United States Prevention, Pesticides EPA 712–C–01–352 

and Toxic Substances 

Agency (7101) 

Environmental Protection July 2001 

Health Effects Test 

Guidelines 

OPPTS 870.8355 

Combined Chronic 

Toxicity/Carcinogenicity 

Testing of Respirable 

Fibrous Particles 
INTRODUCTION 

This guideline is one of a series of test guidelines that have been 

developed by the Office of Prevention, Pesticides and Toxic Substances, 

United States Environmental Protection Agency for use in the testing of 

pesticides and toxic substances, and the development of test data that must 

be submitted to the Agency for review under Federal regulations. 

The Office of Prevention, Pesticides and Toxic Substances (OPPTS) 

has developed this guideline through a process of harmonization that 

blended the testing guidance and requirements that existed in the Office 

of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, 

Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the 

Office of Pesticide Programs (OPP) which appeared in publications of the 

National Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development 

(OECD). 

The purpose of harmonizing these guidelines into a single set of 

OPPTS guidelines is to minimize variations among the testing procedures 

that must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15 

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act 

(7 U.S.C. 136, et seq.). 

Public Draft Access Information: This draft guideline is part of a 

series of related harmonized guidelines that need to be considered as a 

unit. For copies: These guidelines are available electronically from the 

EPA Internet Home Page at http://www.epa.gov/opptsfrs/home/ 

guidelin.htm or in paper by contacting the OPP Public Docket at (703) 

305–5805 or by e-mail: opp-docket@epa.gov. 

To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office 

of Pesticide Programs, Field Operations Division (7506C), Environmental 

Protection Agency, 401 M St. SW., Washington, DC 20460. In person: 

bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending 

electronic mail (e-mail) to: oppdocket@epa.gov. 

Final Guideline Release: This guideline is available from the U.S. 

Government Printing Office, Washington, DC 20402 on disks or paper 

copies: call (202) 512–0132. This guideline is also available electronically 

in PDF (portable document format) from the EPA Internet Home Page 

at http://www.epa.gov/opptsfrs/home/guidelin.htm. 

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OPPTS 870.8355 Combined Chronic Toxicity/Carcinogenicity Testing 

of Respirable Fibrous Particles. 

(a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of the Toxic Substances Control Act (TSCA) (15 U.S.C. 

2601). 

(2) Background. The source materials used in developing this 

OPPTS test guideline are 40 CFR 798.3320 Combined Chronic Toxicity/ 

Oncogenicity, EPA–748–R–96–001 Workshop Report on Chronic Inhalation Toxicity and Carcinogenicity Testing of Respirable Fibrous Particles, 

and EPA’s FIFRA Scientific Advisory Panel Report (Report No. 2000– 

0X, 01/05/2001). 

(b) Purpose. The objective of a combined chronic toxicity/carcinogenicity study is to determine the effects of a fibrous substance in at least 

a rodent species following prolonged and repeated inhalation exposure. 

The fibrous substances to be tested under this guideline will be selected 

based on data from appropriate short-term screening tests indicative of potential health hazard and risk concern. The application of this guideline 

should generate data which identify the majority of chronic toxic and carcinogenic effects and determine dose-response relationships. The design 

and conduct should allow for the detection of neoplastic and non-neoplastic effects of the target tissues. In addition, it may also determine general toxicity and exposure-related morphological (pathology) effects if the 

fiber substance under test is known to have such toxic potential (e.g., based 

on results of pre-chronic studies/analyses). While the guideline will have 

application to testing of organic fibers, additional considerations may be 

necessary for study of organic fibers. 

(c) Definitions. The definitions in section 3 of TSCA and the definitions in 40 CFR Part 792--Good Laboratory Practice Standards (GLP) 

apply to this guideline. The following definitions also apply to this guideline. 

Carcinogenicity is the development of neoplastic lesions as a result 

of the repeated daily exposure of experimental animals to the test substance by the inhalation route of exposure. 

Chronic toxicity is the adverse effects occurring as a result of the 

repeated daily exposure of experimental animals to the test substance by 

the inhalation route of exposure. 

Concentration in a combined chronic toxicity/carcinogenicity study 

of fibrous particles is the amount of test substance administered via inhalation routes for a period of up to 24 months. Concentration of fibrous particles is expressed as absolute number of fibers per cubic centimeter (f/ 

cc). Exposure concentrations should also be expressed by fiber length, e.g., 

World Health Organization (WHO) fibers (greater than 5 μm in length)/ 

cc, fibers with length greater than 10, 15 and 20 μm/cc. Gravimetric con1

centration expressed as milligrams per cubic meter (mg/m3) is used for 

daily monitoring of the generated aerosols in order to achieve the intended 

number of fiber per unit of aerosol volume (f/cc). 

Dose in a combined chronic toxicity/carcinogenicity study of fibrous 

particles is the amount of test substance deposited upon inhalation or the 

amount of the test substance retained in the lung after certain time postexposure. Dose of fibrous particles is expressed as number of fibers per 

lung. Fiber number is expressed by total fibers and by fiber length, e.g., 

WHO fibers (larger than 5 μm in length), fibers larger than 10, 15 and 

20 μm in length. 

Fibrous particles/fibers are generally defined as elongated particles 

with a length-to-diameter ratio (i.e., aspect ratio) equal to or greater than 

3 to 1. This definition is presumed to include particles with varying shapes 

such as rod-like, curly, or acicular (needle-like) shapes, and having different structural units commonly referred to as fibers, fibrils, or whiskers. 

No-observed-adverse-effect-level (NOAEL) is the maximum concentration used in a study which produces no observed adverse effects. 

Respirable means that the particle in question can penetrate to the 

alveolar region upon inhalation. There are considerable differences in fiber 

respirability between laboratory rodents and humans. A ‘‘rat-respirable 

fiber’’ is defined as a fiber having an aerodynamic diameter of less than 

3 μm. A fiber having an aerodynamic diameter of less than 5 μm is respirable by humans. (Aerodynamic diameter, the most important determinant of the respirability of a fiber, is different from its actual, geometric 

diameter. The aerodynamic diameter of a fiber is dependent on its density 

and aspect ratio. For example, fibers having actual diameters of 0.25 to 

2.0 μm would have aerodynamic diameters three to four times their actual 

diameters if their density is 1g/cm3 and their lengths are between 10 and 

150 μm). 

Target organ is any organ of a test animal showing evidence of an 

effect induced by a test substance. 

(d) Test procedure—(1) Animal selection—(i) Species and strain. 

For the study of respirable fibrous particles via the inhalation route, the 

rat should be the first rodent species used because of its susceptibility 

to fiber-induced diseases such as pulmonary fibrosis, lung neoplasms and 

mesothelioma. Commonly used laboratory strains should be employed. The 

strain selected should be susceptible to the carcinogenic or toxic effect 

of fibrous particles. The criteria for a suitable strain include: 

(A) A low background rate of neoplasia. 

(B) A low background rate of pulmonary disease. 

(C) Longevity. 

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(D) A history of laboratory use. 

Since the hamster appears to be more sensitive than the rat with respect to fiber-induced mesothelioma, the hamster should be considered as 

a second species when results of the rat study show pleural toxicity or 

neoplasms and dose response data are needed for risk assessment purposes. 

If other species are used, the tester should provide justification/reasoning 

for the selection. 

(ii) Age/weight. (A) Testing should be started with young healthy 

animals as soon as possible after weaning and acclimatization. 

(B) Dosing should generally begin no later than 8 weeks of age. 

(C) At commencement of the study, the weight variation of animals 

used should not exceed 20 percent of the mean weight for each sex. 

(iii) Sex. (A) Equal numbers of animals of each sex should be used 

at each dose level. 

(B) Females should be nulliparous and nonpregnant. 

(iv) Numbers. (A) At least 100 rodents (50 males and 50 females) 

should be used at each concentration level and concurrent control group. 

At least 40 additional rodents (20 males and 20 females) should be used 

for satellite dose groups and the satellite control group. The purpose of 

the satellite groups are for interim sacrifices for lung burden analysis, 

bronchoalveolar lavage fluid (BALF) analysis and the evaluation of pathology other than neoplasm (e.g., fibrosis). Additional rodents (five to six 

per groups) may also be needed for recovery groups to evaluate fiber persistence and the progression or regression of a given lesion in the absence 

of fiber exposure. 

(B) For a meaningful and valid statistical evaluation of long term 

exposure and for a valid interpretation of negative results, the number of 

animals in any group should not fall below 50 percent at 18 months for 

rats and 15 months for hamsters. Survival in any group should not fall 

below 25 percent at 24 months for rats and 18 months for hamsters. 

(C) To avoid bias, the use of adequate randomization procedures for 

the proper allocation of animals to test and control groups is required. 

(D) Each animal should be assigned a unique identification number. 

Dead animals (and their preserved organs) and tissues, and microscopic 

slides should be identified by reference to the unique numbers assigned. 

(v) Husbandry. (A) Animals should be housed individually during 

exposure in inhalation studies. 

(B) The temperature of the experimental animal rooms should be at 

22 ± 3°C. 

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(C) The relative humidity of the experimental animal rooms should 

be 50 ± 20 percent. 

(D) The daily light cycle should be maintained at 12 h light and 12 

h dark, whether artificial or natural. 

(E) Control and test animals should be fed from the same batch and 

lot. The feed should be analyzed to assure uniform distribution and adequacy of nutritional requirements of the species tested and for impurities 

that might influence the outcome of the test. Animals should be fed and 

watered ad libitum with food replaced at least weekly. 

(F) The study should not be initiated until animals have been allowed 

a period of acclimatization/quarantine to environmental conditions, nor 

should animals from outside sources be placed on test without an adequate 

period of quarantine. 

(2) Control and test substances. (i) One lot of the test substance 

should be used throughout the duration of the study if possible, and the 

research sample should be stored under conditions that maintain its purity 

and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity and physicochemical 

properties (e.g., fiber morphology, dimension, bivariate size distribution, 

aerodynamic diameter, chemistry, density, dissolution rate, surface characteristics, the ability of a fiber to split longitudinally or cross-sectionally) 

of the test fiber, and, if possible, the name and quantities of contaminants 

and impurities. 

(ii) To maximize sensitivity of animal inhalation exposure studies to 

health effects of fibers, the test material should consist of rat-respirable 

fibers which should be enriched with the most potent fraction of long, 

thin fibers or fibers with high aspect ratios. As far as is technically feasible, the aerosol should be cleaned up from non-fibrous particles. The 

aerosol should be characterized in term of fiber and non-fiber/particle size 

and number; fiber number should be expressed by total fibers and by fiber 

length, e.g., WHO fibers (greater than 5 μm in length), fibers greater than 

10, 15 and 20 μm in length. If enriching the test aerosol with long, thin 

fibers is not feasible, the reasons should be clearly stated and justified, 

and the enrichment should be for the longest fibers or fibers with the highest aspect ratios available. The aerosolized fibers should be discharged to 

Boltzmann equilibrium before being delivered to the test species. 

(3) Control groups. A concurrent control group (50 males and 50 

females) and a satellite control group (10 males and 10 females) are required. These groups should be untreated. Animals in the satellite control 

group should be sacrificed at the same time the satellite test group is terminated. A positive control group may not need to be included in every 

study, but each new test system (including use of a different animal species 

and strain) should be validated with a positive control material. 

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(4) Concentration levels selection. (i) For dose-response relationships analysis, at least three concentration levels should be used, in addition to the concurrent control group. Along with other information (decrease in body weight, systemic toxicity etc.), data should be obtained on 

lung burden analysis and BALF analysis in a 90-day subchronic inhalation 

study to assist in establishing the chronic exposure concentration levels. 

A combination of the following parameters should be evaluated: Altered 

alveolar macrophage mediated particle clearance rate, fiber lung burden 

normalized to exposure concentration, cell proliferation, histopathology, 

inflammation (marker enzyme activities, total protein content, total cell 

count, cell differential, and cell viability in lung lavage samples) and lung 

weight. In addition, impairment of clearance should be assessed in a 90-

day inhalation study via challenge with a tagged particle; clearance should 

be assessed after the 90-day exposure period and at a recovery period for 

another 3 months. Exposure concentration levels should be spaced to 

produce a gradation of effects. A rationale for the concentrations selected 

must be provided. 

(ii) The highest fiber concentration to be tested in a chronic inhalation 

study is known as the maximum aerosol concentration or MAC. The MAC 

should be based on the total number of inhaled particles (fibers and nonfibrous particles combined); an appropriate lung burden of critical fibers 

(long and thin) should be achieved. The MAC should be set at a level 

at which some degree of impaired clearance and toxicity are observed. 

All the parameters in the 90-day studies should be considered together, 

rather than individually, in an attempt to define a MAC, and should be 

presented to the agency for evaluation before the chronic study is conducted. 

(iii) The intermediate concentration levels should be spaced to 

produce a gradation of toxic effects. 

(iv) The lowest concentration level should produce minimal or no 

evidence of toxicity. 

(5) Administration of the test substance. Inhalation is the major 

route of human exposure of fibrous particles, and chronic inhalation studies in rodents are deemed appropriate tests for evaluating inhalation hazard 

and risk of fibers to humans. Either nose-only or whole-body exposure 

can be used. If whole-body exposure is used, validation of sufficient fibers 

reaching the gas-exchange region of the lungs must be provided. 

(i) The animals should be exposed to the test substance, for 6 h/day 

on a 7-day per week basis, for a period of at least 24 months in rats. 

However, based primarily on practical considerations, exposure for 6 h/ 

day on a 5-day per week basis is acceptable. Due to shorter life span 

of hamsters, their exposure duration could be shorter, based upon survival/ 

lifetime expectancy. 

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(ii) The animals should be tested in dynamic inhalation equipment 

designed to sustain a minimum air flow of 10 air changes per hour, an 

adequate oxygen content of at least 19 percent, and uniform conditions 

throughout the exposure chamber. Maintenance of slight negative pressure 

inside the chamber will prevent leakage of the test substance into surrounding areas. 

(iii) The selection of a dynamic inhalation chamber should be appropriate for the test substance and test system. Where a whole body chamber 

is used, individual housing must be used to minimize crowding of the 

test animals and maximize their exposure to the test substance. To ensure 

stability of a chamber atmosphere, the total volume occupied by the test 

animals should not exceed 5 percent of the volume of the test chamber. 

The animals should be acclimated and heat stress minimized. 

(iv) The temperature at which the test is performed should be maintained at 22 ± 3°C. The relative humidity should be maintained between 

50 ± 20 percent. 

(v) The rate of air flow should be monitored continuously but recorded at least every 30 minutes. 

(vi) Temperature and humidity should be monitored continuously but 

should be recorded at least every 30 minutes. 

(vii) The system used to generate fibrous aerosols must not cause 

significant breakage and contamination of the test substance. During the 

development of the generating system, fiber/particle size analysis should 

be performed to establish the stability of aerosol concentrations with respect to fiber size. During exposure, analysis should be conducted to determine the consistency of fiber size distribution and the actual concentrations 

of the test substance. The frequency of exposure atmosphere monitoring 

should be daily for mass concentration, weekly for fiber concentration and 

bivariate size distribution. 

(viii) The actual concentrations of the test substance should be measured in the breathing zone. Lung burden analyses should be conducted 

after 3, 6, 12, 18, and 24 months of exposure in the rat and after 3, 6, 

12, and 18 months in the hamster to provide data on biopersistence of 

the test fibers and serve as a better measure of internal dose. Data also 

should be obtained on fiber deposition in the nasal cavity and the fiber 

burden in the thoracic lymph nodes. The fibers should be analyzed for 

number, bivariate size distribution and chemistry. Fiber number should be 

expressed by total fibers and by fiber length, e.g., WHO fibers (greater 

than 5 μm in length), fibers greater than 10, 15 and 20 μm in length. 

For fiber burden analysis, one of the two lungs (left or right) should be 

used, rather than only the accessory lobe. Five to six animals per exposure 

group should be studied at each time point. Lung burden and fiber size 

distribution should be reported as number of fibers per gram of dry lung 

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tissue, as well as number of fibers per gram of wet lung tissue. The burden 

should be extrapolated to the whole lung. The method for lung burden 

analysis (‘‘lung digestion’’) must be validated. It is recommended to include ‘‘recovery’’ groups of animals from exposure at 3, 6, 12, and 18 

months and then hold until 24 months for evaluation. 

(ix) Feed should be withheld during exposure. Water may also be 

withheld during exposure. 

(6) Observation period. The chronic inhalation exposure study with 

fibers should be a lifetime study. The animals should be observed for their 

life span after the exposure duration is completed (at least 24 months for 

rats and 18 months for hamsters); final sacrifice should be carried out 

only when survival of the control group reaches 20 percent. 

(7) Observation of animals. (i) Observations should be at least twice 

each day for morbidity and mortality. Appropriate actions should be taken 

to minimize loss of animals from the study (e.g., necropsy or refrigeration 

of those animals found dead and isolation or sacrifice of weak or moribund 

animals). General clinical observations should be made at least once a day. 

(ii) A careful clinical examination should be made at least once weekly. Observations should be detailed and carefully recorded, preferably 

using explicitly defined scales. Observations should include evaluation of 

skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, 

including tremors and convulsions, changes in the level of motor activity, 

gait and posture, reactivity to handling or sensory stimuli, grip strength 

and stereotypies or bizarre behavior (e.g., self-mutilation, walking 

backwards) if the fiber substance under test is known to have such toxic 

potential (e.g., based on data of pre-chronic studies). 

(iii) Body weights should be recorded individually for all animals: 

Once a week during the first 13 weeks of the study and at least once 

every 4 weeks thereafter unless signs of clinical toxicity suggest more frequent weighing to facilitate monitoring of health status. 

(iv) Moribund animals should be removed and sacrificed when noticed and the time of death should be recorded as precisely as possible. 

At the end of the study period, all survivors should be sacrificed. 

(8) Clinical pathology. If hematological and biochemical effects are 

seen in the subchronic study, hematology, clinical chemistry and urinalyses 

should be performed from 10 animals per sex per group at approximately 

6 month intervals during the first 12 months of the study. If possible, 

these collections should be from the same animals at each interval. Overnight fasting of animals prior to blood sampling is recommended. 

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(i) Hematology. The recommended parameters are: Hemoglobin and 

hematocrit concentrations, red blood cell count, mean corpuscular volume, 

mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, white blood cell count, differential leukocyte count, platelet 

count and a measure of clotting potential, such as prothrombin time or 

thromboplastin time. 

(ii) Clinical chemistry. (A) Parameters which are considered appropriate to all studies are 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 and signs of clinical toxicity. 

(B) The recommended clinical chemistry determinations are potassium, sodium, glucose, total cholesterol, urea nitrogen, creatinine, total 

protein, and albumin. More than two hepatic enzymes (such as alkaline 

phosphatase, alanine aminotransferase, aspartate aminotransferase, sorbitol 

dehydrogenase, or gamma glutamyl transferase.) should also be measured. 

(iii) Urinalyses. The following determinations should be made from 

either individual animals or on a pooled sample per sex per group: Appearance (volume and specific gravity), pH, protein, glucose, ketones, bilirubin, 

occult blood (semiquantitatively), and microscopy of sediment 

(semiquantitatively). 

(9) Bronchoalveolar lavage fluid (BALF) analysis. BALF analysis 

should be conducted at the time of the12 and 24 month sacrifices on subgroups of 5 rats/group; additional time points are optional. Lavage parameters to be determined should include: total cell count, differential cell 

counts (polymorphonuclear leukocytes (PMN), alveolar macrophages, 

lymphocytes, and others), total protein, lactate dehydrogenase and β-glucuronidase as examples of cytoplasmic and lysosomal enzymes. It is recommended to include ‘‘recovery’’ groups of animals from exposure at the 

interim time points and then hold until 24 months for evaluation. 

(10) Lung clearance. It is recommended that animals be tested for 

impaired lung clearance for a pulse of a small spherical particle at 9 and 

18 months and recovery of the animals be followed with sacrifices at the 

same intervals as the animals exposed for 24 months. 

(11) Ophthalmological examination. If changes in eyes are detected 

in the subchronic study, examinations of the eyes should be made on all 

animals using an ophthalmoscope or an equivalent device prior to the administration of the test substance and at termination of the study on 10 

animals per sex in the high-dose and control groups. If changes in eyes 

are detected in the high-dose groups, all animals should be examined. 

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(12) Gross necropsy. (i) A complete gross examination should be 

performed on all animals, including those which died during the experiment or were killed in a moribund condition. 

(ii) The liver, lungs, kidneys, brain, spleen, and gonads should be 

trimmed and weighed wet, as soon as possible after dissection to avoid 

drying. The organs should be weighed from interim sacrifice animals as 

well as from at least 10 animals per sex per group at terminal sacrifice. 

(iii) In inhalation studies of fibers, the entire respiratory tract, including nose, pharynx, larynx, paranasal sinuses, lungs, trachea and pleura 

should be examined and preserved. In addition, since fiber detention may 

occur at the rib cage and diaphragm, these organ tissues should also be 

examined and preserved. 

(iv) Inflation of lungs with a fixative is the optimal method for preservation of these tissues. The proper inflation and fixation of the lungs in 

inhalation studies is essential for appropriate and valid histopathological 

examination. It is recommended the lungs from the scheduled sacrifices 

be inflated with an appropriate fixative to allow the grading of lesions 

and better evaluation of subtle changes. 

(v) The following organs and tissues, or representative samples thereof, should be preserved in a suitable medium for possible future 

histopathological examination: 

(A) Digestive system salivary glands, esophagus, stomach, duodenum, 

jejunum, iIleum, cecum, colon, rectum, liver, pancreas, gallbladder (when 

present). 

(B) Nervous system brain (multiple sections), pituitary, peripheral 

nerves, spinal cord (three levels), eyes (retina, optic nerve). 

(C) Glandular system adrenals, parathyroids, thyroid. 

(D) Cardiovascular/hematopoietic system aorta (thoracic), heart, bone 

marrow, lymph nodes, spleen, thymus. 

(E) Urogenital system kidneys, urinary bladder, prostate, testes/ 

epididymides, seminal vesicles, uterus, ovaries, female mammary gland. 

(F) Other all gross lesions and masses, skin. 

(vi) Information from clinical pathology and other in-life data should 

be considered before microscopic examination, since these data may provide significant guidance to the pathologist. 

(13) Histopathology. (i) The following histopathology should be performed in all animals: 

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(A) Target organs/tissues. Organs/tissues of the respiratory tract represent the target organs/tissues for evaluating effects of inhaled fibers. 

Major effects include pulmonary fibrosis, lung tumors and mesotheliomas. 

Special attention to examination of the lungs of rodents should be made 

for evidence of infection since this provides an assessment of the state 

of health of the animals. 

(B) The rib cage and diaphragm. The rib cage and diaphragm should 

be examined histopathologically for mesothelial lesions. The section of 

diaphragm for histology should be cut in a manner to show the muscular 

as well as the non-muscular portion. 

(ii) (A) If gross lesions are observed on the organs and tissues listed 

under paragraph (d)(12)(ii) or paragraph (d)(12)(v) of this guideline, full 

histopathology should be performed on all animals in the control and high 

dose groups and of all animals that died or were killed during the study. 

(B) If the results show substantial alteration of the animal’s normal 

life span, the induction of effects that might affect a neoplastic response, 

or other effects that might compromise the significance of the data, the 

next lower levels should be examined fully on all animals including those 

which died during the experiment or were killed in a moribund condition. 

(iii) An attempt should be made to correlate gross observations with 

microscopic findings. 

(iv) Tissues and organs designated for microscopic examination 

should be fixed in 10 percent buffered formalin or a recognized suitable 

fixative as soon as necropsy is performed and no less than 48 hours prior 

to trimming. The histology slides from the scheduled sacrifices should, 

in addition to standard hematoxylin and eosin, be stained with a method 

that identifies collagen (fibrosis). 

(v) Histopathological evaluation should incorporate both qualitative 

description of lesions and rigorous quantitation. A consistent approach 

should be taken to record and grade the findings of the lesions using a 

standardized method in contemporary fiber studies. ‘‘Image analysis’’ may 

be used to quantify the severity of lesions in the lungs. 

(e) Data and reporting—(1) Treatment of results. (i) Data should 

be summarized in tabular form, showing for each test group the number 

of animals at the start of the test, the number of animals showing lesions, 

the types of lesions and the percentage of animals displaying each type 

of lesion. 

(ii) All observed results (quantitative and qualitative), should be evaluated by an appropriate statistical method. Any generally accepted statistical methods may be used; the statistical methods including significance 

criteria should be selected during the design of the study. 

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(2) Evaluation of study results. (i) The findings of a combined 

chronic toxicity/carcinogenicity study should be evaluated in conjunction 

with the findings of previous studies and considered in terms of the toxic 

effects, the necropsy and histopathological findings. The evaluation will 

include the relationship between the dose of the test substance and the 

presence, incidence and severity of abnormalities (including behavioral and 

clinical abnormalities), gross lesions, identified target organs, body weight 

changes, effects on mortality and any other general or specific toxic effects. 

(ii) Non-neoplastic and neoplastic endpoints recorded should include, 

but not be limited to, epithelial hyperplasia, alveolar bronchiolization, 

metaplasia, adenomas, mesotheliomas, and carcinomas. A dissecting microscope should be used to examine for mesotheliomas. In distinguishing 

between hyperplasia and mesothelioma, standard diagnostic criteria should 

be applied to identified lesions. Established published guidelines on the 

use of blinding in histopathology should be followed, e.g., those published 

by the Society of American Pathologists. 

(iii) In order for a negative test to be acceptable, it should meet the 

following criteria: No more than 10 percent of any group is lost due to 

autolysis, cannibalism, or management problems, and survival in each 

group is no less than 50 percent at 15 months for hamsters and 18 months 

for rats. Survival should not fall below 25 percent at 18 months for hamsters and 24 months for rats. For acceptance of the results of a chronic 

inhalation exposure study with fibers as negative, the study must have been 

designed and conducted according to the criteria outlined previously, the 

health effects of concern must not be significantly more frequent in the 

exposure groups than in the control group. Sample sizes for toxicity studies 

should be large enough to detect a specific effect level (e.g., a 10% change 

in fibrosis or tumor) with α = P (Type I error) = 0.05 and power = 1-

P (Type II error) ≥ 0.80. 

(iv) The use of historical control data from an appropriate time period 

from the same testing laboratory (i.e., the incidence of tumors and other 

suspect lesions normally occurring under the same laboratory conditions 

and in the same strain of animals employed in the test) is helpful for assessing the significance of changes observed in the current study. 

(3) Test report. (i) In addition to the reporting requirements as specified under 40 CFR part 792, subpart J and 40 CFR part 160, the following 

specific information should be reported: 

(A) Test substance characterization should include: 

(1) Chemical identification. 

(2) Lot or batch number. 

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(3) Physicochemical properties (i.e., fiber morphology, dimension, 

size distribution, aerodynamic diameter, chemistry, density, dissolution 

rate (Kdis), surface characteristics, the ability of a fiber to split longitudinally or cross-sectionally). 

(4) Purity/impurities. 

(B) Test system should contain data on: 

(1) Species and strain of animals used and rationale for selection if 

other than that recommended. 

(2) Age including body weight data and sex. 

(3) Test environment including cage conditions, ambient temperature, 

humidity, and light/dark periods. 

(C) Test procedure should include the following data: 

(1) Method of randomization used. 

(2) Full description of experimental design and procedure. 

(3) Concentration regimen including levels, methods, and volume. 

(D) Test conditions. The following exposure conditions must be reported. 

(1) Description of exposure apparatus including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air and the method of housing 

the animals in a test chamber. 

(2) The equipment for measuring temperature, humidity, and fiber/ 

particulate aerosol concentrations and size should be described. 

(E) Exposure data. These should be tabulated and presented with 

mean values and a measure of variability (e.g. standard deviation) and 

should include: 

(1) Airflow rates through the inhalation equipment. 

(2) Temperature and humidity of air. 

(3) Actual (analytical or gravimetric) concentration in the breathing 

zone. 

(4) Nominal concentration (total amount of test substance fed into 

the inhalation equipment divided by volume of air). 

(5) Fiber and particle size distribution, and calculated mass median 

aerodynamic diameter (MMAD) and geometric standard deviation (GSD). 

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Fiber number reported should also be expressed by fiber length, e.g., WHO 

fibers (>5 μm in length), fibers >10, >15, >20 μm in length. 

(6) Explanation as to why the desired chamber concentration and/ 

or fiber size could not be achieved (if applicable) and the efforts taken 

to comply with this aspect of the guidelines. 

(F) Test results—(1) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for: 

(A) Number of animals exposed. 

(B) Number of animals showing signs of toxicity. 

(C) Number of animals dying. 

(2) Individual animal data. Data should be presented as summary 

(group mean) as well as for individual animals. 

(A) Time of death during the study or whether animals survived to 

termination. 

(B) Time of observation of each abnormal sign and its subsequent 

course. 

(C) Body weight data. 

(D) Feed and water consumption data, when collected. 

(E) Results of ophthalmological examination, when performed. 

(F) Results of hematological tests, when performed. 

(G) Results of clinical chemistry tests, when performed. 

(H) Results of urinalysis tests, when performed. 

(I) Results of lung burden analysis. 

(J) Results of BALF analysis. 

(K) Necropsy findings including absolute/relative organ weight data. 

(L) Detailed description of all histopathological findings. 

Histopathological evaluation should incorporate both qualitative description of lesions and rigorous quantitation. 

(M) Statistical treatment of results where appropriate. 

(N) Historical control data. 

(f) Quality assurance. A system should be developed and maintained 

to assure and document adequate performance of laboratory staff and 

13

equipment. The study must be conducted in compliance with the GLP regulations as described by the Agency (40 CFR parts 160 and 792) and 

the OECD Principles of GLP (ISBN 92-64-12367-9). 

(g) References. The following references should be consulted for additional background information on this guideline. 

(1) Dement, J.M. Overview: Workshop on Fiber Toxicology Research 

Needs. Environmental Health Perspectives 88:261-268 (1990). 

(2) Harington, J.S. Fiber Carcinogenesis: Epidemiologic Observations 

and the Stanton Hypothesis. Journal of National Cancer Institute 67:977-

987 (1981). 

(3) International Union Against Cancer. Carcinogenicity Testing: 

UICC Technical Report Series, Vol.2, Ed. I Berenblum. International 

Union Against Cancer, Geneva (1969). 

(4) ISTRP. International society of Regulatory Toxicology and Pharmacology. Proceedings Symposium on Synthetic Vitreous Fibers: Scientific and Public Policy Issues. Regulatory Toxicology and Pharmacology 

20:S1-S222 (1994). 

(5) Lewis, T.R., Morrow, P.E., McClellan, R.O., Raabe, O.G., Kennedy, G.L., Chhabra, R.S., Schwetz, B.A., Goehl, T.J., and Roycroft, J.H. 

Establishing aerosol exposure concentrations for inhalation toxicity studies. 

Toxicology and Applied Pharmacology 99:377-383 (1989). 

(6) McClellan, R.O., Miller, F.J., Hestersberg, T.H., Warheit, D.B., 

Bunn, W.B., Kane, A.B., Lippmann, M., Mast, R.W., McConnell, E.E. 

and Reinhardt, C.F. Approaches to Evaluating the Toxicity and Carcinogenicity of Man-Made Fibers: Summary of a workshop Held November 

11-13, 1991, Durham, North Carolina. Regulatory Toxicology and Pharmacology 16:321-364 (1992). 

(7) Morrow, P.E., Haseman, J.K., Hobbs, C.H., Driscoll, K.E., Vu, 

V., and Oberdorster, G. Workshop overview: The maximum tolerated dose 

for inhalation bioassays: toxicity vs. overload. Fundamental Applied Toxicology 29:155-167 (1996). 

(8) Organization for Economic Cooperation and Development. Guidelines for Testing of Chemicals, Section 4-Health Effects, Part 453 Combined Chronic Toxicity/Carcinogenicity Studies, Paris (1981). 

(9) Page, N.P. Chronic Toxicity and Carcinogenicity Guidelines. 

Journal of Environmental Pathology and Toxicology 11:161-182 (1977). 

(10) Spurny, K.R., Stober, W., Opiela, H. and Weiss, G. Size-selective Preparation of Inorganic Fibers for Biological Experiments. American 

Industrial Hygiene Association Journal 40:20-37 (1979). 

14

(11) Sontag, J.M., Page, N.P. and Saffiotti, U. Guidelines for Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1 (Bethesda: United 

States Cancer Institute, Division of Cancer Control and Prevention, Carcinogenesis Bioassay Program. 

(12) United States Environmental Protection Agency. Health Effects 

Test Guidelines: Combined Chronic Toxicity/Oncogenicity. 40 CFR 

798.3320 pp. 165–172. 

(13) United States Environmental Protection Agency. Office of Pollution Prevention and Toxics. Workshop on Chronic Inhalation Toxicity and 

Carcinogenicity Testing of Respirable Fibrous Partcles. EPA-748-R-96-

001, January 1996. 

(14) United States Environmental Protection Agency. FIFRA Scientific Advisory Panel Meeting on Test Guidelines for Chronic Inhalation 

Toxicity and Carcinogenicity of Fibrous Partcles, September 26, 2000. 

SAP Report No. 2000-0X, 01/05/2001. 

(15) Vu, V., Barrett, J.C., Roycroft, J., Schuman, L., Dankovic, D., 

Baron, P., Martonen, T., Pepelko, W. and Lai, D. Workshop Report: 

Chronic Inhalation Toxicity and Carcinogenicity Testing of Respirable Fibrous Particles. Regulatory Toxicology and Pharmacology 24:202-212 

(1996). 

(16) World Health Organization (WHO). Part I. Environmental Health 

Criteria 6, Principles and Methods for Evaluating the Toxicity of Chemicals. WHO, Geneva. (1978). 

15