been

demonstrated, or which has shown an

adequate sensitivity to detect agents that

cause structural or numerical

chromosome aberrations. Micronuclei

can be distinguished by a number of

criteria. These include identification of

the presence or absence of a kinetochore

or centromeric DNA in the micronuclei.

The frequency of micronucleated

immature (polychromatic) erythrocytes

is the principal endpoint. The number

of mature (normochromatic)

erythrocytes in the peripheral blood that

contain micronuclei among a given

number of mature erythrocytes can also

be used as the endpoint of the assay

when animals are treated continuously

for 4 weeks or more. This mammalian

in vivo micronucleus test is especially

relevant to assessing mutagenic hazard

in that it allows consideration of factors

of in vivo metabolism, pharmacokinetics

and DNA-repair processes although

these may vary among species, among

tissues and among genetic endpoints.

An in vivo assay is also useful for

further investigation of a mutagenic

effect detected by an in vitro system.

(2) If there is evidence that the test

substance, or a reactive metabolite, will

not reach the target tissue, it is not

appropriate to use this test.

(e) Test method—(1) Principle.

Animals are exposed to the test

substance by an appropriate route. If

bone marrow is used, the animals are

sacrificed at appropriate times after

treatment, the bone marrow extracted,

and preparations made and stained (test

techniques described in the references

under paragraphs (g)(1), (g)(2), and (g)(3)

of this section may be used). When

peripheral blood is used, the blood is

collected at appropriate times after

treatment and smear preparations are
43854 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

made and stained (the test techniques

described in the references under

paragraphs (g)(3), (g)(4), (g)(5), and (g)(6)

of this section may be used). For studies

with peripheral blood, as little time as

possible should elapse between the last

exposure and cell harvest. Preparations

are analyzed for the presence of

micronuclei.

(2) Description—(i) Preparations—(A)

Selection of animal species. Mice or rats

are recommended if bone marrow is

used, although any appropriate

mammalian species may be used. When

peripheral blood is used, mice are

recommended. However, any

appropriate mammalian species may be

used provided it is a species in which

the spleen does not remove

micronucleated erythrocytes or a

species which has shown an adequate

sensitivity to detect agents that cause

structural or numerical chromosome

aberrations. Commonly used laboratory

strains of young healthy animals should

be employed. At the commencement of

the study, the weight variation of

animals should be minimal and not

exceed ± 20% of the mean weight of

each sex.

(B) Housing and feeding conditions.

The temperature in the experimental

animal room should be 22°C (± 3°C).

Although the relative humidity should

be at least 30% and preferably not

exceed 70% other than during room

cleaning, the aim should be 50–60%.

Lighting should be artificial, the

sequence being 12 hrs light, 12 hrs dark.

For feeding, conventional laboratory

diets may be used with an unlimited

supply of drinking water. The choice of

diet may be influenced by the need to

ensure a suitable admixture of a test

substance when administered by this

route. Animals may be housed

individually, or caged in small groups of

the same sex.

(C) Preparation of the animals.

Healthy young adult animals shall be

randomly assigned to the control and

treatment groups. The animals are

identified uniquely. The animals are

acclimated to the laboratory conditions

for at least 5 days. Cages should be

arranged in such a way that possible

effects due to cage placement are

minimized.

(D) Preparation of doses. Solid test

substances shall be dissolved or

suspended in appropriate solvents or

vehicles and diluted, if appropriate,

prior to dosing of the animals. Liquid

test substances may be dosed directly or

diluted prior to dosing. Fresh

preparations of the test substance

should be employed unless stability

data demonstrate the acceptability of

storage.

(ii) Test conditions—(A) Solvent/

vehicle. The solvent/vehicle should not

produce toxic effects at the dose levels

used, and should not be suspected of

chemical reaction with the test

substance. If other than well-known

solvents/vehicles are used, their

inclusion shall be supported with

reference data indicating their

compatibility. It is recommended that

wherever possible, the use of an

aqueous solvent/vehicle should be

considered first.

(B) Controls. (1) Concurrent positive

and negative (solvent/vehicle) controls

shall be included for each sex in each

test. Except for treatment with the test

substance, animals in the control groups

should be handled in an identical

manner to animals of the treatment

groups.

(2) Positive controls shall produce

micronuclei in vivo at exposure levels

expected to give a detectable increase

over background. Positive control doses

should be chosen so that the effects are

clear but do not immediately reveal the

identity of the coded slides to the

reader. It is acceptable that the positive

control be administered by a route

different from the test substance and

sampled at only a single time. In

addition, the use of chemical classrelated positive control chemicals may

be considered, when available.

Examples of positive control substances

include:

Chemical CAS No.

Ethyl methanesulphonate ...................................................................................................... [CAS no. 62–50–0]

Ethyl nitrosourea .................................................................................................................... [CAS no. 759–73–9]

Mitomycin C ........................................................................................................................... [CAS no. 50–07–7]

Cyclophosphamide (monohydrate) ........................................................................................ [CAS no. 50–18–0]

[CAS no. 6055–19–2]

Triethylenemelamine ............................................................................................................. [CAS no. 51–18–3]

(3) Negative controls, treated with

solvent or vehicle alone, and otherwise

treated in the same way as the treatment

groups shall be included for every

sampling time, unless acceptable interanimal variability and frequencies of

cells with micronuclei are demonstrated

by historical control data. If single

sampling is applied for negative

controls, the most appropriate time is

the first sampling time. In addition,

untreated controls should also be used

unless there are historical or published

control data demonstrating that no

deleterious or mutagenic effects are

induced by the chosen solvent/vehicle.

(4) If peripheral blood is used, a pretreatment sample may also be

acceptable as a concurrent negative

control, but only in the short peripheral

blood studies (e.g., one to three

treatment(s)) when the resulting data are

in the expected range for the historical

control.

(3) Procedure—(i) Number and sex of

animals. Each treated and control group

shall include at least 5 analyzable

animals per sex (techniques described

in the reference under paragraph (g)(7)

of this section may be used). If at the

time of the study there are data available

from studies in the same species and

using the same route of exposure that

demonstrate that there are no

substantial differences between sexes in

toxicity, then testing in a single sex will

be sufficient. Where human exposure to

chemicals may be sex-specific, as for

example with some pharmaceutical

agents, the test should be performed

with animals of the appropriate sex.

(ii) Treatment schedule. (A) No

standard treatment schedule (i.e. one,

two, or more treatments at 24 h

intervals) can be recommended. The

samples from extended dose regimens

are acceptable as long as a positive

effect has been demonstrated for this

study or, for a negative study, as long as

toxicity has been demonstrated or the

limit dose has been used, and dosing

continued until the time of sampling.

Test substances may also be

administered as a split dose, i.e., two

treatments on the same day separated by

no more than a few hrs, to facilitate

administering a large volume of

material.
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43855

(B) The test may be performed in two

ways:

(1) Animals shall be treated with the

test substance once. Samples of bone

marrow shall be taken at least twice,

starting not earlier than 24 hrs after

treatment, but not extending beyond 48

hrs after treatment with appropriate

interval(s) between samples. The use of

sampling times earlier than 24 hrs after

treatment should be justified. Samples

of peripheral blood shall be taken at

least twice, starting not earlier than 36

hrs after treatment, with appropriate

intervals following the first sample, but

not extending beyond 72 hrs. When a

positive response is recognized at one

sampling time, additional sampling is

not required.

(2) If two or more daily treatments are

used (e.g. two or more treatments at 24

hr intervals), samples shall be collected

once between 18 and 24 hrs following

the final treatment for the bone marrow

and once between 36 and 48 hrs

following the final treatment for the

peripheral blood (techniques described

in the reference under paragraph (g)(8)

of this section may be used).

(C) Other sampling times may be used

in addition, when relevant.

(iii) Dose levels. If a range finding

study is performed because there are no

suitable data available, it should be

performed in the same laboratory, using

the same species, strain, sex, and

treatment regimen to be used in the

main study (guidance on dose setting is

provided in the reference in paragraph

(g)(9) of this section). If there is toxicity,

three dose levels shall be used for the

first sampling time. These dose levels

shall cover a range from the maximum

to little or no toxicity. At the later

sampling time only the highest dose

needs to be used. The highest dose is

defined as the dose producing signs of

toxicity such that higher dose levels,

based on the same dosing regimen,

would be expected to produce lethality.

Substances with specific biological

activities at low non-toxic doses (such

as hormones and mitogens) may be

exceptions to the dose-setting criteria

and should be evaluated on a case-bycase basis. The highest dose may also be

defined as a dose that produces some

indication of toxicity in the bone

marrow (e.g. a reduction in the

proportion of immature erythrocytes

among total erythrocytes in the bone

marrow or peripheral blood).

(iv) Limit test. If a test at one dose

level of at least 2,000 mg/kg body

weight using a single treatment, or as

two treatments on the same day,

produces no observable toxic effects,

and if genotoxicity would not be

expected based upon data from

structurally related substances, then a

full study using three dose levels may

not be considered necessary. For studies

of a longer duration, the limit dose is

2,000 mg/kg/body weight/day for

treatment up to 14 days, and 1,000 mg/

kg/body weight/day for treatment longer

than 14 days. Expected human exposure

may indicate the need for a higher dose

level to be used in the limit test.

(v) Administration of doses. The test

substance is usually administered by

gavage using a stomach tube or a

suitable intubation cannula, or by

intraperitoneal injection. Other routes of

exposure may be acceptable where they

can be justified. The maximum volume

of liquid that can be administered by

gavage or injection at one time depends

on the size of the test animal. The

volume should not exceed 2 ml/100g

body weight. The use of volumes higher

than these must be justified. Except for

irritating or corrosive substances which

will normally reveal exacerbated effects

with higher concentrations, variability

in test volume should be minimized by

adjusting the concentration to ensure a

constant volume at all dose levels.

(vi) Bone marrow/blood preparation.

Bone marrow cells shall be obtained

from the femurs or tibias immediately

following sacrifice. Cells shall be

removed from femurs or tibias, prepared

and stained using established methods.

Peripheral blood is obtained from the

tail vein or other appropriate blood

vessel. Blood cells are immediately

stained supravitally (the test techniques

described in the references under

paragraphs (g)(4), (g)(5), and (g)(6) of

this section may be used) or smear

preparations are made and then stained.

The use of a DNA specific stain (e.g.

acridine orange (techniques described in

the reference under paragraph (g)(10) of

this section may be used) or Hoechst

33258 plus pyronin-Y) can eliminate

some of the artifacts associated with

using a non-DNA specific stain. This

advantage does not preclude the use of

conventional stains (e.g., Giemsa).

Additional systems (e.g. cellulose

columns to remove nucleated cells (the

test techniques described in the

references under paragraph (g)(12) of

this section may be used)) can also be

used provided that these systems have

been shown to adequately work for

micronucleus preparation in the

laboratory.

(vii) Analysis. The proportion of

immature among total (immature +

mature) erythrocytes is determined for

each animal by counting a total of at

least 200 erythrocytes for bone marrow

and 1,000 erythrocytes for peripheral

blood (techniques described in the

reference under paragraph (g)(13) of this

section maybe used). All slides,

including those of positive and negative

controls, shall be independently coded

before microscopic analysis. At least

2,000 immature erythrocytes per animal

shall be scored for the incidence of

micronucleated immature erythrocytes.

Additional information may be obtained

by scoring mature erythrocytes for

micronuclei. When analyzing slides, the

proportion of immature erythrocytes

among total erythrocytes should not be

less than 20% of the control value.

When animals are treated continuously

for 4 weeks or more, at least 2,000

mature erythrocytes per animal can also

be scored for the incidence of

micronuclei. Systems for automated

analysis (image analysis) and cell

suspensions (flow cytometry) are

acceptable alternatives to manual

evaluation if appropriately justified and

validated.

(f) Data and reporting—(1) Treatment

of results. Individual animal data shall

be presented in tabular form. The

experimental unit is the animal. The

number of immature erythrocytes

scored, the number of micronucleated

immature erythrocytes, and the number

of immature among total erythrocytes

shall be listed separately for each

animal analyzed. When animals are

treated continuously for 4 weeks or

more, the data on mature erythrocytes

should also be given if it is collected.

The proportion of immature among total

erythrocytes and, if considered

applicable, the percentage of

micronucleated erythrocytes shall be

given for each animal. If there is no

evidence for a difference in response

between the sexes, the data from both

sexes may be combined for statistical

analysis.

(2) Evaluation and interpretation of

results. (i) There are several criteria for

determining a positive result, such as a

dose-related increase in the number of

micronucleated cells or a clear increase

in the number of micronucleated cells

in a single dose group at a single

sampling time. Biological relevance of

the results should be considered first.

Statistical methods may be used as an

aid in evaluating the test results (the test

techniques described in the references

paragraphs (g)(14) and (g)(15) of this

section may be used). Statistical

significance should not be the only

determining factor for a positive

response. Equivocal results should be

clarified by further testing preferably

using a modification of experimental

conditions.

(ii) A test substance for which the

results do not meet the criteria

described is considered non-mutagenic

in this test.
43856 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(iii) Although most experiments will

give clearly positive or negative results,

in rare cases the data set will preclude

making a definite judgement about the

activity of the test substance. Results,

may remain equivocal or questionable

regardless of the number of times the

experiment is repeated. Positive results

in the micronucleus test indicate that a

substance induces micronuclei which

are the result of chromosomal damage or

damage to the mitotic apparatus in the

erythroblasts of the test species.

Negative results indicate that, under the

test conditions, the test substance does

not produce micronuclei in the

immature erythrocytes of the test

species.

(iv) The likelihood that the test

substance or its metabolites reach the

general circulation or specifically the

target tissue (e.g. systemic toxicity)

should be discussed.

(3) Test report. In addition to the

reporting requirements as specified

under 40 CFR part 792, subpart J, the

following specific information shall be

reported. Both individual and summary

data should be presented.

(i) Test substance:

(A) Identification data and CAS no., if

known.

(B) Physical nature and purity.

(C) Physiochemical properties

relevant to the conduct of the study.

(D) Stability of the test substance, if

known.

(ii) Solvent/vehicle:

(A) Justification for choice of vehicle.

(B) Solubility and stability of the test

substance in the solvent/vehicle, if

known.

(iii) Test animals:

(A) Species/strain used.

(B) Number, age, and sex of animals.

(C) Source, housing conditions, diet,

etc.

(D) Individual weight of the animals

at the start of the test, including body

weight range, mean and standard

deviation for each group.

(iv) Test conditions:

(A) Positive and negative (vehicle/

solvent) control data.

(B) Data from range-finding study, if

conducted.

(C) Rationale for dose level selection.

(D) Details of test substance

preparation.

(E) Details of the administration of the

test substance.

(F) Rationale for route of

administration.

(G) Methods for verifying that the test

substance reached the general

circulation or target tissue, if applicable.

(H) Conversion from diet/drinking

water test substance concentration parts

per million (ppm) to the actual dose

(mg/kg body weight/day), if applicable.

(I) Details of food and water quality.

(J) Detailed description of treatment

and sampling schedules.

(K) Methods of slide preparation.

(L) Methods for measurement of

toxicity.

(M) Criteria for scoring

micronucleated immature erythrocytes.

(N) Number of cells analyzed per

animal.

(O) Criteria for considering studies as

positive, negative or equivocal.

(v) Results:

(A) Signs of toxicity.

(B) Proportion of immature

erythrocytes among total erythrocytes.

(C) Number of micronucleated

immature erythrocytes, given separately

for each animal.

(D) Mean ± standard deviation of

micronucleated immature erythrocytes

per group.

(E) Dose-response relationship, where

possible.

(F) Statistical analyses and method

applied.

(G) Concurrent and historical negative

control data.

(H) Concurrent positive control data.

(vi) Discussion of the results.

(vii) Conclusion.

(g) References. For additional

background information on this test

guideline, the following references

should be consulted. These references

are available for inspection at the TSCA

Nonconfidential Information Center,

Rm. NE–B607, Environmental

Protection Agency, 401 M St., SW.,

Washington, DC, 12 noon to 4 p.m.,

Monday through Friday, except legal

holidays.

(1) Heddle, J.A. A Rapid In Vivo Test

for Chromosomal Damage. Mutation

Research. 18, 187–190 (1973).

(2) Schmid, W. The Micronucleus

Test. Mutation Research. 31, 9–15

(1975).

(3) Mavournin, K.H., Blakey, D.H.,

Cimino, M.C., Salamone, M.F., and

Heddle, J.A. The In Vivo Micronucleus

Assay in Mammalian Bone Marrow and

Peripheral Blood. A report of the U.S.

Environmental Protection Agency GeneTox Program. Mutation Research. 239,

29–80 (1990).

(4) Hayashi, M., Morita, T., Kodama,

Y., Sofuni, T., and Ishidate, Jr., M. The

Micronucleus Assay with Mouse

Peripheral Blood Reticulocytes Using

Acridine Orange–Coated Slides.

Mutation Research. 245, 245–249

(1990).

(5) The Collaborative Study Group for

the Micronucleus Test (1992).

Micronucleus Test with Mouse

Peripheral Blood Erythrocytes by

Acridine Orange Supravital Staining:

The Summary Report of the 5th

Collaborative Study by CSGMT/JEMS.

MMS. Mutation Research. 278, 83–98.

(6) The Collaborative Study Group for

the Micronucleus Test (CSGMT/

JEMMS.MMS, The Mammalian

Mutagenesis Study Group of the

Environmental Mutagen Society of

Japan) Protocol recommended for the

short-term mouse peripheral blood

micronucleus test. Mutagenesis. 10,

153–159 (1995).

(7) Hayashi, M., Tice, R.R.,

MacGregor, J.T., Anderson, D., Blakey,

D.H., Kirsch-Volders, M., Oleson, Jr.

F.B., Pacchierotti, F., Romagna, F.,

Shimada, H., Sutou, S., and Vannier, B.

In Vivo Rodent Erythrocyte

Micronucleus Assay. Mutation

Research. 312, 293–304 (1994).

(8) Higashikuni, N. and Sutou, S. An

optimal, generalized sampling time of

30 ∂/- 6 h after double dosing in the

mouse peripheral blood micronucleus

test. Mutagenesis. 10, 313–319 (1995).

(9) Fielder, R.J., Allen, J.A., Boobis,

A.R., Botham, P.A., Doe, J., Esdaile, D.J.,

Gatehouse, D.G., Hodson-Walker, G.,

Morton, D.B., Kirkland, D. J., and

Richold, M. Report of British Toxicology

Society/UK Environmental Mutagen

Society Working Group: Dose Setting in

In Vivo Mutagenicity Assays.

Mutagenesis. 7, 313–319 (1992).

(10) Hayashi, M., Sofuni, T., and

Ishidate, Jr., M. An Application of

Acridine Orange Fluorescent Staining to

the Micronucleus Test. Mutation

Research. 120, 241–247 (1983).

(11) MacGregor, J.T., Wehr, C.M., and

Langlois, R.G. A Simple Fluorescent

Staining Procedure for Micronuclei and

RNA in Erythrocytes Using Hoechst

33258 and Pyronin Y. Mutation

Research. 120, 269–275 (1983).

(12) Romagna, F. and Staniforth, C.D.

The automated bone marrow

micronucleus test. Mutation Research.

213, 91–104 (1989).

(13) Gollapudi, B. and McFadden,

L.G. Sample size for the estimation of

polychromatic to normochromatic

eruthrocyte ratio in the bone marrow

micronucleus test. Mutation Research.

347, 97–99 (1995).

(14) Richold, M., Ashby, J., Bootman,

J., Chandley, A., Gatehouse, D.G., and

Henderson, L. Ed. Kirkland, D.J. In Vivo

Cytogenetics Assays. Basic Mutagenicity

Tests, UKEMS Recommended

Procedures. UKEMS Subcommittee on

Guidelines for Mutagenicity Testing.

Report. Part I revised (Cambridge

University Press, Cambridge, New York,

Port Chester, Melbourne, Sydney, 1990)

pp. 115–141.

(15) Lovell, D.P., Anderson, D.,

Albanese, R., Amphlett, G.E., Clare, G.,

Ferguson, R., Richold, M., Papworth,

D.G., and Savage, J.R.K. Ed. D.J.
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43857

Kirkland. Statistical Analysis of In Vivo

Cytogenetic Assays. Statistical

Evaluation of Mutagenicity Test Data.

UKEMS Sub-Committee on Guidelines

for Mutagenicity Testing, Report, Part

III. (Cambridge University Press,

Cambridge, New York, Port Chester,

Melbourne, Sydney, 1989) pp. 184–232.

(16) Heddle, J.A., Salamone, M.F.,

Hite, M., Kirkhart, B., Mavournin, K.,

MacGregor, J.G., and Newell, G.W. The

Induction of Micronuclei as a Measure

of Genotoxicity. Mutation Research.

123: 61–118 (1983).

(17) MacGregor, J.T., Heddle, J.A.,

Hite, M., Margolin, G.H., Ramel C.,

Salamone, M.F., Tice, R.R., and Wild, D.

Guidelines for the Conduct of

Micronucleus Assays in Mammalian

Bone Marrow Erythrocytes. Mutation

Research. 189: 103–112 (1987).

(18) MacGregor, J.T., Wehr, C.M.,

Henika, P.R., and Shelby, M.E. (1990).

The In Vivo Erythrocyte Micronucleus

Test: Measurement at Steady State

Increases Assay Efficiency and Permits

Integration with Toxicity Studies.

Fundamental Applied Toxicology. 14:

513–522.

(19) MacGregor, J.T., Schlegel, R.

Choy, W.N., and Wehr, C.M. Eds. Hayes,

A.W., Schnell, R.C., and Miya, T.S.

Micronuclei in Circulating Erythrocytes:

A Rapid Screen for Chromosomal

Damage During Routine Toxicity

Testing in Mice. Developments in

Science and Practice of Toxicology

(Elsevier, Amsterdam, 1983) pp. 555–

558.

§ 799.9620 TSCA neurotoxicity screening

battery.

(a) Scope. This section is intended to

meet the testing requirements under

section 4 of TSCA. This neurotoxicity

screening battery consists of a

functional observational battery, motor

activity, and neuropathology. The

functional observational battery consists

of noninvasive procedures designed to

detect gross functional deficits in

animals and to better quantify

behavioral or neurological effects

detected in other studies. The motor

activity test uses an automated device

that measures the level of activity of an

individual animal. The

neuropathological techniques are

designed to provide data to detect and

characterize histopathological changes

in the central and peripheral nervous

system. This battery is designed to be

used in conjunction with general

toxicity studies and changes should be

evaluated in the context of both the

concordance between functional

neurological and neuropatholgical

effects, and with respect to any other

toxicological effects seen. This test

battery is not intended to provide a

complete evaluation of neurotoxicity,

and additional functional and

morphological evaluation may be

necessary to assess completely the

neurotoxic potential of a chemical.

(b) Source. The source material used

in developing this TSCA test guideline

is the OPPTS harmonized test guideline

870.6200 (June 1996 Public Draft). This

source is available at the address in

paragraph (g) of this section.

(c) Definitions. The following

definitions apply to this section.

ED is effective dose.

Motor activity is any movement of the

experimental animal.

Neurotoxicity is any adverse effect on

the structure or function of the nervous

system related to exposure to a chemical

substance.

Toxic effect is an adverse change in

the structure or function of an

experimental animal as a result of

exposure to a chemical substance.

(d) Principle of the test method. The

test substance is administered to several

groups of experimental animals, one

dose being used per group. The animals

are observed under carefully

standardized conditions with sufficient

frequency to ensure the detection and

quantification of behavioral and/or

neurologic abnormalities, if present.

Various functions that could be affected

by neurotoxicants are assessed during

each observation period. Measurements

of motor activity of individual animals

are made in an automated device. The

animals are perfused and tissue samples

from the nervous system are prepared

for microscopic examination. The

exposure levels at which significant

neurotoxic effects are produced are

compared to one another and to those

levels that produce other toxic effects.

(e) Test procedures—(1) Animal

selection—(i) Species. In general, the

laboratory rat should be used. Under

some circumstances, other species, such

as the mouse or the dog, may be more

appropriate, although not all of the

battery may be adaptable to other

species.

(ii) Age. Young adults (at least 42 days

old for rats) shall be used.

(iii) Sex. Both males and females shall

be used. Females shall be nulliparous

and nonpregnant.

(2) Number of animals. At least 10

males and 10 females should be used in

each dose and control group for

behavioral testing. At least five males

and five females should be used in each

dose and control group for terminal

neuropathology. If interim

neuropathological evaluations are

planned, the number should be

increased by the number of animals

scheduled to be perfused before the end

of the study. Animals shall be randomly

assigned to treatment and control

groups.

(3) Control groups. (i) A concurrent

(vehicle) control group is required.

Subjects shall be treated in the same

way as for an exposure group except

that administration of the test substance

is omitted. If the vehicle used has

known or potential toxic properties,

both untreated or saline treated and

vehicle control groups are required.

(ii) Positive control data from the

laboratory performing the testing shall

provide evidence of the ability of the

observational methods used to detect

major neurotoxic endpoints including

limb weakness or paralysis, tremor, and

autonomic signs. Positive control data

are also required to demonstrate the

sensitivity and reliability of the activitymeasuring device and testing

procedures. These data should

demonstrate the ability to detect

chemically induced increases and

decreases in activity. Positive control

groups exhibiting central nervous

system pathology and peripheral

nervous system pathology are also

required. Separate groups for peripheral

and central neuropathology are

acceptable (e.g. acrylamide and

trimethyl tin). Positive control data shall

be collected at the time of the test study

unless the laboratory can demonstrate

the adequacy of historical data for this

purpose, i.e. by the approach outlined

in this section.

(4) Dose level and dose selection. At

least three doses shall be used in

addition to the vehicle control group.

The data should be sufficient to produce

a dose-effect curve. The Agency strongly

encourage the use of equally spaced

doses and a rationale for dose selection

that will maximally support detection of

dose-effect relations. For acute studies,

dose selection may be made relative to

the establishment of a benchmark dose

(BD). That is, doses may be specified as

successive fractions, e.g. 0.5, 0.25, ...n of

the BD. The BD itself may be estimated

as the highest nonlethal dose as

determined in a preliminary rangefinding lethality study. A variety of test

methodologies may be used for this

purpose, and the method chosen may

influence subsequent dose selection.

The goal is to use a dose level that is

sufficient to be judged a limit dose, or

clearly toxic.

(i) Acute studies. The high dose need

not be greater than 2 g/kg. Otherwise,

the high dose should result in

significant neurotoxic effects or other

clearly toxic effects, but not result in an

incidence of fatalities that would

preclude a meaningful evaluation of the
43858 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

data. This dose may be estimated by a

BD procedure as described under

paragraph (e)(4) of this section, with the

middle and low dose levels chosen as

fractions of the BD dose. The lowest

dose should produce minimal effect, e.g.

an ED10, or alternatively, no effects.

(ii) Subchronic and chronic studies.

The high dose need not be greater than

1 g/kg. Otherwise, the high dose level

should result in significant neurotoxic

effects or other clearly toxic effects, but

not produce an incidence of fatalities

that would prevent a meaningful

evaluation of the data. The middle and

low doses should be fractions of the

high dose. The lowest dose should

produce minimal effects, e.g. an ED10,

or alternatively, no effects.

(5) Route of exposure. Selection of

route may be based on several criteria

including, the most likely route of

human exposure, bioavailability, the

likelihood of observing effects, practical

difficulties, and the likelihood of

producing nonspecific effects. For many

materials, it should be recognized that

more than one route of exposure may be

important and that these criteria may

conflict with one another. Initially only

one route is required for screening for

neurotoxicity. The route that best meets

these criteria should be selected. Dietary

feeding will generally be acceptable for

repeated exposures studies.

(6) Combined protocol. The tests

described in this screening battery may

be combined with any other toxicity

study, as long as none of the

requirements of either are violated by

the combination.

(7) Study conduct—(i) Time of testing.

All animals shall be weighed on each

test day and at least weekly during the

exposure period.

(A) Acute studies. At a minimum, for

acute studies observations and activity

testing shall be made before the

initiation of exposure, at the estimated

time of peak effect within 8 hrs of

dosing, and at 7 and 14 days after

dosing. Estimation of times of peak

effect may be made by dosing pairs of

rats across a range of doses and making

regular observations of gait and arousal.

(B) Subchronic and chronic studies.

In a subchronic study, at a minimum,

observations and activity measurements

shall be made before the initiation of

exposure and before the daily exposure,

or for feeding studies at the same time

of day, during the 4th, 8th, and 13th

weeks of exposure. In chronic studies, at

a minimum, observations and activity

measurements shall be made before the

initiation of exposure and before the

daily exposure, or for feeding studies at

the same time of day, every 3 months.

(ii) Functional observational battery—

(A) General conduct. All animals in a

given study shall be observed carefully

by trained observers who are unaware of

the animals’ treatment, using

standardized procedures to minimize

observer variability. Where possible, it

is advisable that the same observer be

used to evaluate the animals in a given

study. If this is not possible, some

demonstration of interobserver

reliability is required. The animals shall

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 end-points specified is

encouraged. Examples of observational

procedures using defined protocols may

be found in the references under

paragraphs (g)(5), (g)(6), and (g)(9) of

this section. The functional

observational battery shall include a

thorough description of the subject’s

appearance, behavior, and functional

integrity. This shall be assessed through

observations in the home cage and

while the rat 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, should be developed for those

measures which involve subjective

ranking.

(B) List of measures. The functional

observational battery shall include the

following list of measures:

(1) Assessment of signs of autonomic

function, including but not limited to:

(i) Ranking of the degree of

lacrimation and salivation, with a range

of severity scores from none to severe.

(ii) Presence or absence of

piloerection and exophthalmus.

(iii) Ranking or count of urination and

defecation, including polyuria and

diarrhea. This is most easily conducted

during the open field assessment.

(iv) Pupillary function such as

constriction of the pupil in response to

light or a measure of pupil size.

(v) Degree of palpebral closure, e.g.,

ptosis.

(2) Description, incidence, and

severity of any convulsions, tremors, or

abnormal motor movements, both in the

home cage and the open field.

(3) 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.

(4) Ranking of the subject’s general

level of activity during observations of

the unperturbed subject in the open

field, with a range of severity scores

from unresponsive to hyperactive.

(5) Descriptions and incidence of

posture and gait abnormalities observed

in the home cage and open field.

(6) Ranking of any gait abnormalities,

with a range of severity scores from

none to severe.

(7) Forelimb and hindlimb grip

strength measured using an objective

procedure (the procedure described in

the reference under paragraph (g)(8) of

this section may be used).

(8) Quantitative measure of landing

foot splay (the procedure described in

the reference under paragraph (g)(3) of

this section may be used).

(9) 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 tailpinch, tail-flick, or hot-plate. The

response to a sudden sound, e.g., click

or snap, may be used to assess audition.

(10) Body weight.

(11) 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.

(C) 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:

(1) Count of rearing activity on the

open field.

(2) Ranking of righting ability.

(3) Body temperature.

(4) Excessive or spontaneous

vocalizations.

(5) Alterations in rate and ease of

respiration, e.g., rales or dyspnea.

(6) Sensorimotor responses to visual

or proprioceptive stimuli.

(iii) Motor activity. Motor activity

shall be monitored by an automated

activity recording apparatus. The device

used must be capable of detecting both

increases and decreases in activity, i.e.,

baseline activity as measured by the

device must not be so low as to preclude

detection of decreases nor so high as to

preclude detection of increases in

activity. Each device shall be tested by

standard procedures to ensure, to the

extent possible, reliability of operation
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43859

across devices and across days for any

one device. In addition, treatment

groups must be balanced across devices.

Each animal shall be tested

individually. The test session shall be

long enough for motor activity to

approach asymptotic levels by the last

20% of the session for nontreated

control animals. All sessions shall have

the same duration. Treatment groups

shall be counterbalanced across test

times. Effort should be made to ensure

that variations in the test conditions are

minimal and are not systematically

related to treatment. Among the

variables which can affect motor activity

are sound level, size and shape of the

test cage, temperature, relative

humidity, lighting conditions, odors,

use of the home cage or a novel test

cage, and environmental distractions.

(iv) Neuropathology: Collection,

processing and examination of tissue

samples. To provide for adequate

sampling as well as optimal

preservation of cellular integrity for the

detection of neuropathological

alterations, tissue shall be prepared for

histological analysis using in situ

perfusion and paraffin and/or plastic

embedding procedures. Paraffin

embedding is acceptable for tissue

samples from the central nervous

system. Plastic embedding of tissue

samples from the central nervous

system is encouraged, when feasible.

Plastic embedding is required for tissue

samples from the peripheral nervous

system. Subject to professional

judgment and the type of

neuropathological alterations observed,

it is recommended that additional

methods, such as glial fibrillary acidic

protein (GFAP) immunohistochemistry

and/or methods known as Bodian’s or

Bielchowsky’s silver methods be used in

conjunction with more standard stains

to determine the lowest dose level at

which neuropathological alterations are

observed. When new or existing data

provide evidence of structural

alterations it is recommended that the

GFAP immunoassay also be considered.

A description of this technique can be

found in the reference under paragraph

(g)(10) of this section.

(A) Fixation and processing of tissue.

The nervous system shall be fixed by in

situ perfusion with an appropriate

aldehyde fixative. Any gross

abnormalities should be noted. Tissue

samples taken should adequately

represent all major regions of the

nervous system. The tissue samples

should be postfixed and processed

according to standardized published

histological protocols (protocols

described in the references under

paragraphs (g)(1), (g)(2), or (g)(11) of this

section may be used). Tissue blocks and

slides should be appropriately

identified when stored. Histological

sections should be stained for

hematoxylin and eosin (H&E), or a

comparable stain according to standard

published protocols (some of these

protocols are described in the references

under paragraphs (g)(1) and (g)(11) of

this section).

(B) Qualitative examination.

Representative histological sections

from the tissue samples should be

examined microscopically by an

appropriately trained pathologist for

evidence of neuropathological

alterations. The nervous system shall be

thoroughly examined for evidence of

any treatment-related neuropathological

alterations. Particular attention should

be paid to regions known to be sensitive

to neurotoxic insult or those regions

likely to be affected based on the results

of functional tests. Such treatmentrelated neuropathological alterations

should be clearly distinguished from

artifacts resulting from influences other

than exposure to the test substance. A

stepwise examination of tissue samples

is recommended. In such a stepwise

examination, sections from the high

dose group are first compared with

those of the control group. If no

neuropathological alterations are

observed in samples from the high dose

group, subsequent analysis is not

required. If neuropathological

alterations are observed in samples from

the high dose group, samples from the

intermediate and low dose groups are

then examined sequentially.

(C) Subjective diagnosis. If any

evidence of neuropathological

alterations is found in the qualitative

examination, then a subjective diagnosis

shall be performed for the purpose of

evaluating dose-response relationships.

All regions of the nervous system

exhibiting any evidence of

neuropathological changes should be

included in this analysis. Sections from

all dose groups from each region will be

coded and examined in randomized

order without knowledge of the code.

The frequency of each type and severity

of each lesion will be recorded. After all

samples from all dose groups including

all regions have been rated, the code

will be broken and statistical analysis

performed to evaluate dose-response

relationships. For each type of doserelated lesion observed, examples of

different degrees of severity should be

described. Photomicrographs of typical

examples of treatment-related regions

are recommended to augment these

descriptions. These examples will also

serve to illustrate a rating scale, such as

1+, 2+, and 3+ for the degree of severity

ranging from very slight to very

extensive.

(f) Data reporting and evaluation. The

final test report shall include the

following information:

(1) Description of equipment and test

methods. A description of the general

design of the experiment and any

equipment used shall be provided. This

shall include a short justification

explaining any decisions involving

professional judgment.

(i) A detailed description of the

procedures used to standardize

observations, 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.

Historical control data can be critical in

the interpretation of study findings. The

Agency encourages submission of such

data to facilitate the rapid and complete

review of the significance of effects

seen.

(2) Results. The following information

shall be arranged by test group dose

level.

(i) In tabular form, data for each

animal shall be provided showing:

(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), total

session activity counts, and intrasession

subtotals for each day measured.

(ii) Summary data for each group

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.

(iii) All neuropathological

observations shall be recorded and

arranged by test groups. This data may

be presented in the following

recommended format:

(A) Description of lesions for each

animal. For each animal, data must be

submitted showing its identification

(animal number, sex, treatment, dose,

and duration), a list of structures

examined as well as the locations,

nature, frequency, and severity of

lesions. Inclusion of photomicrographs

is strongly recommended for

demonstrating typical examples of the

type and severity of the

neuropathological alterations observed.

Any diagnoses derived from
43860 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

neurological signs and lesions including

naturally occurring diseases or

conditions, should be recorded.

(B) Counts and incidence of

neuropathological alterations by test

group. Data should be tabulated to

show:

(1) The number of animals used in

each group and the number of animals

in which any lesion was found.

(2) The number of animals affected by

each different type of lesion, the

locations, frequency, and average grade

of each type of lesion.

(3) Evaluation of data. The findings

from the screening battery should be

evaluated in the context of preceding

and/or concurrent toxicity studies and

any correlated functional and

histopathological findings. The

evaluation shall include the relationship

between the doses of the test substance

and the presence or absence, incidence

and severity, of any neurotoxic effects.

The evaluation shall include

appropriate statistical analyses, for

example, parametric tests for

continuous data and nonparametric

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.

(g) References. For additional

background information on this test

guideline, the following references

should be consulted. These references

are available for inspection at the TSCA

Nonconfidential Information Center,

Rm. NE–B607, Environmental

Protection Agency, 401 M St., SW.,

Washington, DC, 12 noon to 4 p.m.,

Monday through Friday, except legal

holidays.

(1) Bennet, H.S. et al. Science and art

in the preparing tissues embedded in

plastic for light microscopy, with

special reference to glycol methacrylate,

glass knives and simple stains. Stain

Technology. 51:71–97 (1976).

(2) Di Sant Agnese, P.A. and De Mesy

Jensen, K. Dibasic staining of large

epoxy sections and application to

surgical pathology. American Journal of

Clinical Pathology. 81:25–29 (1984).

(3) Edwards, P.M. and Parker, V.H. A

simple, sensitive and objective method

for early assessment of acrylamide

neuropathy in rats. Toxicology and

Applied Pharmacology. 40:589–591

(1977).

(4) Finger, F.W. Ed. Myers, R.D.

Measuring Behavioral Activity. Vol. 2.

Methods in Psychobiology (Academic,

NY, 1972) pp.1–19.

(5) Gad, S. A neuromuscular screen

for use in industrial toxicology. Journal

of Toxicology and Environmental

Health. 9:691–704 (1982).

(6) Irwin, S. Comprehensive

observational assessment: Ia. A

systematic quantitative procedure for

assessing the behavioral physiological

state of the mouse.

Psychopharmacologia. 13:222–257

(1968).

(7) Kinnard, E.J. and Watzman, N.

Techniques utilized in the evaluation of

psychotropic drugs on animals activity.

Journal of Pharmaceutical Sciences.

55:995–1012 (1966).

(8) Meyer, O.A. et al. A method for the

routine assessment of fore- and

hindlimb grip strength of rats and mice.

Neurobehavioral Toxicology. 1:233–236

(1979).

(9) Moser V.C. et al. Comparison of

chlordimeform and carbaryl using a

functional observational battery.

Fundamental and Applied Toxicology.

11:189–206 (1988).

(10) O’Callaghan, J.P. Quantification

of glial fibrillary acidic protein:

Comparison of slot-immunobinding

assays with a novel sandwich ELISA.

Neurotoxicology and Teratology.

13:275–281 (1991).

(11) Pender, M.P. A simple method

for high resolution light microscopy of

nervous tissue. Journal of Neuroscience

Methods. 15:213–218 (1985).

(12) Reiter, L.W. Use of activity

measures in behavioral toxicology.

Environmental Health Perspectives.

26:9–20 (1978).

(13) Reiter, L.W. and MacPhail, R.C.

Motor activity: A survey of methods

with potential use in toxicity testing.

Neurobehavorial Toxicology. 1—

Supplement. 1:53–66 (1979).

(14) Robbins, T.W. Eds. Iversen, L.L.,

Iverson, D.S., and Snyder, S.H. A

critique of the methods available for the

measurement of spontaneous motor

activity. Vol 7. Handbook of

Psychopharmacology (Plenum, NY,

1977) pp. 37–82.

§ 799.9780 TSCA immunotoxicity.

(a) Scope. This section is intended to

meet the testing requirements under

section 4 of TSCA. This section is

intended to provide information on

suppression of the immune system

which might occur as a result of

repeated exposure to a test chemical.

While some information on potential

immunotoxic effects may be obtained

from hematology, lymphoid organ

weights and histopathology (usually

done as part of routine toxicity testing),

there are data which demonstrate that

these endpoints alone are not sufficient

to predict immunotoxicity (Luster et al.,

1992, 1993 see paragraphs (j)(8) and

(j)(9) of this section). Therefore, the tests

described in this section are intended to

be used along with data from routine

toxicity testing, to provide more

accurate information on risk to the

immune system. The tests in this

section do not represent a

comprehensive assessment of immune

function.

(b) Source. The source material used

in developing this TSCA test guideline

is the OPPTS harmonized test guideline

870.7800 (June 1996 Public Draft). This

source is available at the address in

paragraph (j) of this section.

(c) Definitions. The following

definitions apply to this section.

Antibodies or immunoglobulins (Ig)

are part of a large family of glycoprotein

molecules. They are produced by B cells

in response to antigens, and bind

specifically to the eliciting antigen. The

different classes of immunoglobulins

involved in immunity are IgG, IgA, IgM,

IgD, and IgE. Antibodies are found in

extracellular fluids, such as serum,

saliva, milk, and lymph. Most antibody

responses are T cell-dependent, that is,

functional T and B lymphocytes, as well

as antigen-presenting cells (usually

macrophages), are required for the

production of antibodies.

Cluster of differentiation (CD) refers to

molecules expressed on the cell surface.

These molecules are useful as distinct

CD molecules are found on different

populations of cells of the immune

system. Antibodies against these cell

surface markers (e.g., CD4, CD8) are

used to identify and quantitate different

cell populations.

Immunotoxicity refers to the ability of

a test substance to suppress immune

responses that could enhance the risk of

infectious or neoplastic disease, or to

induce inappropriate stimulation of the

immune system, thus contributing to

allergic or autoimmune disease. This

section only addresses potential

immune suppression.

Natural Killer (NK) cells are large

granular lymphocytes which

nonspecifically lyse cells bearing tumor

or viral antigens. NK cells are upregulated soon after infection by certain

microorganisms, and are thought to

represent the first line of defense against

viruses and tumors.

T and B cells are lymphocytes which

are activated in response to specific

antigens (foreign substances, usually

proteins). B cells produce antigenspecific antibodies (see the definition

for ‘‘antibodies or immunoglobulins’’),

and subpopulations of T cells are

frequently needed to provide help for

the antibody response. Other types of T

cell participate in the direct destruction

of cells expressing specific foreign
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43861

1 If absorption/distribution/metabolism/excretion

(ADME) data are similar between species, then

either rats or mice may be used for the test

compound in question. If such data are lacking,

both species should be used.

2 Because there is a fairly rapid turnover of many

of the cells in the immune system, 28 days is

considered sufficient for the purposes of the antiSRBC tests.

3 When these optional tests are included, the

phenotypic or NK cell analyses may be performed

at 28 days of exposure, or at a later timepoint if

ADME data suggest that a longer exposure is more

appropriate.

4 The study director shall be aware of strain

differences in response to SRBC. For example, if the

B6C3F1 hybrid mouse is used in the PFC assay, a

response of 800–1,000 PFC/106 spleen cells in

control mice should be the minimally acceptable

PFC response.

(tumor or infectious agent) antigens on

the cell surface.

(d) Principles of the test methods. (1)

In order to obtain data on the functional

responsiveness of major components of

the immune system to a T cell

dependent antigen, sheep red blood

cells (SRBC), rats and/or mice1 shall be

exposed to the test and control

substances for at least 28 days.2 The

animals shall be immunized by

intravenous or intraperitoneal injection

of SRBCs approximately 4 days

(depending on the strain of animal)

prior to the end of the exposure. At the

end of the exposure period, either the

plaque forming cell (PFC) assay or an

enzyme linked immunosorbent assay

(ELISA) shall be performed to determine

the effects of the test substance on the

splenic anti-SRBC (IgM) response or

serum anti-SRBC IgM levels,

respectively.

(2) In the event the test substance

produces significant suppression of the

anti-SRBC response, expression of

phenotypic markers for major

lymphocyte populations (total T and

total B), and T cell subpopulations (T

helpers (CD4) and T cytotoxic/

suppressors (CD8)), as assessed by flow

cytometry, may be performed to

determine the effects of the test

substance on either splenic or

peripheral-blood lymphocyte

populations and T cell subpopulations.

When this study is performed, the

appropriate monoclonal antibodies for

the species being tested should be used.

If the test substance has no significant

effect on the anti-SRBC assay, a

functional test for NK cells may be

performed to test for a chemical’s effect

on non-specific immunity.3 For tests

performed using cells or sera from blood

(ELISA or flow cytometry), it is not

necessary to destroy the animals, since

immunization with SRBCs at 28 days is

not expected to markedly affect the

results of other assays included in

subchronic or longer-term studies (these

tests are discussed in the reference

under paragraph (j)(7) of this section).

The necessity to perform either a

quantitative analysis of the effects of a

chemical on the numbers of cells in

major lymphocyte populations and T

Cell subpopulations by flow cytometry,

or a splenic NK cell activity assay to

assess the effects of the test compound

on non-specific immunity shall be

determined on a case-by-case basis,

depending upon the outcome of the

anti-SRBC assay.

(e) Limit test. If a test at one dose level

of at least 1,000 mg/kg body weight (or

2 mg/L for inhalation route of exposure)

using the procedures described for this

study produces no observable toxic

effects or if toxic effects 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

may indicate the need for a higher dose

level.

(f) Test procedures—(1) Animal

selection—(i) Species and strain. These

tests are intended for use in rats and/or

mice. Commonly used laboratory strains

shall be employed.4 All test animals

shall be free of pathogens, internal and

external parasites. Females shall be

nulliparous and nonpregnant. The

species, strain, and source of the

animals shall be identified.

(ii) Age/weight. (A) Young, healthy

animals shall be employed. At the

commencement of the study, the weight

variation of the animals used shall not

exceed ± 20% of the mean weight for

each sex.

(B) Dosing shall begin when the test

animals are between 6 and 8 weeks old.

(iii) Sex. Either sex may be used in the

study; if one sex is known or believed

to be more sensitive to the test

compound, then that sex shall be used.

(iv) Numbers. (A) At least eight

animals shall be included in each dose

and control group. The number of

animals tested shall yield sufficient

statistical power to detect a 20% change

based upon the interanimal variation

which may be encountered in these

assays.

(B) To avoid bias, the use of adequate

randomization procedures for the

proper allocation of animals to test and

control groups is required.

(C) Each animal shall be assigned a

unique identification number. Dead

animals, their preserved organs and

tissues, and microscopic slides shall be

identified by reference to the animal’s

unique number.

(v) Husbandry. (A) Animals may be

group-caged by sex, but the number of

animals per cage shall not interfere with

clear observation of each animal. The

biological properties of the test

substance or toxic effects (e.g.,

morbidity, excitability) may indicate a

need for individual caging.

(B) The temperature of the

experimental animal rooms shall be at

22 ± 3°C.

(C) The relative humidity of the

experimental animal rooms shall be

between 30 and 70%.

(D) Where lighting is artificial, the

sequence shall be 12 hrs light, 12 hrs

dark.

(E) Control and test animals shall be

maintained on the same type of bedding

and receive feed from the same lot. The

feed shall be analyzed to assure

adequacy of nutritional requirements of

the species tested and for impurities

that might influence the outcome of the

test. Rodents shall be fed and watered

ad libitum with food replaced at least

weekly.

(F) The study shall not be initiated

until the animals have been allowed an

adequate period of acclimatization or

quarantine to environmental conditions.

The period of acclimatization shall be at

least 1 week in duration.

(2) Control and test substances. (i)

The test substance shall be dissolved or

suspended in a suitable vehicle. Ideally,

if a vehicle or diluent is needed, it shall

not elicit toxic effects or substantially

alter the chemical or toxicological

properties of the test substance. It is

recommended that an aqueous solution

should be used. If solubility is a

problem a solution in oil may be used.

Other vehicles may be considered, but

only as a last resort.

(ii) One lot of the test substance shall

be used, if possible, throughout the

duration of the study, and the research

sample shall be stored under conditions

that maintain its purity and stability.

Prior to the initiation of the study, there

shall be a characterization of the test

substance, including the purity of the

test compound and if technically

feasible, the name and quantities of any

known contaminants and impurities.

(iii) If the test or positive control

substance is to be incorporated into feed

or another vehicle, the period during

which the test substance is stable in

such a mixture shall be determined

prior to the initiation of the study. Its

homogeneity and concentration shall

also be determined prior to the

initiation of the study and periodically

during the study. Statistically

randomized samples of the mixture

shall be analyzed to ensure that proper

mixing, formulation, and storage

procedures are being followed, and that

the appropriate concentration of the test
43862 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

or control substance is contained in the

mixture.

(3) Control groups. (i) A concurrent,

vehicle-treated control group is

required.

(ii) A separate untreated control group

is required if the toxicity of the vehicle

is unknown.

(iii) A positive control group with a

known immunosuppressant (e.g.,

cyclophosphamide) shall be included in

the study. A group of at least eight

animals shall be given the

immunosuppressive chemical.

(4) Dose levels. (i) In repeated-dose

toxicity tests, it is desirable to have a

dose-response relationship and a no

observed immunotoxic effect level.

Therefore, at least three dose levels and

a negative control shall be used, unless

a limit test is performed as specified

under paragraph (e) of this section.

(ii) The highest dose level shall not

produce significant stress, malnutrition,

or fatalities, but ideally should produce

some measurable sign of general toxicity

(e.g., a 10% loss of body weight).

(iii) The lowest dose level ideally

shall not produce any evidence of

immunotoxicity.

(5) Administration of the test

substance. (i) The test substance,

vehicle, or positive control substance

shall be administered for at least 28

days for the anti-SRBC assay. The route

of administration of the test material

will usually be oral; however, this shall

be determined by the likely route of

occupational or indoor exposure.

Therefore, under certain conditions, the

dermal or inhalation route of exposure

may be more relevant for the study. All

animals shall be dosed by the same

method during the entire experimental

period.

(ii) If the test substance is

administered by gavage, the animals are

dosed with the test substance ideally on

a 7-days-per-week basis. However,

based primarily on practical

considerations, dosing by gavage on a 5-

days-per-week basis shall be acceptable.

If the test substance is administered in

the drinking water, or mixed directly

into the diet, then exposure shall be on

a 7-days-per-week basis.

(A) For substances of low toxicity, it

is important to ensure that when

administered in the diet, the quantities

of the test substance involved do not

interfere with normal nutrition. When

the test substance is administered in the

diet, either a constant dietary

concentration in parts per million (ppm)

or a constant dose level in terms of the

animal’s body weight shall be used; the

alternative used should be specified.

(B) For a substance administered by

gavage, the dose shall be given at

approximately the same time each day,

and adjusted at intervals (weekly for

mice, twice per week for rats) to

maintain a constant dose level in terms

of the animal’s body weight.

(iii) If the test substance is

administered dermally, use paragraphs

(f)(5)(iii)(A) through (f)(5)(iii)(D) of this

section.

(A) Dose levels and dose selection. (1)

In this test, it is desirable to determine

a dose-response relationship as well as

a NOEL. Therefore, at least three dose

levels plus a control and, where

appropriate, a vehicle control

(corresponding to the concentration of

vehicle at the highest dose level) group

should be used. Doses should be spaced

appropriately to produce test groups

with a range of toxic effects. The data

should be sufficient to produce a doseresponse curve.

(2) The highest dose level should

elicit signs of toxicity but not produce

severe skin irritation or an incidence of

fatality which would prevent a

meaningful evaluation. If application of

the test substance produces severe skin

irritation, the concentration may be

reduced, although this may result in a

reduction in, or absence of, other toxic

effects at the high dose level. If the skin

has been badly damaged early in the

study, it may be necessary to terminate

the study and undertake a new one at

lower concentrations.

(3) The intermediate dose levels

should be spaced to produce a gradation

of toxic effects.

(4) The lowest dose level should not

produce any evidence of toxic effects.

(B) Preparation of animal skin.

Shortly before testing, fur should be

clipped from not less than 10% of the

body surface area for application of the

test substance. In order to dose

approximately 10% of the body surface,

the area starting at the scapulae

(shoulders) to the wing of the ileum

(hipbone) and half-way down the flank

on each side of the animal should be

shaved. Shaving should be carried out

approximately 24 hrs before dosing.

Repeated clipping or shaving is usually

needed at approximately weekly

intervals. When clipping or shaving the

fur, care should be taken to avoid

abrading the skin which could alter its

permeability.

(C) Preparation of test substance. (1)

Liquid test substances are generally

used undiluted, except as indicated in

paragraph (f)(5)(iii)(A)(2) of this section.

(2) Solids should be pulverized when

possible. The substance should be

moistened sufficiently with water or,

when necessary, a suitable vehicle to

ensure good contact with the skin.

When a vehicle is used, the influence of

the vehicle on toxicity of, and

penetration of the skin by, the test

substance should be taken into account.

(3) The volume of application should

be kept constant, e.g. less than 300

<greek-m≤L for the rat; different

concentrations of test solution should be

prepared for different dose levels.

(D) Administration of test substance.

(1) The duration of exposure should be

at least for 90 days.

(2) The animals should be treated

with test substance for at least 6 hrs/day

on a 7-day per week basis. However,

based on practical considerations,

application on a 5-day per week basis is

acceptable. Dosing should be conducted

at approximately the same time each

day.

(3) The test substance should be

applied uniformly over the treatment

site.

(4) The surface area covered may be

less for highly toxic substances. As

much of the area should be covered

with as thin and uniform a film as

possible.

(5) During the exposure period, the

test substance should be held in contact

with the skin with a porous gauze

dressing. The test site should be further

covered with nonirritating tape to retain

the gauze dressing and the test

substance and to ensure that the animals

cannot ingest the test substance.

Restrainers may be used to prevent the

ingestion of the test substance, but

complete immobilization is not

recommended.

(iv) If the test substance is

administered by the inhalation route,

use the procedures under paragraphs

(e)(2), (e)(3), (e)(6), (e)(8), (e)(9), and

(e)(10) of 40 CFR 799.9346. The

exposure time for the anti-SRBC test

shall be at least 28 days.

(6) Observation period. Duration of

the observation period shall be at least

28 days.

(7) Observation of animals. (i)

Observations shall be made at least once

each day for morbidity and mortality.

Appropriate actions shall be taken to

minimize loss of animals to the study

(e.g., necropsy of those animals found

dead and isolation or euthanasia of

weak or moribund animals).

(ii) A careful clinical examination

shall be made at least once a week.

Observations shall be detailed and

carefully recorded, preferably using

explicitly defined scales. Observations

shall include, but not be limited to:

evaluation of skin and fur, eyes and

mucous membranes; respiratory and

circulatory effects; autonomic effects,

such as salivation; central nervous

system effects, including tremors and

convulsions, changes in the level of
Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations 43863

5 If the SRBCs are administered by the

intraperitoneal route, the study director should be

aware that a low percentage of animals may not

respond because the antigen was accidentally

injected into the intestinal tract.

motor activity, gait and posture,

reactivity to handling or sensory

stimuli, grip strength, and stereotypes or

bizarre behavior (e.g., self-mutilation,

walking backwards).

(iii) Signs of toxicity shall be recorded

as they are observed, including the time

of onset, degree and duration.

(iv) Food and water consumption

shall be determined weekly.

(v) Animals shall be weighed

immediately prior to dosing, weekly

(twice per week for rats) thereafter, and

just prior to euthanasia.

(vi) Any moribund animals shall be

removed and euthanized when first

noticed. Necropsies shall be conducted

on all moribund animals, and on all

animals that die during the study.

(vii) The spleen and thymus shall be

weighed in all animals at the end of the

study.

(g) Immunotoxicity tests—(1)

Functional tests. Either a splenic PFC

assay or an ELISA shall be used to

determine the response to antigen

administration.

(i) Antibody plaque-forming cell (PFC)

assay. If the antibody PFC assay is

performed, the criteria listed under

paragraphs (g)(1)(i)(A) through

(g)(1)(i)(F) of this section shall be

adhered to. Assays described in the

references under paragraphs (j)(2) and

(j)(4) of this section may be used.

(A) The T cell-dependent antigen,

SRBC, shall be injected intravenously or

intraperitoneally, usually at 24 days

after the first dosing with the test

substance.5 Although the optimum

response time is usually 4 days after

immunization, some strains of test

animal may deviate from this time

point. The strain to be used shall be

evaluated for the optimum day for PFC

formation after immunization.

(B) The activity of each new batch of

complement shall be determined. For

any given study, the SRBCs shall be

from a single sheep, or pool of sheep, for

which the shelf life and dose for

optimum response has been determined.

(C) Modifications of the PFC assay

described in paragraph (g)(1)(i) of this

section exist and may prove useful;

however, the complete citation shall be

made for the method used, any

modifications to the method shall be

reported, and the source and, where

appropriate, the activity or purity of

important reagents shall be given.

Justification or rationale shall be

provided for each protocol modification.

Discussions of modifications of the PFC

assay are available in the references

under paragraphs (j)(5),(j)(6), and (j)(10)

of this section

(D) Samples shall be randomized and

shall be coded for PFC analysis, so that

the analyst is unaware of the treatment

group of each sample examined.

(E) Spleen cell viability shall be

determined.

(F) The numbers of IgM PFC per

spleen, and the number of IgM PFC per

106 spleen cells shall be reported.

(ii) Immunoglobulin quantification.

As an alternative to a PFC assay, the

effects of the test substance on the

antibody response to antigen may be

determined by an Enzyme-Linked

Immunosorbent Assay (ELISA).

Comparison between the PFC and

ELISA assays for immunotoxicity

assessment are discussed in the

references under paragraphs (j)(5), (j)(6),

and (j)(10) of this section. Test animals

shall be immunized with SRBCs as for

the PFC assay. IgM titers in the serum

of each test animal shall be determined

(usually 4 days after immunization). As

with the PFC assay, the optimum dose

of SRBCs and optimum time for

collection of the sera shall be

determined for the species and strain of

animal to be tested. Several methods are

described in the reference under

paragraph (j)(11) of this section).

(iii) Natural killer (NK) cell activity.

The methods described in the reference

under paragraph (j)(3) of this section

may be used to demonstrate the effects

of at least 28 days of exposure to a test

substance on spontaneous cytotoxic

activity. In this assay, splenocytes from

treated and untreated test animals are

incubated with 51Cr-labeled YAC-1

lymphoma cells. The amount of

radiolabel released from the target cells

after incubation with the effector cells

for four hrs is used as a measure of NK

cytolysis. The following points shall be

adhered to when using the NK cell

assay:

(A) Assay controls shall be included

to account for spontaneous release of

radiolabel from target cells in the

absence of effector cells, and also for the

determination of total release of

radiolabel.

(B) Target cells other than YAC-1

lymphoma cells may be appropriate for

use in the assay. In all cases, target cell

viability shall be determined.

(C) Modifications of the protocol exist

that may prove useful. However,

complete citation shall be made to the

method used. Modifications shall be

reported, and where appropriate, the

source, activity, and/or purity of the

reagents should be given. Justification or

rationale shall be provided for each

protocol modification.

(2) Enumeration of splenic or

peripheral blood total B cells, total T

cells, and T cell subpopulations. The

phenotypic analysis of total B cell, total

T cell, and T cell subpopulations from

the spleen or peripheral blood by flow

cytometry should be performed after at

least 28 days of dosing; this may be

performed at a later timepoint, if ADME

data suggest that a longer exposure is

more appropriate. If an exposure period

longer than 28 days is used, then these

tests may be performed in conjunction

with subchronic (ninety day oral,

dermal, or inhalation) toxicity studies,

when these studies are required.

Methods described in the references

under paragraphs (j)(1) and (j)(5) of this

section may be used.

(h) Data and reporting—(1) Treatment

of results—(i) Data shall be summarized

in tabular form, showing for each test

group the number of animals at the start

of the test, the number of animals

showing effects, the types of effects and

the percentage of animals displaying

each type of effect.

(ii) All observed results, quantitative

and incidental, shall be evaluated by an

appropriate statistical method. Any

generally accepted statistical methods

may be used; the statistical methods

including significance criteria shall be

selected during the design of the study.

(2) Evaluation of study results. The

findings of an immunotoxicity study

shall be evaluated in conjunction with

the findings of preceding studies and

considered in terms of other toxic

effects. The evaluation shall include the

relationship between the dose of the test

substance and the presence or absence,

and the incidence and severity of

abnormalities, including behavioral and

clinical abnormalities, gross lesions,

identified target organs, body weight

changes, effects on mortality and any

other general or specific toxic effects. A

properly conducted test shall provide a

satisfactory estimation of a no-observedeffect level. It may indicate the need for

an additional study and provide

information on the selection of dose

levels.

(3) Test report. In addition to the

reporting requirements as specified

under 40 CFR part 792, subpart J, the

following specific information shall be

reported. Both individual and summary

data should be presented.

(i) The test substance characterization

shall include:

(A) Chemical identification.

(B) Lot or batch number.

(C) Physical properties.

(D) Purity/impurities.

(E) Identification and composition of

any vehicle used.
43864 Federal Register / Vol. 62, No. 158 / Friday, August 15, 1997 / Rules and Regulations

(ii) The test system shall contain data

on:

(A) Species, strain, and rationale for

selection of animal species, if other than

that recommended.

(B) Age, body weight data, and sex.

(C) Test environment including cage

conditions, ambient temperature,

humidity, and light/dark periods.

(D) When inhalation is the route of

exposure, a description of the exposure

equipment and data shall be included as

follows:

(1) Description of test conditions; the

following exposure conditions shall be

reported:

(i) Description of exposure apparatus

including design, type, volume, source

of air, system for generating aerosols,

method of conditioning air, treatment of

exhaust air and the method of housing

the animals in a test chamber.

(ii) The equipment for measuring

temperature, humidity, and particulate

aerosol concentrations and size should

be described.

(2) Exposure data shall be tabulated

and presented with mean values and a

measure of variability (e.g., standard

deviation) and include:

(i) Airflow rates through the

inhalation equipment.

(ii) Temperature and humidity of air.

(iii) Actual (analytical or gravimetric)

concentration in the breathing zone.

(iv) Nominal concentration (total

amount of test substance fed into the

inhalation equipment divided by

volume of air).

(v) Particle size distribution,

calculated mass median aerodynamic

diameter (MMAD) and geometric

standard deviation (GSD).

(vi) Explanation as to why the desired

chamber concentration and/or particle

size could not be achieved (if

applicable) and the efforts taken to

comply with this aspect of the section.

(E) Identification of animal diet.

(iii) The test procedure shall include

the following data:

(A) Method of randomization used.

(B) Full description of experimental

design and procedure.

(C) Dose regimen including levels,

methods, and volume.

(iv) Test results should include the

following data:

(A) Group animal toxic response data

shall be tabulated by species, strain, sex,

and exposure level for:

(1) Number of animals exposed.

(2) Number of animals showing signs

of toxicity.

(3) Number of animals dying.

(B) Individual animal data shall be

presented, as well as summary (group

mean data).

(C) Date of death during the study or

whether animals survived to

termination.

(D) Date of observation of each

abnormal sign and its subsequent

course.

(E) Absolute and relative spleen and

thymus weight data.

(F) Feed and water consumption data,

when collected.

(G) Results of immunotoxicity tests.

(H) Necropsy findings of animals that

were found moribund and euthanized or

died during the study.

(I) Statistical treatment of results,

where appropriate.

(i) Quality control. A system shall be

developed and maintained to assure and

document adequate performance of

laboratory staff and equipment. The

study shall be conducted in compliance

with the 40 CFR Part 792—Good

Laboratory Practice.

(j) References. For additional

background information on this test

guideline, the following references

should be consulted. These references

are available for inspection at the TSCA

Nonconfidential Information Center,

Rm. NE–B607, Environmental

Protection Agency, 401 M St., SW.,

Washington, DC, 12 noon to 4 p.m.,

Monday through Friday, except legal

holidays.

(1) Cornacoff, J.B., Graham, C.S., and

LaBrie, T.K. Eds. Burleson, G.R., Dean,

J.H., and Munson, A.E. Phenotypic

identification of peripheral blood

mononuclear leukocytes by flow

cytometry as an adjunct to

immunotoxicity evaluation. Vol. 1.

Methods in Immunotoxicology (WileyLiss, Inc., New York, 1995) pp. 211–226.

(2) Cunningham, A.J. A method of

increased sensitivity for detecting single

antibody-forming cells. Nature.

207:1106–1107 (1965).

(3) Djeu, Julie Y. Eds. Burleson, G.R.,

Dean, J.H., and Munson, A.E. Natural

Killer Activity. Methods in

Immunotoxicology. pp. 437–449 (1995).

(4) Holsapple, M.P. Eds. Burleson,

G.R., Dean, J.H., and Munson, A.E. The

plaque-forming cell (PFC) response in

Immunotoxicology: An approach to

monitoring the primary effector function

of B lymphocytes. Vol. 1. Methods in

Immunotoxicology (Wiley-Liss, Inc.,

New York, 1995) pp. 71–108.

(5) Ladics, G.S. and Loveless, S.E. Cell

surface marker analysis of splenic

lymphocyte populations of the CD rat

for use in immunotoxicological studies.

Toxicology Methods. 4: 77–91 (1994).

(6) Ladics, G.S., Smith, C., Heaps, K.,

and Loveless, S.E. Evaluation of the

humoral immune response of CD rats

following a 2-week exposure to the

pesticide carbaryl by the oral, dermal, or

inhalation routes. Journal of Toxicology

Environmental Health. 42:143–156

(1994).

(7) Ladics., G.S., Smith, C., Heaps, K.,

Elliot, G.S., Slone, T.W., and Loveless,

S.E. Possible incorporation of an

immunotoxicological functional assay

for assessing humoral immunity for

hazard identification purposes in rats on

standard toxicology study. Toxicology.

96:225–238 (1995).

(8) Luster, M.I., Portier, C., Pait, D.G.,

White, K.L., Jr., Gennings, C., Munson,

A.E., and Rosenthal, G.J. Risk

assessment in immunotoxicology I.

Sensitivity and predictability of

immune tests. Fundamental Applied

Toxicology. 18:200–210 (1992).

(9) Luster, M.I., Portier, C., Pait, D.G.,

Rosenthal, G.J. Germolec. D.R., Corsini,

E., Blaylock, B.L., Pollock, P., Kouchi,

Y., Craig, W., White, D.L., Munson, A.E.,

and Comment, C.E. Risk Assessment in

Immunotoxicology II. Relationships

Between Immune and Host Resistance

Tests. Fundamental Applied

Toxicology. 21:71–82 (1993).

(10) Temple, L., Kawabata, T. T.,

Munson, A. E., and White, Jr., K. L.

Comparison of ELISA and plaqueforming cell assays for measuring the

humoral immune response to SRBC in

rats and mice treated with

benzo[a]pyrene or cyclophosphamide.

Fundamental Applied Toxicology.

21:412–419 (1993).

(11) Temple, L., Butterworth, L.,

Kawabata, T.T., Munson, A.E., and

White, Jr., K.L. Eds. Burleson, G.R.,

Dean, J.H., and Munson, A.E. ELISA to

Measure SRBC Specific Serum IgM:

Method and Data Evaluation. Vol. 1.

Methods in Immunotoxicology (WileyLiss, Inc., New York, 1995) pp. 137–157.

[FR Doc. 97–21413 Filed 8–14–97; 8:45 am]

BILLING CODE 6560–50–F