[Code of Federal Regulations]
[Title 40, Volume 14]
[Revised as of July 1, 2003]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR79.62]

[Page 551-555]
 
                   TITLE 40--PROTECTION OF ENVIRONMENT
 
         CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
 
PART 79--REGISTRATION OF FUELS AND FUEL ADDITIVES--Table of Contents
 
            Subpart F--Testing Requirements for Registration
 
Sec. 79.62  Subchronic toxicity study with specific health effect assessments.

    (a) Purpose--(1) General toxicity. This subchronic inhalation study 
is designed to determine a concentration-response relationship for 
potential toxic effects in rats resulting from continuous or repeated 
inhalation exposure to vehicle/engine emissions over a period of 90 
days. A subgroup of perfusion-fixed animals is required, in addition to 
the main study population, for more exacting organ and tissue histology. 
This test will provide screening information on target organ toxicities 
and on concentration levels useful for running chronic studies and 
establishing exposure criteria. Initial information on effective 
concentrations/exposures of the test atmosphere may be determined from 
the literature of previous studies or through concentration range-
finding trials prior to starting this study. This health effects 
screening test is not capable of directly determining those effects 
which have a long latency period for development (e.g., carcinogenicity 
and life-shortening), though it may permit the detremination of a no-
observed-adverse-effect level, or NOAEL.
    (2) Specific health effects assessments (HEAs). These supplemental 
studies are designed to determine the potential for reproductive/
teratologic, carcinogenic, mutagenic, and neurotoxic health effect 
outcomes from vehicle/engine emission exposures. They are done in 
combination with the subchronic toxicity study and paragraph (c) of this 
section or may be done separately as outlined by the appropriate test 
guideline.
    (i) Fertility assessment/teratology. The fertility assessment is an 
in vivo study designed to provide information on potential health 
hazards to the fetus arising from the mother's repeated exposure to 
vehicle/engine emissions before and during her pregnancy. By including a 
mating of test animals, the study provides preliminary data on the 
effects of repeated vehicle/engine emissions exposure on gonadal 
function, conception, and fertility. The fertility assessment/teratology 
guideline is found in Sec. 79.63.
    (ii) Micronucleus (MN) Assay. The MN assay is an in vivo cytogenetic 
test which gives information on potential carcinogenic and/or mutagenic 
effects of exposure to vehicle/engine emissions. The MN assay detects 
damage to the chromosomes or mitotic apparatus of cells in the tissues 
of a test subject exposed repeatedly to vehicle/engine emissions. The 
assay is based on an increase in the frequency of micronucleated 
erythrocytes found in bone marrow from treated animals compared to that 
of control animals. The guideline for the MN assay is found in 
Sec. 79.64.
    (iii) Sister Chromatid Exchange (SCE) Assay. The SCE assay is an in 
vivo analysis which gives information on potential mutagenic and/or 
carcinogenic effects of exposure to vehicle/engine emissions. The assay 
detects the ability of a chemical to enhance the exchange of DNA between 
two sister chromatids of a duplicating chromosome. This assay uses 
peripheral blood lymphocytes isolated from an exposed rodent test 
species and grown to confluence in cell culture. The guideline for the 
SCE assay is found in Sec. 79.65.
    (iv) Neurotoxicity (NTX) measures. NTX measures include (A) 
histopathology of specified central and peripheral nervous system 
tissues taken from emission-exposed rodents, and (B) an assay of brain 
tissue levels of glial fibrillary acidic protein (GFAP), a major 
filament protein of astrocytes, from emission-exposed rodents. The 
guidelines for the neurohistopathology and GFAP studies are found in 
Sec. 79.66 and Sec. 79.67, respectively.
    (b) Definitions. For the purposes of this section, the following 
definitions apply:
    No-observed-adverse-effect-level (NOAEL) means the maximum 
concentration used in a test which produces no observed adverse effects. 
A NOAEL is expressed in terms of weight or volume of test substance 
given daily per unit volume of air ([mu]g/L or ppm).

[[Page 552]]

    Subchronic inhalation toxicity means the adverse effects occurring 
as a result of the continuous or repeated daily exposure of experimental 
animals to a chemical by inhalation for part (approximately 10 percent) 
of a life span.
    (c) Principle of the test method. As long as none of the 
requirements of any study are violated by the combination, one or more 
HEAs may be combined with the general toxicity study through concurrent 
exposures of their study populations and/or by sharing the analysis of 
the same animal subjects. Requirements duplicated in combined studies 
need not be repeated. Guidelines for combining HEAs with the general 
toxicity study are as follows.
    (1) Fertility assessment. (i) The number of study animals in the 
test population is increased when the fertility assessment is run 
concurrently with the 90-day toxicity study. A minimum of 40 females per 
test group shall undergo vaginal lavage daily for two weeks before the 
start of the exposure period. The resulting wet smears are examined to 
cull those animals which are acyclic. Twenty-five females shall be 
randomly assigned to a for-breeding group with the balance of females 
assigned to a group for histopathologic examination.
    (ii) All test groups are exposed over a period of 90 days to various 
concentrations of the test atmosphere for a minimum of six hours per 
day. After seven weeks of exposures, analysis of vaginal cell smears 
shall resume on a daily basis for the 25 for-breeding females and shall 
continue for a period of four weeks or until each female in the group is 
confirmed pregnant. Following the ninth week of exposures, each for-
breeding female is housed overnight with a single study male. Matings 
shall continue for as long as two weeks, or until pregnancy is confirmed 
(pregnancy day 0). Pregnant females are only exposed through day 15 of 
their pregnancy while daily exposures continue throughout the course of 
the study for non-pregnant females and study males.
    (iii) On pregnancy day 20, pregnant females are sacrificed and their 
uteri are examined. Pregnancy status and fetal effects are recorded as 
described in Sec. 79.63. At the end of the exposure period, all males 
and non-pregnant females are sacrificed and necropsied. Testes and 
epididymal tissue samples are taken from five perfusion-fixed test 
subjects and histopathological examinations are carried out on the 
remainder of the non-pregnant females and study males.
    (2) Carcinogenicity/mutagenicity(C/M) assessment. When combined with 
the subchronic toxicity study, the main study population is used to 
perform both the in vivo MN and SCE assays. Because of the constant 
turnover of the cells to be analyzed in these assays, a separate study 
population may be used for this assessment. A study population needs 
only to be exposed a minimum of four weeks. At exposure's end, ten 
animals per exposure and control groups are anaesthetized and heart 
punctures are performed on all members. After separating blood 
components, individual lymphocyte cell cultures are set up for SCE 
analysis. One femur from each study subject is also removed and the 
marrow extracted. The marrow is smeared onto a glass slide, and stained 
for analysis of micronuclei in erythrocytes.
    (3) Neurotoxicity (NTX) measures. (i) When combined with this 
subchronic toxicity study, test animals designated for whole-body 
perfusion fixation/lung histology and exposed as part of the main animal 
population are used to perform the neurohistology portion of these 
measures. After the last exposure period, a minimum of ten animals from 
each exposure group shall be preserved in situ with fixative. Sections 
of brain, spinal cord, and proximal sciatic or tibial nerve are then 
cut, processed further in formalin, and mounted for viewing under a 
light microscope. Fibers from the sciatic or tibial nerve sample are 
teased apart for further analysis under the microscope.
    (ii) GFAP assay. After the last exposure period, a minimum of ten 
rodents from each exposure group shall be sacrificed, and their brains 
excised and divided into regions. The tissue samples are then applied to 
filter paper, washed with anti-GFAP antibody, and visualized with a 
radio-labelled Protein A. The filters are quantified for degree of

[[Page 553]]

immunoreactivity between the antibody and GFAP in the tissue samples. A 
non-radioactive ELISA format is also referenced in the GFAP guideline 
cited in paragraph (a)(2)(iv) of this section. Note: Because the GFAP 
assay requires fresh, i.e., non-preserved, brain tissue, the number of 
test animals may need to be increased to provide an adequate number of 
test subjects to complete the histopathology requirements of both the 
GFAP and the general toxicity portion of the 90-day inhalation study.
    (iii) The start of the exposure period for the NTX measures study 
population may be staggered from that of the main study group to more 
evenly distribute the analytical work required in both study 
populations. The exposures would remain the same in all other respects.
    (d) Test procedures--(1) Animal selection--(i) Species and sex. The 
rat is the recommended species. If another rodent species is used, the 
tester shall provide justification for its selection. Both sexes shall 
be used in any assessment unless it is demonstrated that one sex is 
refractory to the effects of exposure.
    (ii) Age and number. Rats shall be at least ten weeks of age at the 
beginning of the study exposure. The number of animals necessary for 
individual health effect outcomes is as follows:
    (A) Thirty rodents per concentration level/group, fifteen of each 
sex, shall be used to satisfy the reporting requirements of the 90-day 
toxicity study. Ten animals per concentration level/group shall be 
designated for whole body perfusion with fixative (by gravity) for lung 
studies, and neurohistology and testes studies, as appropriate.
    (B) Thirty-five rodents, 25 females and ten males, shall be added 
for each test concentration or control group when combining a 90-day 
toxicity study with a fertility assessment.
    (C) The tester shall provide a group of 10 animals (five animals per 
sex per experimental/control groups) in addition to the main test 
population when performing the GFAP neurotoxicity HEA.
    (2) Recovery group. The manufacturer shall include an group of 20 
animals (10 animals per sex) in the test population, exposing them to 
the highest concentration level for the entire length of the study's 
exposure period. This group shall then be observed for reversibility, 
persistence, or delayed occurrence of toxic effects during a post-
exposure period of not less than 28 days.
    (3) Inhalation exposure. (i) All data developed within this study 
shall be in accordance with good laboratory practice provisions under 
Sec. 79.60.
    (ii) The general conduct of this study shall be in accordance with 
the vehicle emissions inhalation exposure guideline in Sec. 79.61.
    (4) Observation of animals. (i) All toxicological (e.g., weight 
loss) and neurological signs (e.g., motor disturbance) shall be recorded 
frequently enough to observe any abnormality, and not less than weekly 
for all study animals. Animals shall be weighed weekly.
    (ii) The following is a minimal list of measures that shall be 
noted:
    (A) Body weight;
    (B) Subject's reactivity to general stimuli such as removal from the 
cage or handling;
    (C) Description, incidence, and severity of any convulsions, 
tremors, or abnormal motor movements in the home cage;
    (D) Descriptions and incidence of posture and gait abnormalities 
observed in the home cage;
    (E) 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.
    (iii) Any animal which dies during the test is necropsied as soon as 
possible after discovery.
    (5) Clinical examinations. (i) The following examinations shall be 
performed on the twenty animals designated as the 90-day study 
population, exclusive of pregnant dams and those study animals targeted 
for perfusion by gravity:
    (A) The following hematology determinations shall be carried out at 
least two times during the test period (after 30 days of exposure and 
just prior to

[[Page 554]]

terminal sacrifice at the end of the exposure period): hematocrit, 
hemoglobin concentration, erythrocyte count, total and differential 
leukocyte count, and a measure of clotting potential such as prothrombin 
time, thromboplastin time, or platelet count.
    (B) Clinical biochemistry determinations on blood shall be carried 
out at least two times during the test period, after 30 days of exposure 
and just prior to terminal sacrifice at the end of the exposure period, 
on all groups of animals including concurrent controls. Clinical 
biochemical testing shall include assessment of electrolyte balance, 
carbohydrate metabolism, and liver and kidney function. The selection of 
specific tests will be influenced by observations on the mode of action 
of the substance. In the absence of more specific tests, the following 
determinations may be made: calcium, phosphorus, chloride, sodium, 
potassium, fasting glucose (with period of fasting appropriate to the 
species), serum alanine aminotransferase, serum aspartate 
aminotransferase, sorbitol dehydrogenase, gamma glutamyl transpeptidase, 
urea nitrogen, albumen, blood creatinine, methemoglobin, bile acids, 
total bilirubin, and total serum protein measurements. Additional 
clinical biochemistry shall be employed, where necessary, to extend the 
investigation of observed effects, e.g., analyses of lipids, hormones, 
acid/base balance, and cholinesterase activity.
    (ii) The following examinations shall initially be performed on the 
high concentration and control groups only:
    (A) Ophthalmological examination, using an ophthalmoscope or 
equivalent suitable equipment, shall be made prior to exposure to the 
test substance and at the termination of the study. If changes in the 
eyes are detected, all animals shall be examined.
    (B) Urinalysis is not required on a routine basis, but shall be done 
when there is an indication based on expected and/or observed toxicity.
    (iii) Preservation by whole-body perfusion of fixative into the 
anaesthetized animal for lung histology of ten animals from the 90-day 
study population for each experimental and control group.
    (6) Gross pathology. With the exception of the whole body perfusion-
fixed test animals cited in paragraph (d)(1)(ii)(A) of this section, all 
rodents 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. Gross 
pathology shall be performed on the following organs and tissues:
    (i) The liver, kidneys, lungs, adrenals, brain, and gonads, 
including uterus, ovaries, testes, epididymides, seminal vesicles (with 
coagulating glands), and prostate, constitute the group of target organs 
for histology and shall be weighed as soon as possible after dissection 
to avoid drying. In addition, for other than rodent test species, the 
thyroid with parathyroids, when present, shall also be weighed as soon 
as possible after dissection to avoid drying.
    (ii) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for possible future 
histopathological examination: All gross lesions; lungs--which shall be 
removed intact, weighed, and treated with a suitable fixative to ensure 
that lung structure is maintained (perfusion with the fixative is 
considered to be an effective procedure); nasopharyngeal tissues; brain-
-including sections of medulla/pons, cerebellar cortex, and cerebral 
cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum 
with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; 
pancreas; reproductive organs: uterus; cervix; ovaries; vagina; testes; 
epididymides; prostate; and, if present, seminal vesicles; aorta; 
(skin); gall bladder (if present); esophagus; stomach; duodenum; 
jejunum; ileum; cecum; colon; rectum; urinary bladder; representative 
lymph node; (mammary gland); (thigh musculature); peripheral nerve/
tissue; (eyes); (femur--including articular surface); (spinal cord at 
three levels--cervical, midthoracic, and lumbar); and (zymbal and 
exorbital lachrymal glands).
    (7) Histopathology. Histopathology shall be performed on the 
following organs and tissues from all rodents:
    (i) All gross lesions.

[[Page 555]]

    (ii) Respiratory tract and other organs and tissues, listed in 
paragraph (d)(6)(ii) of this section (except organs/tissues in 
parentheses), of all animals in the control and high dose groups.
    (iii) The tissues mentioned in parentheses, listed in paragraph 
(d)(6)(ii) of this section, if indicated by signs of toxicity or target 
organ involvement.
    (iv) Lungs of animals in the low and intermediate dose groups shall 
also be subjected to histopathological examination, primarily for 
evidence of infection since this provides a convenient assessment of the 
state of health of the animals.
    (v) Lungs and trachea of the whole-body perfusion-fixed test animals 
cited in paragraph (d)(1)(ii)(A) of this section are examined for 
inhaled particle distribution.
    (e) Interpretation of results. All observed results, quantitative 
and incidental, shall be evaluated by an appropriate statistical method. 
The specific methods, including consideration of statistical power, 
shall be selected during the design of the study.
    (f) Test report. In addition to the reporting requirements as 
specified under Secs. 79.60 and 79.61(e), the following individual 
animal data information shall be reported:
    (1) Date of death during the study or whether animals survived to 
termination.
    (2) Date of observation of each abnormal sign and its subsequent 
course.
    (3) Individual body weight data, and group average body weight data 
vs. time.
    (4) Feed consumption data, when collected.
    (5) Hematological tests employed and all results.
    (6) Clinical biochemistry tests employed and all results.
    (7) Necropsy findings.
    (8) Type of stain/fixative and procedures used in preparing tissue 
samples.
    (9) Detailed description of all histopathological findings.
    (10) Statistical treatment of the study results, where appropriate.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted.
    (1) 40 CFR 798.2450, Inhalation toxicity.
    (2) 40 CFR 798.2675, Oral Toxicity with Satellite Reproduction and 
Fertility Study.
    (3) General Statement of Work for the Conduct of Toxicity and 
Carcinogenicity Studies in Laboratory Animals (revised April, 1987/
modifications through January, 1990) appendix G, National Toxicology 
Program--U.S. Dept. of Health and Human Services (Public Health 
Service), P.O. Box 12233, Research Triangle Park, NC 27709.

[59 FR 33093, June 27, 1994, as amended at 63 FR 63793, Nov. 17, 1998]