40 C.F.R. §§ 79.62–79.68

7 sections in range

§79.62. Subchronic toxicity study with specific health effect assessments.

40 C.F.R. § 79.62

(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 § 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 § 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 § 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 § 79.66 and § 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 (µg/L or ppm).

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 § 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 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 a 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 § 79.60.

(ii)

The general conduct of this study shall be in accordance with the vehicle emissions inhalation exposure guideline in § 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 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.

(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 §§ 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.

Notes, amendments, and revision history

Amendments

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

Source

Source: 59 FR 33093, June 27, 1994, unless otherwise noted.

Authority

Authority: 42 U.S.C. 7414, 7524, 7545 and 7601.

Source

Source: 40 FR 52011, Nov. 7, 1975, unless otherwise noted.

Amendments

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

§79.63. Fertility assessment/teratology.

40 C.F.R. § 79.63

(a)

Purpose. Fertility assessment/teratology is an in vivo study designed to provide information on potential health hazards to the fetus arising from the mother's repeated inhalation 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. Since this is a one-generation test that ends with examination of full-term fetuses, but not of live pups, it is not capable of determining effects on reproductive development which would only be detected in viable offspring of treated parents.

(b)

Definitions. For the purposes of this section, the following definitions apply:

Developmental toxicity means the ability of an agent to induce in utero death, structural or functional abnormalities, or growth retardation after contact with the pregnant animal.

Estrous cycle means the periodic recurrence of the biological phases of the female reproductive system which prepare the animal for conception and the development of offspring. The phases of the estrous cycle for a particular animal can be characterized by the general condition of the cells present in the vagina and the presence or absence of various cell types.

Vaginal cytology evaluation means the use of wet vaginal cell smears to determine the phase of a test animal's estrous cycle and the potential for adverse exposure effects on the regularity of the animal's cycle. In the rat, common cell types found in the smears correlate well with the various stages of the estrous cycle and to changes occurring in the reproductive tract.

(c)

Principle of the test method.

(1)

For a two week period before exposures start, daily vaginal cell smears are examined from a surplus of female test animals to identify and cull those females which are acyclic. After culling, testers shall randomly assign at each exposure concentration (including unexposed) a minimum of twenty-five females for breeding and fifteen non-bred females for later histologic evaluation. Test animals shall be exposed by inhalation to graduated concentrations of the test atmosphere for a minimum of six hours per day over the next 13 weeks. Males and females in both test and control groups are mated after nine weeks of exposure. Exposures for pregnant females continue through gestation day 15, while exposures for males and all non-pregnant females shall continue for the full exposure period.

(2)

Beginning two weeks before the start of the mating period, daily vaginal smears resume for all to-be-bred females to characterize their estrous cycles. This will continue for four weeks or until a rat's pregnancy is confirmed, i.e., day 0, by the presence of sperm in the cell smear. On pregnancy day 20, shortly before the expected date of delivery, each pregnant female is sacrificed, her uterus removed, and the contents examined for embryonic or fetal deaths, and live fetuses. At the end of the exposure period, males and all non-pregnant females shall be weighed, and various organs and tissues, as appropriate, shall be removed and weighed, fixed with stain, and sectioned for viewing under a light microscope.

(3)

This assay may be done separately or in combination with the subchronic toxicity study, pursuant to the provisions in § 79.62.

(d)

Limit test. If a test at one dose level of the highest concentration that can be achieved while maintaining a particle size distribution with a mass median aerodynamic diameter (MMAD) of 4 micrometers (µm) or less, using the procedures described in section 79.60 of this part produces no observable toxic effects and if toxicity would not be expected based upon data of structurally related compounds, then a full study using three dose levels might not be necessary. Expected human exposure though may indicate the need for a higher dose level.

(e)

Test procedures—

(1)

Animal selection—

(i)

Species and strain. The rat is the preferred species. Strains with low fecundity shall not be used and the candidate species shall be characterized for its sensitivity to developmental toxins. If another rodent species is used, the tester shall provide justification for its selection.

(ii)

Animals shall be a minimum of 10 weeks old at the start of the exposure period.

(iii)

Number and sex. Each test and control group shall have a minimum of 25 males and 40 females. In order to ensure that sufficient pups are produced to permit meaningful evaluation of the potential developmental toxicity of the test substance, twenty pregnant test animals are required for each exposure and control level.

(2)

Observation period. The observation period shall be 13 weeks, at a minimum.

(3)

Concentration levels and concentration selection.

(i)

To select the appropriate concentration levels, a pilot or trial study may be advisable. Since pregnant animals have an increased minute ventilation as compared to non-pregnant animals, it is recommended that the trial study be conducted in pregnant animals. Similarly, since presumably the minute ventilation will vary with progression of pregnancy, the animals should be exposed during the same period of gestation as in the main study. It is not always necessary, though, to carry out a trial study in pregnant animals. Comparisons between the results of a trial study in non-pregnant animals, and the main study in pregnant animals will demonstrate whether or not the test substance is more toxic in pregnant animals. In the trial study, the concentration producing embryonic or fetal lethalities or maternal toxicity should be determined.

(ii)

The highest concentration level shall induce some overt maternal toxicity such as reduced body weight or body weight gain, but not more than 10 percent maternal deaths.

(iii)

The lowest concentration level shall not produce any grossly observable evidence of either maternal or developmental toxicity.

(4)

Inhalation exposure.

(i)

All data developed within this study shall be in accordance with good laboratory practice provisions under § 79.60.

(ii)

The general conduct of this study shall be in accordance with the vehicle emissions inhalation exposure guideline in § 79.61.

(iii)

Pregnant females shall be exposed to the test atmosphere on each and every day between (and including) the first and fifteenth day of gestation.

(f)

Test performance—

(1)

Study conduct. Directions specific to this study are:

(i)

The duration of exposure shall be at least six hours daily, allowing appropriate additional time for chamber equilibrium.

(ii)

Where an exposure chamber is used, its design shall minimize crowding of the test animals. This is best accomplished by individual caging.

(iii)

Pregnant animals shall not be subjected to beyond the minimum amount of stress. Since whole-body exposure appears to be the least stressful mode of exposure, it is the preferred method. In general oronasal or head-only exposure, which is sometimes used to avoid concurrent exposure by the dermal or oral routes, is not recommended because of the associated stress accompanying the restraining of the animals. However, there may be specific instances where it may be more appropriate than whole-body exposure. The tester shall provide justification/reasoning for its selection.

(iv)

Measurements shall be made at least every other day of food consumption for all animals in the study. Males and females shall be weighed on the first day of exposure and 2-3 times per week thereafter, except for pregnant dams.

(v)

The test animal housing, mating, and exposure chambers shall be operated on a twenty-four hour lighting schedule, with twelve hours of light and twelve hours of darkness. Test animal exposure shall only occur during the light portion of the cycle.

(vi)

Signs of toxicity shall be recorded as they are observed including the time of onset, degree, and duration.

(vii)

Females showing signs of abortion or premature delivery shall be sacrificed and subjected to a thorough macroscopic examination.

(viii)

Animals that die or are euthanized because of morbidity will be necropsied promptly.

(2)

Vaginal cytology.

(i)

For a two week period before the mating period starts, each female in the to-be-bred population shall undergo a daily saline vaginal lavage. Two wet cell smears from this lavage shall be examined daily for each subject to determine a baseline pattern of estrus. Testers shall avoid excessive handling and roughness in obtaining the vaginal cell samples, as this may induce a condition of pseudo-pregnancy in the test animals.

(ii)

This will continue for four weeks or until day 0 of a rat's pregnancy is confirmed by the presence of sperm in the cell smear.

(3)

Mating and fertility assessment.

(i)

Beginning nine weeks after the start of exposure, each exposed and control group female (exclusive of the histology group females) shall be paired during non-exposure hours with a male from the same exposure concentration group. Matings shall continue for a period of two weeks, or until all mated females are determined to be pregnant. Mating pairs shall be clearly identified.

(ii)

Each morning, including weekends, cages shall be examined for the presence of a sperm plug. When found, this shall mark gestation day 0 and pregnancy shall be confirmed by the presence of sperm in the day's wet vaginal cell smears.

(iii)

Two weeks after mating is begun, or as females are determined to be pregnant, bred animals are returned to pre-mating housing. Daily exposures continues through gestation day 15 for all pregnant females or through the balance of the exposure period for non-pregnant females and all males.

(iv)

Those pairs which fail to mate shall be evaluated in the course of the study to determine the cause of the apparent infertility. This may involve such procedures as additional opportunities to mate with a proven fertile partner, histological examination of the reproductive organs, and, in males, examination of the spermatogenic cycles. The stage of estrus for each non-pregnant female in the breeding group will be determined at the end of the exposure period.

(4)

All animals in the histology group shall be subject to histopathologic examination at the end of the study's exposure period.

(g)

Treatment of results.

(1)

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.

(2)

Data and reporting. In addition to the reporting requirements specified under §§ 79.60 and 79.61, the final test report must include the following information:

(i)

Gross necropsy.

(A)

All animals shall be subjected to a full necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. Special attention shall be directed to the organs of the reproductive system.

(B)

The liver, kidneys, adrenals, pituitary, uterus, vagina, ovaries, testes, epididymides and seminal vesicles (with coagulating glands), and prostate shall be weighed wet, as soon as possible after dissection, to avoid drying.

(i) At the time of sacrifice on gestation day 20 or at death during the study, each dam shall be examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.

(ii) The contents of the uterus shall be examined for embryonic or fetal deaths and the number of viable fetuses. Gravid uterine weights need not be obtained from dead animals where decomposition has occurred. The degree of resorption shall be described in order to help estimate the relative time of death.

(iii) The number of corpora lutea shall be determined in each pregnant dam.

(iv) Each fetus shall be weighed, all weights recorded, and mean fetal weights determined.

(v) Each fetus shall be examined externally and the sex determined.

(vi) One-half of the rat fetuses in each litter shall be examined for skeletal anomalies, and the remaining half shall be examined for soft tissue anomalies, using appropriate methods.

(ii)

Histopathology.

(A)

Histopathology on vagina, uterus, ovaries, testes, epididymides, seminal vesicles, and prostate as appropriate for all males and histology group females in the control and high concentration groups and for all animals that died or were euthanized during the study. If abnormalities or equivocal results are seen in any of these organs/tissues, the same organ/tissue from test animals in lower concentration groups shall be examined.

(B)

All gross lesions in all study animals shall be examined.

(C)

As noted under mating procedures, reproductive organs of animals suspected of infertility shall be subject to microscopic examination.

(D)

The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for future histopathological examination: all gross lesions; vagina; uterus; ovaries; testes; epididymides; seminal vesicles; prostate; liver; and kidneys/adrenals.

(3)

Evaluation of results.

(i)

The findings of a developmental toxicity study shall be evaluated in terms of the observed effects and the exposure levels producing effects. It is necessary to consider the historical developmental toxicity data on the species/strain tested.

(ii)

There are several criteria for determining a positive result for reproductive/teratologic effects; a statistically significant dose-related decrease in the weight of the testes for treated subjects over control subjects, a decrease in neonatal viability, a significant change in the presence of soft tissue or skeletal abnormalities, or an increased rate of embryonic or fetal resorption or death. Other criteria, e.g., lengthening of the estrous cycle or the time spent in any one stage of estrus, changes in the proportion of viable male vs female fetuses or offspring, the number and type of cells in vaginal smears, or pathologic changes found during gross or microscopic examination of male or female reproductive organs may be based upon detection of a reproducible and statistically significant positive response for that evaluation parameter. A positive result indicates that, under the test conditions, the test substance does induce reproductive organ or fetal toxicity in the test species.

(iii)

A test substance which does not produce either a statistically significant dose-related change in the reproductive organs or cycle or a statistically significant and reproducible positive response at any one of the test points may not induce reproductive organ toxicity in this test species, but further investigation , e.g., to establish absorption and bioavailability of the test substance, should be considered.

(h)

Test report. In addition to the reporting requirements as specified under 40 CFR 79.60 and the vehicle emissions inhalation toxicity guideline as published in 40 CFR 79.61, the following specific information shall be reported:

(1)

Individual animal data.

(i)

Time of death during the study or whether animals survived to termination.

(ii)

Date of onset and duration of each abnormal sign and its subsequent course.

(iii)

Feed and body weight data.

(iv)

Necropsy findings.

(v)

Male test subjects.

(A)

Testicle weight, and body weight— testicle weight ratio.

(B)

Detailed description of all histopathological findings, especially for the testes and the epididymides.

(vi)

Female test subjects.

(A)

Uterine weight data.

(B)

Beginning and ending collection dates for vaginal cell smears.

(C)

Estrous cycle length compared within and between groups including mean cycle length for groups.

(D)

Percentage of time spent in each stage of cycle.

(E)

Stage of estrus at time of mating/sacrifice and proportion of females in estrus between concentration groups.

(F)

Detailed description of all histopathological findings, especially for uterine/ovary samples.

(vii)

Pregnancy and litter data. Toxic response data by exposure level, including but not limited to, indices of fertility and time-to-mating, including the number of days until mating and the number of full or partial estrous cycles until mating.

(A)

Number of pregnant animals,

(B)

Number and percentage of live fetuses, resorptions.

(viii)

Fetal data.

(A)

Numbers of each sex.

(B)

Number of fetuses with any soft tissue or skeletal abnormalities.

(2)

Type of stain/fixative and procedures used in preparing tissue samples.

(3)

Statistical treatment of the study results.

(i)

References. For additional background information on this test guideline, the following references should be consulted.

(1)

40 CFR 798.2675, Oral Toxicity with Satellite Reproduction and Fertility Study.

(2)

40 CFR 798.4350, Inhalation Developmental Toxicity Study.

(3)

Chapin, R.E. and J.J. Heindel (1993) Methods in Toxicology, Vol. 3, Parts A and B: Reproductive Toxicology, Academic Press, Orlando, FL.

(4)

Gray, L.E., et al. (1989) “A Dose-Response Analysis of Methoxychlor-Induced Alterations of Reproductive Development and Function in the Rat” Fund. App. Tox. 12, 92-108.

(5)

Leblond, C.P. and Y. Clermont (1952) “Definition of the Stages of the Cycle of the Seminiferous Epithelium of the Rat.” Ann. N. Y. Acad. Sci. 55:548-73.

(6)

Morrissey, R.E., et al. (1988) “Evaluation of Rodent Sperm, Vaginal Cytology, and Reproductive Organ Weight Data from National Toxicology Program 13-week Studies.” Fundam. Appl. Toxicol. 11:343-358.

(7)

Russell, L.D., Ettlin, R.A., Sinhattikim, A.P., and Clegg, E.D (1990) Histological and Histopathological Evaluation of the Testes, Cache River Press, Clearwater, FL.

Notes, amendments, and revision history

Amendments

[59 FR 33093, June 27, 1994, as amended at 61 FR 36513, July 11, 1996]

Source

Source: 59 FR 33093, June 27, 1994, unless otherwise noted.

Authority

Authority: 42 U.S.C. 7414, 7524, 7545 and 7601.

Source

Source: 40 FR 52011, Nov. 7, 1975, unless otherwise noted.

Amendments

[59 FR 33093, June 27, 1994, as amended at 61 FR 36513, July 11, 1996]

§79.64. In vivo micronucleus assay.

40 C.F.R. § 79.64

(a)

Purpose. The micronucleus assay is an in vivo cytogenetic test which uses erythrocytes in the bone marrow of rodents to detect chemical damage to the chromosomes or mitotic apparatus of mammalian cells. As the erythroblast develops into an erythrocyte (red blood cell), its main nucleus is extruded and may leave a micronucleus in the cell body; a few micronuclei form under normal conditions in blood elements. This 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 visualization of micronuclei is facilitated in these cells because they lack a main nucleus.

(b)

Definitions. For the purposes of this section the following definitions apply:

Micronuclei mean small particles consisting of acentric fragments of chromosomes or entire chromosomes, which lag behind at anaphase of cell division. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm.

Polychromatic erythrocyte (PCE) means an immature red blood cell that, because it contains RNA, can be differentiated by appropriate staining techniques from a normochromatic erythrocyte (NCE), which lacks RNA. In one to two days, a PCE matures into a NCE.

(c)

Test method—

(1)

Principle of the test method.

(i)

Groups of rodents are exposed by the inhalation route for a minimum of 6 hours/day over a period of not less than 28 days to three or more concentrations of a test substance in air. Groups of animals are sacrificed at the end of the exposure period and femoral bone marrow is extracted. The bone marrow is then smeared onto glass slides, stained, and PCEs are scored for micronuclei. Researchers may need to run a trial at the highest tolerated concentration of the test atmosphere to optimize the sample collection time for micronucleated cells.

(ii)

This assay may be done separately or in combination with the subchronic toxicity study, pursuant to the provisions in § 79.62.

(2)

Species and strain.

(i)

The rat is the recommended test animal. Other rodent species may be used in this assay, but use of that species will be justified by the tester.

(ii)

If a strain of mouse is used in this assay, the tester shall sample peripheral blood from an appropriate site on the test animal, e.g., the tail vein, as a source of normochromatic erythrocytes. Results shall be reported as outlined later in this guideline with “normochromatic” interchanged for “polychromatic”, where specified.

(3)

Animal number and sex. At least five female and five male animals per experimental/sample and control group shall be used. The use of a single sex or a smaller number of animals shall be justified.

(4)

Positive control group. A single concentration of a compound known to produce micronuclei in vivo is adequate as a positive control if it shows a significant response at any one time point; additional concentration levels may be used. To select an appropriate concentration level, a pilot or trial study may be advisable. Initially, one concentration of the test substance may be used, the maximum tolerated dose or that producing some indication of toxicity, e.g., a drop in the ratio of polychromatic to normochromatic erythrocytes. Intraperitoneal injection of 1,2-dimethyl-benz-anthracene or benzene are examples of positive control exposures. A concentration of 50-80 percent of an LD50 may be a suitable guide.

(d)

Test performance—

(1)

Inhalation exposure.

(i)

All data developed within this study shall be in accordance with good laboratory practice provisions under § 79.60.

(ii)

The general conduct of this study shall be in accordance with the vehicle emissions inhalation exposure guideline in § 79.61.

(2)

Preparation of slides and sampling times. Within twenty-four hours of the last exposure, test animals will be sacrificed. One femur from each test animal will be removed and placed in fetal bovine serum. The bone marrow is removed, cells processed, and two bone marrow smears are made for each animal on glass microscope slides. The slides are stained with acridine- orange (AO) or another appropriate stain (Giemsa + Wright's, etc.) and examined under a microscope.

(3)

Analysis. Slides shall be coded for study before microscopic analysis. At least 1,000 first-division erythrocytes per animal shall be scored for the incidence of micronuclei. Sexes will be analyzed separately.

(e)

Data and report—

(1)

Treatment of results. In addition to the reporting requirements specified under §§ 79.60 and 79.61, the final test report must include the criteria for scoring micronuclei. Individual data shall be presented in a tabular form including both positive and negative controls and experimental groups. The number of polychromatic erythrocytes scored, the number of micronucleated erythrocytes, the percentage of micronucleated cells, and, where applicable, the percentage of micronucleated erythrocytes shall be listed separately for each experimental and control animal. Absolute numbers shall be included if percentages are reported.

(2)

Interpretation of data.

(i)

There are several criteria for determining a positive response, one of which is a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations.

(ii)

A test substance which does not produce either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.

(3)

Test evaluation.

(i)

Positive results in the micronucleus test provide information on the ability of a chemical to induce micronuclei in erythrocytes of the test species under the conditions of the test. This damage may have been the result of chromosomal damage or damage to the mitotic apparatus.

(ii)

Negative results indicate that under the test conditions the test substance does not produce micronuclei in the bone marrow of the test species.

(f)

Test report. In addition to the reporting recommendations as specified under § 79.60, the following specific information shall be reported:

(1)

Test atmosphere concentration(s) used and rationale for concentration selection.

(2)

Rationale for and description of treatment and sampling schedules, toxicity data, negative and positive controls.

(3)

Historical control data (negative and positive), if available.

(4)

Details of the protocol used for slide preparation.

(5)

Criteria for identifying micronucleated erythrocytes.

(6)

Micronucleus analysis by animal and by group for each concentration (sexes analyzed separately).

(i)

Ratio of polychromatic to normochromatic erythrocytes.

(ii)

Number of polychromatic erythrocytes with micronuclei.

(iii)

Number of polychromatic erythrocytes scored.

(7)

Statistical methodology chosen for test analysis.

(g)

References. For additional background information on this test guideline, the following references should be consulted.

(1)

40 CFR 798.5395, In Vivo, Mammalian Bone Marrow Cytogenetics Tests: Micronucleus Assay.

(2)

Cihak, R. “Evaluation of Benzidine by the Micronucleus Test.” Mutation Research, 67: 383-384 (1979).

(3)

Evans, H.J. “Cytological Methods for Detecting Chemical Mutagens.” Chemical Mutagens: Principles and Methods for Their Detection, Vol. 4. Ed. A. Hollaender (New York and London: Plenum Press, 1976) pp. 1-29.

(4)

Heddle, J.A., et al. “The Induction of Micronuclei as a Measure of Genotoxicity. A Report of the U.S. Environmental Protection Agency Gene-Tox Program.” Mutation Research, 123:61-118 (1983).

(5)

Preston, J.R. et al. “Mammalian In Vivo and In Vitro Cytogenetics Assays: Report of the Gene-Tox Program.” Mutation Research, 87:143-188 (1981).

(6)

Schmid, W. “The micronucleus test for cytogenetic analysis”, Chemical Mutagens, Principles and Methods for their Detection. Vol. 4 Hollaender A, (Ed. A ed. (New York and London: Plenum Press, (1976) pp. 31-53.

(7)

Tice, R.E., and Al Pellom “User's guide: Micronucleus assay data management and analysis system”, NTIS Order no. PB-90-212-598AS.

Notes, amendments, and revision history

Source

Source: 59 FR 33093, June 27, 1994, unless otherwise noted.

Authority

Authority: 42 U.S.C. 7414, 7524, 7545 and 7601.

Source

Source: 40 FR 52011, Nov. 7, 1975, unless otherwise noted.

§79.65. In vivo sister chromatid exchange assay.

40 C.F.R. § 79.65

(a)

Purpose. The in vivo sister chromatid exchange (SCE) assay detects the ability of a chemical to enhance the exchange of DNA between two sister chromatids of a duplicating chromosome. The most commonly used assays employ mammalian bone marrow cells or peripheral blood lymphocytes, often from rodent species.

(b)

Definitions. For the purposes of this section, the following definitions apply:

C-metaphase means a state of arrested cell growth typically seen after treatment with a spindle inhibitor, i.e., colchicine.

Sister chromatid exchange means a reciprocal interchange of the two chromatid arms within a single chromosome. This exchange is visualized during the metaphase portion of the cell cycle and presumably requires the enzymatic incision, translocation and ligation of at least two DNA helices.

(c)

Test method—

(1)

Principle of the test method.

(i)

Groups of rodents are exposed by the inhalation route for a minimum of 6 hours/day over a period of not less than 28 days to three or more concentrations of a test substance in air. Groups of animals are sacrificed at the end of the exposure period and blood lymphocyte cell cultures are prepared from study animals. Cell growth is suspended after a time and cells are harvested, fixed and stained before scoring for SCEs. Researchers may need to run a trial at the highest tolerated concentration of the test atmosphere to optimize the sample collection time for second division metaphase cells.

(ii)

This assay may be done separately or in combination with the subchronic toxicity study, pursuant to the provisions in § 79.62.

(2)

Description.

(i)

The method described here employs peripheral blood lymphocytes (PBL) of laboratory rodents exposed to the test atmosphere.

(ii)

Within twenty-four hours of the last exposure, test animal lymphocytes are obtained by heart puncture and duplicate cell cultures are started for each animal. Cultures are grown in bromo-deoxyuridine (BrdU), and then a spindle inhibitor (e.g., colchicine) is added to arrest cell growth. Cells are harvested, fixed, and stained and their chromosomes are scored for SCEs.

(3)

Species and strain. The rat is the recommended test animal. Other rodent species may be used in this assay, but use of that species will be justified by the tester.

(4)

Animal number and sex. At least five female and five male animals per experimental and control group shall be used. The use of a single sex or different number of animals shall be justified.

(5)

Positive control group. A single concentration of a compound known to produce SCEs in vivo is adequate as a positive control if it shows a significant response at any one time point; additional concentration levels may be used. To select an appropriate concentration level, a pilot or trial study may be advisable. Initially, one concentration of the test substance may be used, the maximum tolerated dose or that producing some indication of toxicity as evidenced by animal morbidity (including death) or target cell toxicity. Intraperitoneal injection of 1,2-dimethyl-benz-anthracene or benzene are examples of positive control exposures. A concentration of 50-80 percent of an LD50 would also be a suitable guide.

(6)

Inhalation exposure.

(i)

All data developed within this study shall be in accordance with good laboratory practice provisions under § 79.60.

(ii)

The general conduct of this study shall be in accordance with the vehicle emissions inhalation exposure guideline in § 79.61.

(d)

Test performance—

(1)

Treatment. At the conclusion of the exposure period, all test animals are anaesthetized and heart punctures are performed. Lymphocytes are isolated over a Ficoll gradient and replicate cell cultures are started for each animal. After some 21 hours, the cells are treated with BrdU and returned to incubation. The following day, a spindle inhibitor (e.g., colchicine) is added to arrest cell growth in c-metaphase. Cells are harvested 4 hours later and second-division metaphase cells are washed and fixed in methanol:acetic acid, stained, and chromosome preparations are scored for SCEs.

(2)

Staining method. Staining of slides to reveal SCEs can be performed according to any of several protocols. However, the fluorescence plus Giemsa method is recommended.

(3)

Number of cells scored.

(i)

A minimum of 25 well-stained, second-division metaphase cells shall be scored for each animal for each cell type.

(ii)

At least 100 consecutive metaphase cells shall be scored for the number of first, second, and third division metaphases for each animal for each cell type.

(iii)

At least 1000 consecutive PBL's shall be scored for the number of metaphase cells present.

(iv)

The number of cells to be analyzed per animal shall be based upon the number of animals used, the negative control frequency, the pre-determined sensitivity and the power chosen for the test. Slides shall be coded before microscopic analysis.

(e)

Data and report—

(1)

Treatment of results. In addition to the reporting requirements specified under §§ 79.60 and 61, data shall be presented in tabular form, providing scores for both the number of SCE for each metaphase. Differences among animals within each group shall be considered before making comparisons between treated and control groups.

(2)

Statistical evaluation. Data shall be evaluated by appropriate statistical methods.

(3)

Interpretation of results.

(i)

There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of SCE. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test concentrations.

(ii)

A test substance which does not produce either a statistically significant dose-related increase in the number of SCE or a statistically significant and reproducible positive response at any one of the test concentrations is considered not to induce rearrangements of DNA segments in this system.

(iii)

Both biological and statistical significance shall be considered together in the evaluation.

(4)

Test evaluation.

(i)

A positive result in the in vivo SCE assay for either, or both, the lung or lymphocyte cultures indicates that under the test conditions the test substance induces reciprocal interchanges of DNA in duplicating chromosomes from lung or lymphocyte cells of the test species.

(ii)

Negative results indicate that under the test conditions the test substance does not induce reciprocal interchanges in lung or lymphocyte cells of the test species.

(5)

Test report. In addition to the reporting recommendations as specified under §§ 79.60 and 79.61, the following specific information shall be reported:

(i)

Test concentrations used, rationale for concentration selection, negative and positive controls;

(ii)

Toxic response data by concentration;

(iii)

Schedule of administration of test atmosphere, BrdU, and spindle inhibitor;

(iv)

Time of harvest after administration of BrdU;

(v)

Identity of spindle inhibitor, its concentration and timing of treatment;

(vi)

Details of the protocol used for cell culture and slide preparation;

(vii)

Criteria for scoring SCE;

(viii)

Replicative index, i.e., [percent 1st division + (2 × percent 2nd division) + (3 × percent 3rd division) metaphases]/100; and

(ix)

Mitotic activity, i.e., # of metaphases/1000 cells.

(f)

References. For additional background information on this test guideline, the following references should be consulted.

(1)

40 CFR 798.5915, In vivo Sister Chromatid Exchange Assay.

(2)

Kato, H. “Spontaneous Sister Chromatid Exchanges Detected by a BudR-Labeling Method.” Nature, 251:70-72 (1974).

(4)

Kligerman, A. D., et al. “Sister Chromatid Exchange Analysis in Lung and Peripheral Blood Lymphocytes of Mice Exposed to Methyl Isocyanate by Inhalation.” Environmental Mutagenesis 9:29-36 (1987).

(5)

Kligerman, A.D., et al., “Cytogenetic Studies of Rodents Exposed to Styrene by Inhalation”, IARC Monographs no. 127 “Butadiene and Styrene: Assesment of Health Hazards” (Sorsa, et al., eds), pp 217-224, 1993.

(6)

Kligerman, A., et al., “Cytogenetic Studies of Mice Exposed to Styrene by Inhalation.”, Mutation Research, 280:35-43, 1992.

(7)

Wolff, S., and P. Perry. “Differential Giemsa Staining of Sister Chromatids and the Study of Sister Chromatid Exchanges Without Autoradiography.” Chromosoma 48: 341-53 (1974).

Notes, amendments, and revision history

Source

Source: 59 FR 33093, June 27, 1994, unless otherwise noted.

Authority

Authority: 42 U.S.C. 7414, 7524, 7545 and 7601.

Source

Source: 40 FR 52011, Nov. 7, 1975, unless otherwise noted.

§79.66. Neuropathology assessment.

40 C.F.R. § 79.66

(a)

Purpose.

(1)

The histopathological and biochemical techniques in this guideline are designed to develop data in animals on morphologic changes in the nervous system associated with repeated inhalation exposures to motor vehicle emissions. These tests are not intended to provide a detailed evaluation of neurotoxicity. Neuropathological evaluation should be complemented by other neurotoxicity studies, e.g. behavioral and neurophysiological studies and/or general toxicity testing, to more completely assess the neurotoxic potential of an exposure.

(2)

[Reserved]

(b)

Definition. Neurotoxicity (NTX) or a neurotoxic effect is an adverse change in the structure or function of the nervous system following exposure to a chemical substance.

(c)

Principle of the test method.

(1)

Laboratory rodents are exposed to one of several concentration levels of a test atmosphere for at least six hours daily over a period of 90 days. At the end of the exposure period, the animals are anaesthetized, perfused in situ with fixative, and tissues in the nervous system are examined grossly and prepared for microscopic examination. Starting with the highest dosage level, tissues are examined under the light microscope for morphologic changes, until a no-observed-adverse-effect level is determined. In cases where light microscopy has revealed neuropathology, the NOAEL may be confirmed by electron microscopy.

(2)

The tests described herein may be combined with any other toxicity study, as long as none of the requirements of either are violated by the combination. Specifically, this assay may be combined with a subchronic toxicity study, pursuant to provisions in § 79.62.

(d)

Limit test. If a test at one dose level of the highest concentration that can be achieved while maintaining a particle size distribution with a mass median aerodynamic diameter (MMAD) of 4 micrometers (µm) or less, using the procedures described in paragraph (a) of this section, produces no observable toxic effects and if toxicity would not be expected based upon data of structurally related compounds, then a full study using three dose levels might not be necessary. Expected human exposure though may indicate the need for a higher dose level.

(e)

Test procedures—

(1)

Animal selection—

(i)

Species and strain. Testing shall be performed in the species being used in other NTX tests. A standard strain of laboratory rat is recommended. The choice of species shall take into consideration such factors as the comparative metabolism of the chemical and species sensitivity to the toxic effects of the test substance, as evidenced by the results of other studies, the potential for combined studies, and the availability of other toxicity data for the species.

(ii)

Age. Animals shall be at least ten weeks of age at the start of exposure.

(iii)

Sex. Both sexes shall be used unless it is demonstrated that one sex is refractory to the effects of exposure.

(2)

Number of Animals. A minimum of ten animals per group shall be used. The tissues from each animal shall be examined separately.

(3)

Control Groups.

(i)

A concurrent control group, exposed to clean, filtered air only, is required.

(ii)

The laboratory performing the testing shall provide positive control data, e.g., results from repeated acrylamide exposure, as evidence of the ability of their histology procedures to detect neurotoxic endpoints. Positive control data shall be collected at the time of the test study unless the laboratory can demonstrate the adequacy of historical data for the planned study.

(iii)

A satellite group of 10 female and 10 male test subjects shall be treated with the highest concentration level for the duration of the exposure and observed thereafter for reversibility, persistence, or delayed occurrence of toxic effects during a post-treatment period of not less than 28 days.

(4)

Inhalation exposure.

(i)

All data developed within this study shall be in accordance with good laboratory practice provisions under § 79.60.

(ii)

The general conduct of this study shall be in accordance with the vehicle emissions inhalation exposure guideline in § 79.61.

(5)

Study conduct—

(i)

Observation of animals. 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.

(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; and

(E)

(iii)

Sacrifice of animals—

(A)

General. The goal of the techniques outlined for sacrifice of animals and preparation of tissues is preservation of tissue morphology to simulate the living state of the cell.

(B)

Perfusion technique. Animals shall be perfused in situ by a generally recognized technique. For fixation suitable for light or electronic microscopy, saline solution followed by buffered 2.5 percent glutaraldehyde or buffered 4.0 percent paraformaldehyde, is recommended. While some minor modifications or variations in procedures are used in different laboratories, a detailed and standard procedure for vascular perfusion may be found in the text by Zeman and Innes (1963), Hayat (1970), and Spencer and Schaumburg (1980) under paragraph (g) of this section. A more sophisticated technique is described by Palay and Chan-Palay (1974) under paragraph (g) of this section. In addition, the lungs shall be instilled with fixative via the trachea during the fixation process in order to preserve the lungs and achieve whole-body fixation.

(C)

Removal of brain and cord. After perfusion, the bony structure (cranium and vertebral column) shall be exposed. Animals shall then be stored in fixative-filled bags at 4 °C for 8-12 hours. The cranium and vertebral column shall be removed carefully by trained technicians without physical damage of the brain and cord. Detailed dissection procedures may be found in the text by Palay and Chan-Palay (1974) under paragraph (g) of this section. After removal, simple measurement of the size (length and width) and weight of the whole brain (cerebrum, cerebellum, pons-medulla) shall be made. Any abnormal coloration or discoloration of the brain and cord shall also be noted and recorded.

(D)

Sampling. Cross-sections of the following areas shall be examined: The forebrain, the center of the cerebrum, the midbrain, the cerebellum, and the medulla oblongata; the spinal cord at the cervical swelling (C3-C6), and proximal sciatic nerve (mid-thigh and sciatic notch) or tibial nerve (at knee). Other sites and tissue elements (e.g., gastrocnemius muscle) shall be examined if deemed necessary. Any observable gross changes shall be recorded.

(iv)

Specimen storage. Tissue samples from both the central and peripheral nervous system shall be further immersion fixed and stored in appropriate fixative (e.g., 10 percent buffered formalin for light microscopy; 2.5 percent buffered gluteraldehyde or 4.0 percent buffered paraformaldehyde for electron microscopy) for future examination. The volume of fixative versus the volume of tissues in a specimen jar shall be no less than 25:1. All stored tissues shall be washed with buffer for at least 2 hours prior to further tissue processing.

(v)

Histopathology examination—

(A)

Fixation. Tissue specimens stored in 10 percent buffered formalin may be used for this purpose. All tissues must be immersion fixed in fixative for at least 48 hours prior to further tissue processing.

(B)

Dehydration. All tissue specimens shall be washed for at least 1 hour with water or buffer, prior to dehydration. (A longer washing time is needed if the specimens have been stored in fixative for a prolonged period of time.) Dehydration can be performed with increasing concentration of graded ethanols up to absolute alcohol.

(C)

Clearing and embedding. After dehydration, tissue specimens shall be cleared with xylene and embedded in paraffin or paraplast. Multiple tissue specimens (e.g. brain, cord, ganglia) may be embedded together in one single block for sectioning. All tissue blocks shall be labelled showing at least the experiment number, animal number, and specimens embedded.

(D)

Sectioning. Tissue sections, 5 to 6 microns in thickness, shall be prepared from the tissue blocks and mounted on standard glass slides. It is recommended that several additional sections be made from each block at this time for possible future needs for special stainings. All tissue blocks and slides shall be filed and stored in properly labeled files or boxes.

(E)

Histopathological techniques. The following general testing sequence is proposed for gathering histopathological data:

(1) General staining. A general staining procedure shall be performed on all tissue specimens in the highest treatment group. Hematoxylin and eosin (H&E) shall be used for this purpose. The staining shall be differentiated properly to achieve bluish nuclei with pinkish background.

(2) Peripheral nerve teasing. Peripheral nerve fiber teasing shall be used. Detailed staining methodology is available in standard histotechnological manuals such as AFIP (1968), Ralis et al. (1973), and Chang (1979) under paragraph (g) of this section. The nerve fiber teasing technique is discussed in Spencer and Schaumberg (1980) under paragraph (g) of this section. A section of normal tissue shall be included in each staining to assure that adequate staining has occurred. Any changes shall be noted and representative photographs shall be taken. If a lesion(s) is observed, the special techniques shall be repeated in the next lower treatment group until no further lesion is detectable.

(F)

Examination. All stained microscopic slides shall be examined with a standard research microscope. Examples of cellular alterations (e.g., neuronal vacuolation, degeneration, and necrosis) and tissue changes (e.g., gliosis, leukocytic infiltration, and cystic formation) shall be recorded and photographed.

(f)

Data collection, reporting, and evaluation. In addition to information meeting the requirements stated under 40 CFR 79.60 and 79.61, the following specific information shall be reported:

(1)

Description of test system and test methods.

(i)

A description of the general design of the experiment shall be provided. This shall include a short justification explaining any decisions where professional judgment is involved such as fixation technique and choice of stains; and

(ii)

Positive control data from the laboratory performing the test that demonstrate the sensitivity of the procedures being used. Historical data may be used if all essential aspects of the experimental protocol are the same.

(2)

Results. All observations shall be recorded and arranged by test groups. This data may be presented in the following recommended format:

(i)

Description of signs and lesions for each animal. For each animal, data must be submitted showing its identification (animal number, treatment, dose, duration), neurologic signs, location(s) nature of, frequency, and severity of lesion(s). A commonly-used scale such as 1 + , 2 + , 3 + , and 4 + for degree of severity ranging from very slight to extensive may be used. Any diagnoses derived from neurologic signs and lesions including naturally occurring diseases or conditions, shall also be recorded;

(ii)

Counts and incidence of lesions, by test group. Data shall be tabulated to show:

(A)

The number of animals used in each group, the number of animals displaying specific neurologic signs, and the number of animals in which any lesion was found; and

(B)

The number of animals affected by each different type of lesion, the average grade of each type of lesion, and the frequency of each different type and/or location of lesion.

(iii)

Evaluation of data.

(A)

An evaluation of the data based on gross necropsy findings and microscopic pathology observations shall be made and supplied. The evaluation shall include the relationship, if any, between the animal's exposure to the test atmosphere and the frequency and severity of any lesions observed; and

(B)

The evaluation of dose-response, if existent, for various groups shall be given, and a description of statistical method must be presented. The evaluation of neuropathology data shall include, where applicable, an assessment in conjunction with any other neurotoxicity studies, electrophysiological, behavioral, or neurochemical, which may be relevant to this study.

(g)

References. For additional background information on this test guideline, the following references should be consulted.

(1)

40 CFR 798.6400, Neuropathology.

(2)

AFIP Manual of Histologic Staining Methods. (New York: McGraw-Hill (1968).

(3)

Chang, L.W. A Color Atlas and Manual for Applied Histochemistry. (Springfield, IL: Charles C. Thomas, 1979).

(4)

Dunnick, J.K., et.al. Thirteen-week Toxicity Study of N-Hexane in B6C3F1 Mice After Inhalation Exposure (1989) Toxicology, 57, 163-172.

(5)

Hayat, M.A. “Vol. 1. Biological applications,” Principles and techniques of electron microscopy. (New York: Van Nostrand Reinhold, 1970).

(6)

Palay S.L., Chan-Palay, V. Cerebellar Cortex: Cytology and Organization. (New York: Springer-Verlag, 1974).

(7)

Ralis, H.M., Beesley, R.A., Ralis, Z.A. Techniques in Neurohistology. (London: Butterworths, 1973).

(8)

Sette, W. “Pesticide Assessment Guidelines, Subdivision F, Neurotoxicity Test Guidelines.” Report No. 540/09-91-123 U.S. Environmental Protection Agency 1991 (NTIS # PB91-154617).

(9)

Spencer, P.S., Schaumburg, H.H. (eds). Experimental and Clinical Neurotoxicology. (Baltimore: Williams and Wilkins, 1980).

(10)

Zeman, W., Innes, J.R.M. Craigie's Neuroanatomy of the Rat. (New York: Academic, 1963).

Notes, amendments, and revision history

Amendments

[59 FR 33093, June 27, 1994, as amended at 63 FR 63793, Nov. 17, 1999]

Source

Source: 59 FR 33093, June 27, 1994, unless otherwise noted.

Authority

Authority: 42 U.S.C. 7414, 7524, 7545 and 7601.

Source

Source: 40 FR 52011, Nov. 7, 1975, unless otherwise noted.

Amendments

[59 FR 33093, June 27, 1994, as amended at 63 FR 63793, Nov. 17, 1999]

§79.67. Glial fibrillary acidic protein assay.

40 C.F.R. § 79.67

(a)

Purpose. Chemical-induced injury of the nervous system, i.e., the brain, is associated with astrocytic hypertrophy at the site of damage (see O'Callaghan, 1988 in paragraph (e)(3) in this section). Assays of glial fibrillary acidic protein (GFAP), the major intermediate filament protein of astrocytes, can be used to document this response. To date, a diverse variety of chemical insults known to be injurious to the central nervous system have been shown to increase GFAP. Moreover, increases in GFAP can be seen at concentrations below those necessary to produce cytopathology as determined by routine Nissl stains (standard neuropathology). Thus it appears that assays of GFAP represent a sensitive approach for documenting the existence and location of chemical-induced injury of the central nervous system. Additional functional, histopathological, and biochemical tests are necessary to assess completely the neurotoxic potential of any chemical. This biochemical test is intended to be used in conjunction with neurohistopathological studies.

(b)

Principle of the test method.

(1)

This guideline describes the conduct of a radioimmunoassay for measurement of the amount of GFAP in the brain of vehicle emission-exposed and unexposed control animals. It is based on modifications (O'Callaghan & Miller 1985 in paragraph (e)(5), O'Callaghan 1987 in paragraph (e)(1) of this section) of the dot-immunobinding procedure described by Jahn et al. (1984) in paragraph (e)(2) of this section. Briefly, brain tissue samples from study animals are assayed for total protein, diluted in dot-immunobinding buffer, and applied to nitrocellulose sheets. The spotted sheets are then fixed, blocked, washed and incubated in anti-GFAP antibody and [I 125] Protein A. Bound protein A is then quantified by gamma spectrometry. In lieu of purified protein standards, standard curves are constructed from dilution of a single control sample. By comparing the immunoreactivity of individual samples (both control and exposed groups) with that of the sample used to generate the standard curve, the relative immunoreactivity of each sample is obtained. The immunoreactivity of the control groups is normalized to 100 percent and all data are expressed as a percentage of control. A variation on this radioimmunoassay procedure has been proposed (O'Callaghan 1991 in paragraph (e)(4) of this section) which uses a “sandwich” of GFAP, anti-GFAP, and a chromophore in a microtiter plate format enzyme-link immunosorbent assay (ELISA). The use of this variation shall be justified.

(2)

This assay may be done separately or in combination with the subchronic toxicity study, pursuant to the provisions of § 79.62.

(c)

Test procedure—

(1)

Animal selection—

(i)

Species and strain. Test shall be performed on the species being used in concurrent testing for neurotoxic or other health effect endpoints. This will generally be a species of laboratory rat. The use of other rodent or non-rodent species shall be justified.

(ii)

Age. Based on other concurrent testing, young adult rats shall be used. Study rodents shall not be older than ten weeks at the start of exposures.

(iii)

Number of animals. A minimum of ten animals per group shall be used. The tissues from each animal shall be examined separately.

(iv)

Sex. Both sexes shall be used unless it is demonstrated that one sex is refractory to the effects.

(2)

Materials. The materials necessary to perform this study are [I 125] Protein A (2-10 µCi/µg), Anti-sera to GFAP, nitrocellulose paper (0.1 or 0.2 µm pore size), sample application template (optional; e.g., “Minifold II”, Schleicher & Schuell, Keene, NH), plastic sheet incubation trays.

(3)

Study conduct.

(i)

All data developed within this study shall be in accordance with good laboratory practice provisions under § 79.60.

(ii)

Tissue Preparation. Animals are euthanized 24 hours after the last exposure and the brain is excised from the skull. On a cold dissecting platform, the following six regions are dissected freehand: cerebellum; cerebral cortex; hippocampus; striatum; thalamus/hypothalamus; and the rest of the brain. Each region is then weighed and homogenized in 10 volumes of hot (70-90 °C) 1 percent (w/v) sodium dodecyl sulfate (SDS). Homogenization is best achieved through sonic disruption. A motor driven pestle inserted into a tissue grinding vessel is a suitable alternative. The homogenized samples can then be stored frozen at −70 °C for at least 4 years without loss of GFAP content.

(iii)

Total Protein Assay. Aliquots of the tissue samples are assayed for total protein using the method of Smith et al. (1985) in paragraph (e)(7) of this section. This assay may be purchased in kit form (e.g., Pierce Chemical Company, Rockford, IL).

(iv)

Sample Preparation. Dilute tissue samples in sample buffer (120 mM KCl, 20 mM NaCl, 2 mM MgCl2), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per ml (5 µg/20 µl).

(v)

Preparation of Standard Curve. Dilute a single control sample in sample buffer to give at least five standards, between 1 and 10 µg total protein per 20 µl. The suggested values of total protein per 20 µl sample buffer are 1.25, 2.50, 3.25, 5.0, 6.25, 7.5, 8.75, and 10.0 µg.

(vi)

Preparation of Nitrocellulose Sheets. Nitrocellulose sheets of 0.1 or 0.2 micron pore size are rinsed by immersion in distilled water for 5 minutes and then air dried.

(vii)

Sample Application. Samples can be spotted onto the nitrocellulose sheets free-hand or with the aid of a template. For free-hand application, draw a grid of squares approximately 2 centimeters by 2 centimeters (cm) on the nitrocellulose sheets using a soft pencil. Spot 5-10 µl portions to the center of each square for a total sample volume of 20 µl. For template aided sample application a washerless microliter capacity sample application manifold is used. Position the nitrocellulose sheet in the sample application device as recommended by the manufacturer and spot a 20 µl sample in one application. Do not wet the nitrocellulose or any support elements prior to sample application. Do not apply vacuum during or after sample application. After spotting samples (using either method), let the sheets air dry. The sheets can be stored at room temperature for several days after sample application.

(viii)

Standard Incubation Conditions. These conditions have been described by Jahn et al. (1984) in paragraph (e)(2) of this section. All steps are carried out at room temperature on a flat shaking platform (one complete excursion every 2-3 seconds). For best results, do not use rocking or orbital shakers. Perform the following steps in enough solution to cover the nitrocellulose sheets to a depth of 1 cm.

(A)

Incubate 20 minutes in fixer (25 percent (v/v) isopropanol, 10 percent (v/v) acetic acid).

(B)

Discard fixer, wash several times in deionized water to eliminate the fixer, and then incubate for 5 minutes in Tris-buffered saline (TBS): 200 mM NaCL, 60 mM Tris-HCl to pH 7.4.

(C)

Discard TBS and incubate 1 hour in blocking solution (0.5 percent gelatin (w/v)) in TBS.

(D)

Discard blocking solution and incubate for 2 hours in antibody solution (anti-GFAP antiserum diluted to the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine GFAP, which cross reacts with GFAP from rodents and humans, can be obtained commercially (e.g., Dako Corp.) and used at a dilution of 1:500.

(E)

Discard antibody solution, and wash in 4 changes of TBS for 5 minutes each time. Then wash in TBS for 10 minutes.

(F)

Discard TBS and incubate in blocking solution for 30 minutes.

(G)

Discard blocking solution and incubate for 1 hour in Protein A solution ([I 125]-labeled Protein A diluted in blocking solution containing 0.1 percent Triton X-100, sufficient to produce 2000 counts per minute (cpm) per 10 µl of Protein A solution).

(H)

Remove Protein A solution (it may be reused once). Wash in 0.1 percent Triton X-100 in TBS (TBSTX) for 5 minutes, 4 times. Then wash in TBSTX for 2-3 hours for 4 additional times. An overnight wash in a larger volume can be used to replace the last 4 washes.

(I)

Hang sheets to air-dry. Cut out squares or spots and count radioactivity in a gamma counter.

(ix)

Expression of data. Compare radioactivity counts for samples obtained from control and treated animals with counts obtained from the standard curve. By comparing the immunoreactivity (counts) of each sample with that of the standard curve, the relative amount of GFAP in each sample can be determined and expressed as a percent of control.

(d)

Data Reporting and Evaluation—

(1)

Test Report. In addition to information meeting the requirements stated under 40 CFR 79.60, the following specific information shall be reported:

(i)

Body weight and brain region weights at time of sacrifice for each subject tested;

(ii)

Indication of whether each subject survived to sacrifice or time of death;

(iii)

Data from control animals and blank samples; and

(iv)

Statistical evaluation of results;

(2)

Evaluation of Results.

(i)

Results shall be evaluated in terms of the extent of change in the amount of GFAP as a function of treatment and dose. GFAP assays (of any brain region) from a minimum of 6 samples typically will result in a standard error of the mean of ±5 percent. In this case, a chemically-induced increase in GFAP of 115 percent of control is likely to be statistically significant.

(ii)

The results of this assay shall be compared to and evaluated with any relevant behavioral and histopathological data.

(e)

References. For additional background information on this test guideline the following references should be consulted.

(1)

Brock, T.O and O'Callaghan, J.P. 1987. Quantitative changes in the synaptic vesicle proteins, synapsin I and p38 and the astrocyte specific protein, glial fibrillary acidic protein, are associated with chemical-induced injury to the rat central nervous system, J. Neurosci. 7:931-942.

(2)

Jahn, R., Schiebler, W. Greengard, P. 1984. A quantitative dot-immunobinding assay for protein using nitrocellulose membrane filters. Proc. Natl. Acad. Sci. U.S.A. 81:1684-1687.

(3)

O'Callaghan, J.P. 1988. Neurotypic and gliotypic protein as biochemical markers of neurotoxicity. Neurotoxicol. Teratol. 10:445-452.

(4)

O'Callaghan, J.P. 1991. Quantification of glial fibrillary acidic protein: comparison of slot-immunobinding assays with a novel sandwich ELISA. Neurotoxicol. Teratol. 13:275-281.

(5)

O'Callaghan, J.P. and Miller, D.B. 1985. Cerebellar hypoplasia in the Gunn rat is associated with quantitative changes in neurotypic and gliotypic proteins. J. Pharmacol. Exp. Ther. 234:522-532.

(6)

Sette, W.F. “Pesticide Assessment Guidelines, Subdivision ‘F’, Hazard Evaluation: Human and Domestic Animals, Addendum 10, Neurotoxicity, Series 81, 82, and 83” US-EPA, Office of Pesticide Programs, EPA-540/09-91-123, March 1991.

(7)

Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C. 1985. Measurement of protein using bicinchoninic acid. Annal. Biochem. 150:76-85.

Notes, amendments, and revision history

Source

Source: 59 FR 33093, June 27, 1994, unless otherwise noted.

Authority

Authority: 42 U.S.C. 7414, 7524, 7545 and 7601.

Source

Source: 40 FR 52011, Nov. 7, 1975, unless otherwise noted.

§79.68. Salmonella typhimurium reverse mutation assay.

40 C.F.R. § 79.68

(a)

Purpose. The Salmonella typhimurium histidine (his) reversion system is a microbial assay which measures his− → his + reversion induced by chemicals which cause base changes or frameshift mutations in the genome of the microorganism Salmonella typhimurium.

(b)

Definitions. For the purposes of this section, the following definitions apply:

Base pair mutagen means an agent which causes a base change in DNA. In a reversion assay, this change may occur at the site of the original mutation or at a second site in the chromosome.

Frameshift mutagen is an agent which causes the addition or deletion of single or multiple base pairs in the DNA molecule.

Salmonella typhimurium reverse mutation assay detects mutation in a gene of a histidine-requiring strain to produce a histidine independent strain of this organism.

(c)

Reference substances. These may include, but need not be limited to, sodium azide, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene, congo red, benzopurpurin 4B, trypan blue or direct blue 1.

(d)

Test method—

(1)

Principle. Motor vehicle combustion emissions from fuel or additive/base fuel mixtures are, first, filtered to trap particulate matter and, then, passed through a sorbent resin to trap semi-volatile gases. Bacteria are separately exposed to the extract from both the filtered particulates and the resin-trapped organics. Assays are conducted using both test mixtures with and without a metabolic activation system and exposed cells are plated onto minimal medium. After a suitable period of incubation, revertant colonies are counted in test cultures and compared to the number of spontaneous revertants in unexposed control cultures.

(2)

Description. Several methods for performing the test have been described. The procedures described here are for the direct plate incorporation method and the azo-reduction method. Among those used are:

(i)

Direct plate incorporation method;

(ii)

Preincubation method;

(iii)

Azo-reduction method;

(iv)

Microsuspension method; and

(v)

Spiral assay.

(3)

Strain selection—

(i)

Designation. Five tester strains shall be used in the assay. At the present time, TA1535, TA1537, TA98, and TA100 are designated as tester strains. The fifth strain will be chosen from the pool of Salmonella strains commonly used to determine the degree to which nitrated organic compounds, i.e., nitroarenes, contribute to the overall mutagenic activity of a test substance. TA98/1,8-DNP6 or other suitable Rosenkranz nitro-reductase resistant strains will be considered acceptable. The choice of the particular strain is left to the discretion of the researcher. However, the researcher shall justify the use of the selected bacterial tester strains.

(ii)

Preparation and storage of bacterial tester strains. Recognized methods of stock culture preparation and storage shall be used. The requirement of histidine for growth shall be demonstrated for each strain. Other phenotypic characteristics shall be checked using such methods as crystal violet sensitivity and resistance to ampicillin. Spontaneous reversion frequency shall be in the range expected as reported in the literature and as established in the laboratory by historical control values.

(iii)

Bacterial growth. Fresh cultures of bacteria shall be grown up to the late exponential or early stationary phase of growth (approximately 108-109 cells per ml).

(4)

Exogenous metabolic activation. Bacteria shall be exposed to the test substance both in the presence and absence of an appropriate exogenous metabolic activation system. For the direct plate incorporation method, the most commonly used system is a cofactor-supplemented postmitochondrial fraction prepared from the livers of rodents treated with enzyme-inducing agents, such as Aroclor 1254. For the azo-reduction method, a cofactor- supplemented postmitochondrial fraction (S-9) prepared from the livers of untreated hamsters is preferred. For this method, the cofactor supplement shall contain flavin mononucleotide, exogenous glucose 6-phosphate dehydrogenase, NADH and excess of glucose-6-phosphate.

(5)

Control groups—

(i)

Concurrent controls. Concurrent positive and negative (untreated) controls shall be included in each experiment. Positive controls shall ensure both strain responsiveness and efficacy of the metabolic activation system.

(ii)

Strain specific positive controls shall be included in the assay. Examples of strain specific positive controls are as follows:

(A)

Strain TA1535, TA100— sodium azide;

(B)

TA98— 2-nitrofluorene (without activation), 2-anthramine (with activation);

(C)

TA1537— 9-aminoacridine; and

(D)

TA98/1,8-DNP<I>6</I>— benzo(a)pyrene (with activation).

The papers by Claxton et al., 1991 and 1992 in paragraph (g) in this section will provide helpful information for the selection of positive controls.

(iii)

Positive controls to ensure the efficacy of the activation system. The positive control reference substances for tests including a metabolic activation system shall be selected on the basis of the type of activation system used in the test. 2-Aminoanthracene is an example of a positive control compound in plate-incorporation tests using postmitochondrial fractions from the livers of rodents treated with enzyme-inducing agents such as Aroclor-1254. Congo red is an example of a positive control compound in the azo-reduction method. Other positive control reference substances may be used.

(iv)

Class-specific positive controls. The azo-reduction method shall include positive controls from the same class of compounds as the test agent wherever possible.

(6)

Sampling the test atmosphere.

(i)

Extracts of test emissions are collected on Teflon ®-coated glass fiber filters using an exhaust dilution setup. The particulates are extracted with dichloromethane (DCM) using Soxhlet extraction techniques. Extracts in DCM can be stored at dry ice temperatures until use.

(ii)

Gaseous hydrocarbons passing through the filter are trapped by a porous, polymer resin, like XAD-2/styrene-divinylbenzene, or an equivalent product. Methylene chloride is used to extract the resin and the sample is evaporated to dryness before storage or use.

(iii)

Samples taken from this material are then used to expose the cells in this assay. Final concentration of extracts in solvent/vehicle, or after solvent exchange, shall not interfere with cell viability or growth rate. The paper by Stump (1982) in paragraph (g) of this section is useful for preparing extracts of particulate and semi-volatile organic compounds from diesel and gasoline exhaust stream.

(iv)

Exposure concentrations.

(A)

The test should initially be performed over a broad range of concentrations. Among the criteria to be taken into consideration for determining the upper limits of test substance concentration are cytotoxicity and solubility. Cytotoxicity of the test chemical may be altered in the presence of metabolic activation systems. Toxicity may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the background lawn or by the degree of survival of treated cultures. Relatively insoluble samples shall be tested up to the limits of solubility. The upper test chemical concentration shall be determined on a case by case basis.

(B)

Generally, a maximum of 5 mg/plate for pure substances is considered acceptable. At least 5 different concentrations of test substance shall be used with adequate intervals between test points.

(C)

When appropriate, a single positive response shall be confirmed by testing over a narrow range of concentrations.

(e)

Test performance. All data developed within this study shall be in accordance with good laboratory practice provisions under § 79.60.

(1)

Direct plate incorporation method. When testing with metabolic activation, test solution, bacteria, and 0.5 ml of activation mixture containing an adequate amount of postmitochondrial fraction shall be added to the liquid overlay agar and mixed. This mixture is poured over the surface of a selective agar plate. Overlay agar shall be allowed to solidify before incubation. At the end of the incubation period, revertant colonies per plate shall be counted. When testing without metabolic activation, the test sample and 0.1 ml of a fresh bacterial culture shall be added to 2.0 ml of overlay agar.

(2)

Azo-reduction method. When testing with metabolic activation, 0.5 ml of activation mixture containing 150 µl of postmitochondrial fraction and 0.1 ml of bacterial culture shall be added to a test tube kept on ice. 0.1 ml of test solution shall be added, and the tubes shall be incubated with shaking at 30 °C for 30 minutes. At the end of the incubation period, 2.0 ml of agar shall be added to each tube, the contents mixed and poured over the surface of a selective agar plate. Overlay agar shall be allowed to solidify before incubation. At the end of the incubation period, revertant colonies per plate shall be counted. For tests without metabolic activation, 0.5 ml of buffer shall be used in place of the 0.5 ml of activation mixture. All other procedures shall be the same as those used for the test with metabolic activation.

(3)

Other methods/modifications may also be appropriate.

(4)

Media. An appropriate selective medium with an adequate overlay agar shall be used.

(5)

Incubation conditions. All plates within a given experiment shall be incubated for the same time period. This incubation period shall be for 48-72 hours at 37 °C.

(6)

Number of cultures. All plating shall be done at least in triplicate.

(f)

Data and report—

(1)

Treatment of results. Data shall be presented as number of revertant colonies per plate, revertants per kilogram (or liter) of fuel, and as revertants per kilometer (or mile, or brake-horsepower/hour, as appropriate) for each replicate and dose. These same measures shall be recorded on both the negative and positive control plates. The mean number of revertant colonies per plate, revertants per kilogram (or liter) of fuel, and revertants per kilometer (or mile, or brake-horsepower/hour), as well as individual plate counts and standard deviations shall be presented for the test substance, positive control, and negative control plates.

(2)

Statistical evaluation. Data shall be evaluated by appropriate statistical methods. Those methods shall include, at a minimum, means and standard deviations of the reversion data.

(3)

Interpretation of results.

(i)

There are several criteria for determining a positive result, one of which is a statistically significant dose-related increase in the number of revertants. Another criterion may be based upon detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations.

(ii)

A test substance which does not produce either a statistically significant dose-related increase in the number of revertants or a statistically significant and reproducible positive response at any one of the test points is considered nonmutagenic in this system.

(iii)

Both biological and statistical significance shall be considered together in the evaluation.

(4)

Test evaluation.

(i)

Positive results from the Salmonella typhimurium reverse mutation assay indicate that, under the test conditions, the test substance induces point mutations by base changes or frameshifts in the genome of this organism.

(ii)

Negative results indicate that under the test conditions the test substance is not mutagenic in Salmonella typhimurium.

(5)

Test report. In addition to the reporting recommendations as specified under 40 CFR 79.60, the following specific information shall be reported:

(i)

Sampling method(s) used and manner in which cells are exposed to sample solution;

(ii)

Bacterial strains used;

(iii)

Metabolic activation system used (source, amount and cofactor); details of preparation of postmitochondrial fraction;

(iv)

Concentration levels and rationale for selection of concentration range;

(v)

Description of positive and negative controls, and concentrations used, if appropriate;

(vi)

Individual plate counts, mean number of revertant colonies per plate, number of revertants per kilometer (or mile, or brake-horsepower/hour), and standard deviation; and

(vii)

Dose-response relationship, if applicable.

(g)

References. For additional background information on this test guideline, the following references should be consulted.

(1)

40 CFR 798.5265, The Salmonella typhimurium reverse mutation asay.

(2)

Ames, B.N., McCann, J., Yamasaki, E. “Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test,” Mutation Research 31:347-364 (1975).

(3)

Huisingh, J.L., et al.,“Mutagenic and Carcinogenic Potency of Extracts of Diesel and Related Environmental Emissions: Study Design, Sample Generation, Collection, and Preparation”. In: Health Effects of Diesel Engine Emissions, Vol. II, W.E. Pepelko, R., M., Danner and N. A. Clarke (Eds.), US EPA, Cincinnati, EPA-600/9-80-057b, pp. 788-800 (1980).

(4)

[Reserved]

(5)

Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., Nestmann, E., Zeiger, E. “Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity” Mutation Research 189(2):83-91 (1987).

(6)

Claxton, L., Houk, V.S., Allison, J.C., Creason, J., “Evaluating the relationship of metabolic activation system concentrations and chemical dose concentrations for the Salmonella Spiral and Plate Assays” Mutation Research 253:127-136 (1991).

(7)

Claxton, L., Houk, V.S., Monteith, L.G., Myers, L.E., Hughes, T.J., “Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: I. Without exogenous activation.” Mutation Research 253:137-147 (1991).

(8)

Claxton, L., Houk, V.S., Warner, J.R., Myers, L.E., Hughes, T.J., “Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: II. With exogenous activation.” Mutation Research 253:149-159 (1991).

(9)

Claxton, L., Creason, J., Lares, B., Augurell, E., Bagley, S., Bryant, D.W., Courtois, Y.A., Douglas, G., Clare, C.B., Goto, S., Quillardet, P., Jagannath, D.R., Mohn, G., Neilsen, P.A., Ohnishi, Y., Ong, T., Pederson, T.C., Shimizu, H., Nylund, L., Tokiwa, H., Vink, I.G.R., Wang, Y., Warshawsky, D., “Results of the IPCS Collaborative Study on Complex Mixtures” Mutation Research 276:23-32 (1992).

(10)

Claxton, L., Douglas, G., Krewski, D., Lewtas, J., Matsushita, H., Rosenkranz, H., “Overview, conclusions, and recommendations of the IPCS Collaborative Study on Complex Mixtures” Mutation Research 276:61-80 (1992).

(11)

Houk, V.S., Schalkowsky, S., and Claxton, L.D., “Development and Validation of the Spiral Salmonella Assay: An Automated Approach to Bacterial Mutagenicity Testing” Mutation Research 223:49-64 (1989).

(12)

Jones, E., Richold, M., May, J.H., and Saje, A. “The Assessment of the Mutagenic Potential of Vehicle Engine Exhaust in the Ames Salmonella Assay Using a Direct Exposure Method” Mutation Research 97:35-40 (1985).

(13)

Maron, D., and Ames, B. N., Revised methods for the Salmonella mutagenicity test, Mutation Research, 113:173-212 (1983).

(14)

Prival, M.J., and Mitchell, V.D. “Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver S-9,” Mutation Research 97:103-116 (1982).

(15)

Rosenkranz, H.S., et.al. “Nitropyrenes: Isolation, identification, and reduction of mutagenic impurities in carbon black and toners” Science 209:1039-43 (1980).

(16)

Stump, F., Snow, R., et.al., “Trapping gaseous hydrocarbons for mutagenic testing” SAE Technical Paper Series, No. 820776 (1982).

(17)

Vogel, H.J., Bonner, D.M. “Acetylornithinase of E. coli: partial purification and some properties,” Journal of Biological Chemistry. 218:97-106 (1956).

Notes, amendments, and revision history

Amendments

[59 FR 33093, June 27, 1994, as amended at 61 FR 36513, July 11, 1996]

Source

Source: 59 FR 33093, June 27, 1994, unless otherwise noted.

Authority

Authority: 42 U.S.C. 7414, 7524, 7545 and 7601.

Source

Source: 40 FR 52011, Nov. 7, 1975, unless otherwise noted.

Amendments

[59 FR 33093, June 27, 1994, as amended at 61 FR 36513, July 11, 1996]

40 C.F.R. §§ 79.62–79.68

§79.62. Subchronic toxicity study with specific health effect assessments.

Amendments

Source

Authority

Source

Amendments

§79.63. Fertility assessment/teratology.

Amendments

Source

Authority

Source

Amendments

§79.64. In vivo micronucleus assay.

Source

Authority

Source

§79.65. In vivo sister chromatid exchange assay.

Source

Authority

Source

§79.66. Neuropathology assessment.

Amendments

Source

Authority

Source

Amendments

§79.67. Glial fibrillary acidic protein assay.

Source

Authority

Source

§79.68. Salmonella typhimurium reverse mutation assay.

Amendments

Source

Authority

Source

Amendments

Cite this provision

Incoming references