Combined Chronic Toxicity/Carcinogenicity Testing of Respirable Fibrous Particle Final Test Guideline; Notice of Availability

AGENCY:

Environmental Protection Agency (EPA).

ACTION:

Notice of availability.

SUMMARY:

EPA has established a unified library for test guidelines issued by the Office of Prevention, Pesticides and Toxic Substances (OPPTS) for use in testing chemical substances to develop data for submission to EPA under the Toxic Substances Control Act (TSCA), the Federal Food, Drug, and Cosmetic Act (FFDCA), or the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). These test guidelines represent an Agency effort that began in 1991 to harmonize the test guidelines within OPPTS, as well as to harmonize the OPPTS test guidelines with those of the Organization for Economic Cooperation and Development (OECD). The process for developing and amending these test guidelines includes public participation and the extensive involvement of the scientific community, including peer review by the Scientific Advisory Panel (SAP) and the Scientific Advisory Board (SAB) and other expert scientific organizations. With this notice, EPA is announcing the availability of the final test guideline for OPPTS 870.8355 Combined Chronic Toxicity/Carcinogenicity Testing of Respirable Fibrous Particles.

FOR FURTHER INFORMATION CONTACT:

Barbara Cunningham, Director, Office of Program Management and Evaluation, Office of Pollution Prevention and Toxics (7401), Environmental Protection Agency, 1200 Pennsylvania Ave., NW., Washington, DC 20460; telephone number: (202) 554-1404; e-mail address: TSCA-Hotline@epa.gov.

SUPPLEMENTARY INFORMATION:

I. Does this Action Apply to Me?

This action is directed to the public in general. Although this action may be of particular interest to those persons who are or may be required to conduct testing of chemical substances under TSCA, FFDCA, or FIFRA, the Agency has not attempted to describe all the specific entities that may be affected by this action. If you have any questions regarding the applicability of this action to a particular entity, consult the person under FOR FURTHER INFORMATION CONTACT.

II. How Can I Get Additional Information, Including Copies of this Document or Other Related Documents?

1. Electronically. You may obtain electronic copies of this document, and certain other related documents that might be available electronically, from the EPA Internet Home Page at http://www.epa.gov/. To access this document, on the Home Page select “Laws and Regulations,” “Regulations and Proposed Rules,” and then look up the entry for this document under the “Federal Register—Environmental Documents.” You can also go directly to the Federal Register listings at http://www.epa.gov/fedrgstr/. You may also obtain copies of test guidelines from the EPA Internet Home Page at http://www.epa.gov/opptsfrs/home/guidelin.htm.

2. In person. The Agency has established an official record for this final guideline under docket control number OPP-00723. The official record consists of the documents specifically referenced in this action, any public comments received during an applicable comment period, and other information related to this action, including any information claimed as confidential business information (CBI). This official record includes the documents that are physically located in the docket, as well as the documents that are referenced in those documents. The public version of the official record does not include any information claimed as CBI. The public version of the official record, which includes printed, paper versions of any electronic comments submitted during an applicable comment period, is available for inspection in the Public Information and Records Integrity Branch (PIRIB), Rm.119, Crystal Mall #2, 1921 Jefferson Davis Hwy., Arlington, VA, from 8:30 a.m. to 4 p.m., Monday through Friday, excluding legal holidays. The PIRIB telephone number is (703) 305-5805.

III. What Action is EPA Taking?

EPA is announcing the availability of the final test guideline for OPPTS 870.8355 Combined Chronic Toxicity/Carcinogenicity Testing of Respirable Fibrous Particles.

EPA recognizes that the current health effects test guidelines for chronic inhalation toxicity and/or carcinogenicity studies on chemicals are not specific enough for the testing of fibrous substances. These guidelines have to be modified to take into account testing issues which are unique to fibrous particles. Although a number of test systems and/or protocols have been utilized by the scientific community for evaluating the fibrogenic and carcinogenic potential of fibrous particles, there has been considerable debate about the scientific validity and utility of available test methods. Thus, there is a need for EPA to develop a standardized health effects test guideline for fibrous substances that can be used by EPA in future rulemaking, negotiated enforceable consent agreements, or voluntary action to obtain the necessary toxicologic information for risk assessment purposes.

The objective of this combined chronic toxicity/carcinogenicity testing of respirable fibrous particles is to determine the effects of a fibrous substance identified to be of potential health concern in at least a rodent species following prolonged and repeated inhalation exposure. The application of this guideline should generate data which identify the majority of chronic toxic and carcinogenic effects and determine dose-response relationships.

EPA recognizes concerns have been expressed about data development using animal models. While no comments were received from the animal advocacy community, it is important to note that EPA is committed to avoiding unnecessary or duplicative animal testing. As part of this commitment, the Agency plays an important role in the Federal Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) (http://iccvam.niehs.nih.gov/ home.htm) whose goals are: (1) To encourage the reduction of the number of animals used in testing; (2) to seek opportunities to replace test methods requiring animals with alternative test methods when acceptable alternative methods are available; and (3) to refine existing test methods to optimize animal use when there is no substitute for animal testing. Further, where testing is needed to develop scientifically adequate data, the Agency is committed to reducing the number of animals used for testing, including, whenever possible, by incorporating in vitro (non-animal) test methods or other alternative approaches that have been scientifically validated and have received regulatory acceptance. EPA considers these goals and commitments to be important considerations in developing health effects data; however, they must be balanced with the essential need to conduct scientifically sound chemical hazard/risk assessments in support of the Agency's mission to protect human health and the environment studies.

IV. Response to Public Comments

In the notice of availability for the proposed test guideline published in the Federal Register of July 28, 1999 (64 FR 40871) (FRL-6078-6), EPA requested comments from the public on the proposed test guideline which was developed using the current EPA/OPPTS health effects test guideline for combined chronic toxicity and carcinogenicity (Ref. 1) as a template and based on the comments and recommendations made by a 1995 Workshop Panel on a number of scientific issues specific for fiber testing (Refs. 2 and 4). A number of comments on various issues related to the test substance and the study design were received from three U.S. fiber manufacturer associations, a European fiber industry association, a U.S. Government agency, a U.S. consultant, and a European scientist. All public comments were evaluated and a revised draft guideline with some of the public comments incorporated was prepared. At an EPA FIFRA SAP meeting on September 26, 2000, the draft guideline as well as a number of issues raised by the public commenters were reviewed and evaluated by the SAP. In addition to providing EPA with comments on the draft guideline, the SAP provided EPA an opinion on a number of issues raised by the public commenters (Ref. 3). Following are the major public comments received and EPA's responses to these comments.

Comment 1. A number of commenters expressed that with the recent development of short-term in vitro and in vivo assay systems for predicting the biopersistence and toxicity of fibers, it is frequently possible to make informed statements about the likely hazard and risk posed by a fiber. Therefore, EPA should make use of these new developments and consider a tiered testing approach; the circumstances under which long-term rodent inhalation studies of a fiber are required should be specified in the guideline.

Response. In the 1995 Workshop, the panel had identified a number of in vitro and in vivo short-term screening tests that could be used to develop a minimum data set for making decisions about the potential health hazards of fibers and for prioritizing the need for further testing in a chronic inhalation study. EPA is aware of the recent development of short-term in vitro and in vivo assay systems for predicting the biopersistence and toxicity of fibers and has initiated a plan to develop a tiered testing approach for identifying potential hazardous fibers for long-term inhalation studies. In response to this comment, the following statement indicating the use of short-term tests to identify potential hazardous fibers for long-term inhalation studies has been added in the “Purpose” paragraph of the guideline: “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.”

Comment 2. A commenter indicated that there are no verified reports showing evidence of serious adverse health effects caused by organic fibers in either occupational or consumer settings and suggested that EPA should expressly exclude organic fibers from the guideline.

Response. As indicated in the response to comment 1, a fibrous substance will be tested for chronic toxicity/carcinogenicity under this guideline only if it is identified to be of potential human health concern. The SAP believed that the guideline will have application to testing of some organic fibers. Since the guideline was derived primarily from experience with inorganic fibers and there are differences between organic and inorganic fibers, the following statement has been added to the test guideline as recommended by the SAP: “While the guideline will have application to testing of organic fibers, additional considerations may be necessary for study of organic fibers.”

Comment 3. A couple of commenters expressed that it would be very inefficient for the industry to have to conduct more than one study in order to satisfy regulatory guidelines in both Europe and the United States and suggested that EPA should work closely with the Organization for Economic Cooperation and Development (OECD) to develop a harmonized testing guideline for the chronic toxicity and carcinogenicity of fibers.

Response. The EPA fiber testing guideline was developed based largely on the recommendations made by a panel of international experts. There are fundamental differences in the purpose and objective of long-term toxicity tests in Europe and the United States. In Europe, the primary purpose of the tests is to collect data for hazard identification while the United States requires the data for both hazard identification and dose-response assessment. Nonetheless, in the development of the EPA fiber testing guideline, attempts have been made to harmonize with available OECD guidelines to the extent possible.

Comment 4. This comment deals with the definition and criteria of test materials suitable for rat inhalation studies. The guideline specifies that for rat inhalation studies, the fiber samples used should be rat-respirable. A rat-respirable fiber is defined as a fiber with an aspect ratio of at least 3:1 and an aerodynamic diameter of less than 3 μm. A comment suggested that the definition of a “rat-respirable fiber” should be modified to be “a fiber with a geometric mean diameter (GMD) ≤0.8 μm” since actual measurement of the aerodynamic diameter of fibrous materials by traditional sampling techniques is not always reliable.

Response. The definition of fibers is adopted from the recommendation of a Workshop Panel and is widely accepted in the field. The Workshop Panel recommended that respirability should be defined on the basis of experimental data, rather than calculated data. For many types of fibers, what reaches the rat lung has been well characterized; an upper limit of 3 μm aerodynamic diameter is deemed effective in capturing rat-respirable fibers. The guideline defines a “rat-respirable fiber” using the aerodynamic diameter rather than the geometric diameter because the aerodynamic diameter (and not the geometric diameter) is the major determinant for the respirability of fibrous particles. The SAP also concluded that a definition of “rat-respirable fiber” as having an aerodynamic diameter of 3 μm would be preferable to a definition of GMD of ≤ 0.8 μm.

Comment 5. In the definition section of the guideline, the “concentration” of fibrous particles was expressed as the absolute number of fibers/cc. A comment suggested that “Aerosol concentration should also be expressed as the total number of WHO f/cc.” A WHO (World Health Organization) fiber is a fiber with a diameter < 3 μm and a length > 5 μm.

Response. On this issue, the SAP concluded that the definition of concentration of fibrous particles should start with the absolute number of fibers/cc. However, it emphasized that exposure concentrations should also be expressed as WHO fibers/cc as well as fibers greater than 20 μm/cc and smaller size categories. Accordingly, the following statement has been added into this final test guideline: “Exposure concentrations should also be expressed by fiber length, e.g., WHO fibers (greater than 5 μm in length)/cc, fibers with length greater than 10, 15 and 20 μm/cc.

Comment 6. There are a couple of comments on the selection of animal species for fiber testings. A commenter expressed that the rat inhalation model is not sensitive enough to reliably predict the cancer risk of fibers for human. The basis of the argument is that to cause similar risks from asbestos by chronic inhalation experiments with rats, the fiber concentration per milliliter (mL) measured with the same method as in the workplace atmosphere has to be about 100 times higher. Another commenter felt that although the hamster is capable of maximizing the number of mesotheliomas that may be induced by a fiber, the cost of such information outweighs its usefulness, and that the use of the model is controversial because hamsters are unusually sensitive to the development of mesothelioma.

Response. The rat bioassay model has proven to be a fairly good predictor of carcinogenic potential of fibers in humans. Both the Workshop Panel and the SAP concluded that for identifying the carcinogenic potential of fibers via the inhalation route, the rat is clearly the species of choice because of its susceptibility to fiber-induced pulmonary fibrosis, lung neoplasms and mesothelioma. While the rat model is quite effective at identifying the carcinogenic potential of fibers, as the commenter pointed out, it is of less value as a measure of carcinogenic potency. EPA uses testing not only for qualitative determination of carcinogenic potential, but also for quantitative risk assessment, and has historically required the use of two species. Although only the rat model has a sufficient database to be recommended for inhalation exposure studies with fibers, both the Workshop Panel and the SAP expressed that the hamster could be used if mesothelioma was the endpoint of interest because the hamster is more sensitive than the rat for this endpoint. However, the panels commented that the use of the hamster should not be a mandatory requirement because the hamster does not seem to develop lung tumors and lifetime hamster studies are fraught with technical problems. Hence, regarding animal species selection, the final test guideline recommends the rat to be the first species used and since the hamster is more sensitive than the rat with respect of 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.

Comment 7. A number of commenters expressed opinions that: “The use of both sexes of rats does not appear warranted, either in expense or the use of additional animals; male and female rats do not appear to differ in their response to the pathogenic effects of fibers.”

Response. It is true that presently, there is no evidence of a sex difference in response to inhaled fibers, in contrast to non-fibrous particles where female rats appear to be more sensitive (thus a single sex is adequate). As a commenter of the previous comment (comment 6) pointed out, the rat appears to be less sensitive than the human (with regard to tumorigenesis of asbestos), and this provides another reason for using both sexes since the use of both sexes would increase the total number of animals (typically from 50 to 100) and thus the sensitivity of the chronic bioassay. On this issue, the Workshop Panel concluded that testing in both sexes should be encouraged. Most of the SAP members also felt that both sexes of rats should be required for the chronic toxicity and carcinogenicity testing of fibers. Therefore, the final guideline maintains that “Equal numbers of animals of each sex should be used at each dose level.”

Comment 8. There are a few comments on the characterization of the exposure aerosol. For instance, one commenter suggested that: “The system used to generate fibrous aerosols must not cause significant breakage and contamination of the test substance.” Another commenter suggested to change the sizes of fibers required to be enriched in the exposure aerosol fibers to maximize the sensitivity of animal inhalation exposure studies: “An exposure aerosol should be enriched with the following fiber size fractions: rat respirable fibers with an aspect ratio of at least 3:1 and an aerodynamic diameter less than 2.4 μm, and include an appropriate number of fibers with lengths of at least 15 μm.”

Response. The final test guideline has adopted the suggestion from the commenter that “The system used to generate fibrous aerosols must not cause significant breakage and contamination of the test substance.” Further, it specifies that: “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.” With regard to the fibers to be enriched in the exposure aerosol to maximize the sensitivity of animal inhalation exposure studies, the guideline first specifies that the aerosol should be characterized in terms of fiber and non-fiber/particle size and number; the number of fiber should be expressed by fiber length, e.g., WHO fibers (greater than 5 μm in length), fibers greater than 10, 15 and 20 μm in length. It is realized that different fiber types may have different size characteristics and it may not always be feasible to generate long, thin fibers (e.g., longer that 20 μm, thinner than 1 μm) as was specified in the draft guideline. Therefore, the final test guideline no longer specifies the fiber size requirement and simply states that: “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. ..... 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.”

Comment 9. This comment relates to the selection of the highest fiber concentration level, known as the Maximal Aerosol Concentration or MAC, to be tested in a chronic inhalation exposure study. A commenter expressed concern that while the use of an insufficient number of long fibers would produce false negative results, using too large a mass exposure would cause pulmonary overload and produce false positive results. Therefore, it would be more useful operationally if the word “appropriate” is defined in the guideline: “An appropriate lung burden of critical fibers (long and thin) should be achieved” (for setting the MAC). Another commenter felt that the definition of MAC should be expanded to include the following statement: “The MAC is the highest concentration of test substance that will not cause a significant impairment (retardation) of particle clearance based on assessment of clearance in a 90-day subchronic inhalation study.”

Response. The final guideline has specified that the MAC, the highest fiber concentration to be tested in a chronic study, should be set at a level at which some degree of impaired clearance and toxicity (as determined by parameters observed during lung burden analysis and bronchoalveolar lavage fluid (BALF) analysis in a 90-day subchronic inhalation study) are observed. The SAP concluded that there is no universal level of long fibers that would serve as an appropriate lung burden for all types of fibers. Important differences in the biopersistence and other key physicochemical properties account for the difficulty in setting a single appropriate burden. Present information is insufficient to set levels for what constitutes “significant impairment” of particle clearance. Both the Workshop Panel and the SAP concluded that a weight-of-evidence approach, based on all data of the 90-day subchronic inhalation studies, should be used for establishing whether a given exposure meets or exceeds the MAC.

Comment 10. On fiber concentration level selection, a commenter suggested that “The lower exposures should be defined as some fractions of the highest exposure.” Another commenter expressed that: “A single exposure level would suffice if it is anticipated that the MAC will not show carcinogenic activity.”

Response. The guideline does not attempt to provide specific fractions/numbers to define the lower exposure levels. The lower levels should be appropriately spaced to produce a gradation of toxic effects. A rationale for the concentrations selected must be provided. The objective of a combined chronic toxicity/carcinogenicity study is to determine the effects of a fibrous substance identified to be of potential health concern. If a fibrous material is not anticipated to have any health hazard concern, a chronic inhalation study need not be performed. When a fibrous substance is to be tested, for dose-response analysis, at least three concentrations levels should be used.

Comment 11. A commenter expressed that “recovery” animals for satellite dose groups in lung burden analysis and BALF analysis can provide important information on the interpretation of the test results and suggested to remove a minimal of 5 animals from each dose group at 3, 6, 12, and 18 months of exposure and then hold until 24 months at which they will be evaluated for the kinetics of fiber buildup/removal from the lungs and progression/regression of lesions.

Response. The final guideline has adopted the suggestions of this comment which is endorsed by the SAP.

Comment 12. A couple of commenters expressed that BALF analysis data are useful in the 90-day studies and are not of any value in the lifetime studies.

Response. Most members of the SAP felt that BALF analysis at interim time points (3, 6, 12, 18 and 24 months) are very useful for an overall evaluation of the fiber toxicity and proper interpretation of the study results. However, it was also felt that a decision to include all time points should be left to the individual study design, and some of the interim time points (e.g., 3, 6 and 18 months) could be omitted. On the basis of this, the final guideline requires BALF analysis be conducted only at the 12 and 24 month sacrifices and makes other time points optional.

Comment 13. On clinical pathology (hematology and serum chemistry) and ophthalmology evaluation, there were comments that these determinations provide little toxicological value in the assessment of fiber hazard/risk and should be omitted.

Response. The final guideline has adopted the suggestions of these comments which are endorsed by the SAP. However, evaluation of these parameters is required when clinical changes are seen in subchronic studies.

Comment 14. On histopathology, a commenter suggested that: “The histology slides from the scheduled sacrifices should also be stained with Masson-Goldner's trichrome stain that identifies collagen (fibrosis),” and that: “The scheduled sacrifices should be recorded according to the Wagner scoring method.”

Response. In response to this comment, the following statement has been added into the final test guideline: “The histology slides from the scheduled sacrifices should, in addition to standard hematoxylin and eosin, be stained with a method that identifies collagen (fibrosis).” The SAP commented that there are many stains for collagen and no single stain should be specified. The Wagner scoring system has proved useful for providing a consistent and systemic reference for parenchymal disease. It also can be of value when attempting to compare the pathogenic effects of one fiber to another. However, use of the Wagner scoring system to evaluate progression of fibrosis has the disadvantage of being purely qualitative. The SAP suggested to take a consistent approach to record and grade the findings of airway disease and to use “image analysis” to assess severity of lesions. The recommendations of the SAP were incorporated into the final guideline.

V. How Was this Test Guideline Developed?

On May 8-10, 1995, a Workshop on chronic inhalation toxicity and carcinogenicity testing of respirable fibrous particles was held in Chapel Hill, North Carolina. The Workshop was sponsored by the Office of Pollution Prevention and Toxics, Environmental Protection Agency, in collaboration with the National Institute of Environmental Health Sciences (NIEHS), the National Institute for Occupational Safety and Health (NIOSH), and the Occupational Safety and Health Administration (OSHA). The goal of the Workshop was to obtain input from the scientific community on a number of issues related to fiber testing. The Workshop Panel, which was composed of 19 international expert scientists in inhalation toxicology, reviewed, evaluated, and commented on the scientific issues of the Workshop. After extensive discussion and debate of the Workshop issues, the Workshop Panel provided a number of recommendations specific for the design and conduct of chronic inhalation studies of fibers (Refs. 2 and 4).

Using the current EPA/OPPTS health effects test guideline for combined chronic toxicity and carcinogenicity (Ref. 1) as a template and based on the comments and recommendations made by the Workshop Panel on a number of scientific issues specific for fiber testing, EPA/OPPT developed a proposed guideline for combined chronic toxicity and carcinogenicity testing of fibrous particles. In July 1999, the proposed guideline was announced in the Federal Register for public comments (64 FR 40871, July 28, 1999). A number of comments on various issues related to the test substance and study design were received. All public comments were evaluated and a revised draft guideline with some of the public comments incorporated was prepared.

On September 26, 2000, the EPA FIFRA SAP held a meeting to review the EPA draft guideline, advise on a number of issues raised by the public commenters on the study protocol, and provide EPA with an opinion about the scientific validity of the test guideline. The final test guideline for OPPTS 870.8355 Combined Chronic Toxicity/ Carcinogenicity Testing of Respirable Fibrous Particles has incorporated many of the comments and recommendations made by the SAP (Ref. 3).

VI. Are there Any Applicable Voluntary Consensus Standards that EPA Should Consider?

This notice of availability does not involve a proposed regulatory action that would require the Agency to consider voluntary consensus standards pursuant to section 12(d) of the National Technology Transfer and Advancement Act of 1995 (NTTAA), Public Law 104-113, section 12(d) (15 U.S.C. 272 note). Section 12(d) directs EPA to use voluntary consensus standards in its regulatory activities unless to do so would be inconsistent with applicable law or otherwise impractical. Voluntary consensus standards are technical standards (e.g., materials specifications, test methods, sampling procedures, and business practices) that are developed or adopted by voluntary consensus standards bodies. The NTTAA requires EPA to provide an explanation to Congress, through Office of Management and Budget (OMB), when the Agency decides not to use available and applicable voluntary consensus standards when the NTTAA directs the Agency to do so.

In the notice of availability for the proposed test guideline published in the Federal Register of July 28, 1999 (64 FR 40871), EPA specifically sought comment on the availability of any applicable voluntary consensus standards that should be considered during the development of the final test guideline or any future regulatory action that EPA may take under TSCA. No response to this request was received.

VII. References

The following references are cited in this document by the number indicated:

1. United States Environmental Protection Agency. Health Effects Test Guidelines: Combined Chronic Toxicity/Oncogenicity. 40 CFR 798.3320. pp. 165-172.

2. 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.

3. 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.

4. 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).

List of Subjects

• Environmental protection
• Chemical testing
• Test guideline