Tiered testing for
chemical hazard assessment A hierarchical, four-1er:el system of increasingly complex and time-consuming tests is requiredjor efficient, reliable, and economical ecaluation of new chemicals
Judith M. Hushon Robert J. Clerman M I T R E Corporation McLean, Vu. 22102 Burkhard 0. Wagner OECD Encironrvient Directorate Paris, France During the past decade it has become increasingly evident that a number of chemicals in comnierce pose a potential threat to human health a n d / o r t h e environment. M a n ) countries have moved to ban production or curtail use of such substances as PCBs, chlorofluorocarbons. vinql chloride, and Tris-all existing c he in ica Is. There has also been growing pressure from the public sector to control the entry into the marketplace of new chemical substances. In response. several countries have been developing and implementing legislation to regulate new chemicals; among these are Canada, Denmark, the Federal Republic of Germany, France. Japan, Siceden, Switzerland. and the U.S. (Table I ) . In general. each piece of legislation has been developed independently of the laws of neighboring
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countries. The European Communities ;iIso passed. in June of this year, a chemicals' directive, which will be reflected in the toxic substances laws of the member countries within two )ears of its passage. There is ;in effort now under way b) the Organiration for Economic Cooperation and Development (OECD), headyunrtcred in Paris. to implement t h e various I a u s i n a harmonired manner. For this purpose, the O E C D initiated a chemicals testing program i n 1977 to develop consistent d 'I, t a requircmcnts and testing methods to be uscd for predictive purposes in assessing potential effects of new chemicals bcforc they arc marketed. This should fLicilitatc international trade. one of the goal.; of the O E C D . W i t h i n t hi s f rii ni e u or k . test i ng g II id e I i n es for c h c in i ca I ha ra r d as scssnient are being developed b) expert groups. These guidelines \ \ i l l generally recommend a set of testing methods and schemes for establishing testing priorities in a cost cffective manner. Coni pl e in en t a r y efforts ;I re ;i I so being made t o develop principles of good 1abor:itor) practice to assure the q u a I i t y a nd i n tercoin pa ra b i 1it ) of testing data. A tiered scheme is emerging a s the most vinble approach to developing thc t e s t d a t a needed for hiiiard :~xscssment. It \4as in response to this s i t u a tion that the tiered-testing system prescnted below wiis designed (and rccomrncridcd) by M I T R E to the
Federal Republic of Germany's Environmental Agency; i t is not meant to represent the current views of that agency. T h e results are published i n a two-volume set entitled /nformation Required for Toxic Substances Regii la t ion. P B - 2 8 80 2 3 .
Test method selection In all of the existing or proposed programs for regulating new chemicals. two general categories of information a r e required of the manufacturer. T h e first relates to a substance's Linticipated production. market distribution, use patterns, and disposal; this t \ p e of information is gathered as part of the market/use characterization process. T h e second class of information covers a substance's physical. chemical. biological. and environmental properties: for new chemicals, these data are generated by tests. In order to decide which tests should bc required of a nianufacturer/importer to predict a new chemical's fate and effects, a survel of test methods \+:is undertaken. This survey resulted in the identification of over 150 test methods covering the following ;i r e;\ s : physical/cheniical properties mobility and transport degradabilit) biological accumulation acute and chronic toxicity ni u t ;!ge n i c i t y , carcinogenicity , tcrxtogcnicity.
0013-936X/79/0913-1202$01.00/0
@
1979 American Chemical Society
effects testing should bt: covered with minimun-1 of over1:ip (Table 2). Tests should he included that represent each of the major exposure environments: t e r r e5t r i a I , a q u a tic, and a t m os p h e r i c. I n addition, animal. plant. microbial, a n d human populations should be represented in the effect:, tests. Finally. the tiered-testing s p t c n i should be applicable to ;I v:iriet! of chemicals. To achieve uniformit! across the rn.in! categoric. included in the \cheriic. the decision options should be clearlq specified for each set o f required te.ts. Ever> cff1,)rt should be made to e q u a l i x the difficulty of the tezts. the degree of quantitation. and
the accuracy of the hazard prediction a t each level. A standardized reporting format is desirable, including specification and justification of any deviation from standard procedures. Overall accuracy in hazard prediction is a function of the accuracy. reliability, and sensitivity of thr: individual tests in the tiered system. U n fortunately, theie is a high degree of uncertainty associated with the interpretation of many test results. This uncertainty can be mitigated to some extent by including duplicative tests a t certain points in the tiered scheme. Where no individual test accurately predicts a hazard, a battery of t.e>ts is
TABLE 1
Canada France Federal Republic of Germany Japan Sweden Switzerland
us. Denmark European Communities
Environmental Contaminants Act Control of Chemical Products Act Chemicals Act
1974 1977 Pending
Chemical Substances Control Act Act on Products Hazardous to Man and the Environment Law on Trade in Toxic Substances
1973 1973
Toxic Substances Control Act Toxic Chemicals Act Directive for the Sixth Modificationof the Council Directive of 27 June 1967 on the Approximation of the Laws of Member States Relating to the Classification, Packaging, and Labeling of Dangerous Substances
1969 (effective 1972) 1976 Pending
1979
TABLE 2
Water solubility Partition coefficient Dissociationconstant Volatility Biodegradation Soil Water Photodegradation Air Liquid Soil surface Solid surface Chemical degradation Hydrolysis Oxidation
Aquatic Terrestrial Human
Point mutations DNA repair Chromosome aberrations Cultured cell mutagenesis In vivo mutagenesis Cell transformation Chronic testing Teratogenicity
Adsorption Soils Particles in air Detritus Leaching Terrestrial Aquatic Airborne Human health
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mandated. This battery approach is best exemplified in carcinogenicity screening, which is discussed later. Given the rapid developments in chemical hazard assessment during the last few years, it is essential that a tiered system be flexible in design so as to be able to incorporate new techniques, and reflect subsequent policy decisions. It should be possible to integrate new interpretations of existing tests into the tiered plan, and to refine older methods.
Criteria for test placement The tiered-testing system discussed below is structured around four levels of increasingly complex tests. At each level, tests are categorized according to the properties they measure. Subcategories are based on the exposure medium (air, water, or soil) and the organisms exposed. Four levels or tiers are suggested (box). The major criteria for placing a test a t a particular level are: time required level of training necessary estimated cost per compound tested predictive or confirmatory nature of the test accuracy and sensitivity how “crucial” the test is for hazard assessment difficulty of performing the test or analyzing the results. For a test to be placed a t the first level, it must be a sensitive indicator, yet pass those compounds which clearly do not pose a hazard. Also, the results from first-level tests should predict the nature of the hazard for substances that require further testing. Ideally, a test should not produce false negatives, and permit only a limited number of false positives. Higher level tests generally provide more accurate data than those from lower level tests, and serve to confirm or refute early hazard perdictions. Results from these higher level tests are generally expressed quantitatively, and test conditions more closely approximate anticipated natural exposure conditions. At Level 111, for example, microcosms or other elaborate simulation systems are used to model field exposure. I n many cases these complicated and costly tests are not necessary to assess hazayd. Screening tests, predominant a t Levels 0 and I , are not merely low-cost substitutes for higher level tests; they a r e a means of determining when further testing is necessary, and indicating the particular test(s) required. A positive result (failure) from an early test should not preclude a manufacturer 1204
Environmental Science & Technology
from proceeding to a higher level test to rule out a false positive, or to clarify a substance’s environmental, or human health risk. As more efficient and accurate screening methods are developed, they will increase the reliability of predictions based on results from Levels 0 and 1 in the tiered system, and limit the need for more costly tests a t higher
levels. Level 0 tests should have the greatest impact on the development of new chemicals since they will be done routinely, and a chemical will have to pass this level to enter the market.
Flow through the system The flow of testing from one level to another is fundamentally dependent on a chemical’s anticipated health and ecological effects, environmental fate, and human-exposure levels. The environmental fate and human exposure levels are initially estimated from physical/chemical properties, anticipated production levels, and use and disposal patterns (Figure 1). This initial exposure estimate is used in selecting Level I test categories. The preliminary assessment of health and environmental effects is based on acute toxicity tests, and a mutagenicity/carcinogenicity screen a t Level 0. Results from these toxicity tests are used in establishing dosages, routes of administration, and test organisms a t Level I. Progress to higher levels in all testing categories is triggered by results of earlier tests in the form of a refined exposure and effects assessment. Once the basic structure and design criteria for a tiered system have been decided upon, the key steps i n the flow of testing must be identified. One of the distinguishing characterizations of tiered testing is that the same decision processes apply a t Levels 1-111. In
Figure 2, this approach is presented as a series of decision options within an iterative loop for additional testing. The preliminary hazard assessment, combining available exposure and effects data, determines the appropriate test categories a t Level I. It is important to note that the decision to continue testing does not necessarily mean progress to the next level; testing in new categories may be required a t the same level. Therefore, the flow of testing can be within as well as between levels. This hazard assessment ultimately results in a decision to regulate, to allow use without further testing, or to continue testing. If testing is to be continued, a reassessment of exposure and effects data is made, test categories are selected, and the hazard assessment process is repeated.
Test categories Tests for mobility and transport are predictive of a substance’s distribution and fate in the environment. A chemical’s location in the environment determines its availability for biological and chemical interactions, including toxicity, bioaccumulation, and degradation. Mobility and transport tests a r e designed primarily to predict partitioning among three media: air, water, and soil. Two physical/chemical properties that greatly affect a substance’s mobility in the environment are its water solubility and volatility (vapor pressure). These and other distributional characteristics are used to select potentially applicable mobility and transport tests. Tests for adsorption in soils and soil-water suspensions are included along with tests for leachability (desorption). Adsorption to detritus particles indicates potential entrance into aquatic and terrestrial food webs, and adsorption to atmospheric particles indicates wide-ranging transport with potential inhalation exposure. A substance’s tendency to degrade is particularly important in determining its environmental fate and potential toxic effects. Degradability tests indicate the conditions under which degradation is likely to occur, and allow for identification of significant breakdown products. Substances that a r e highly resistant to breakdown merit thorough toxicity and bioaccumulation testing as they present a high potential for exposure. A measurement of microbial biodegradation is likely to be included as a mandatory test under most testing programs. Additional biodegradation, chemical degradation, and photodeg-
radation tests may be required.:These include tests of biodegradation in soil and water. Chemical degradation can be divided into tests for hydrolysis in aquatic and nonarid soil environments, a n d oxidation in aquatic and atmospheric environments. Photodegradation tests are categorized according to the medium in which the reaction occurs: air, water, soil surface, or solid surface. Biological accumulation poses a hazard to both the organisms accumulating a chemical, and the consumers feeding on these organisms. Toxic heavy metal and pesticide contaminations of fish and shellfish a r e good examples of the hazards associated with bioaccumulation. Increasing body burdens over time can have debilitating effects, perhaps eventually resulting in mortality. T h e octanol/water partition coefficient is generally considered to be a reliable indicator of bioaccumulation potential. T h e octanol/water partitioning results, coupled with anticipated exposure information, permit selection of an appropriate bioaccumulation test. Tests for accumulation by microorganisms, plants, and animals from soil, water, and air are included. Biomagnification, the process by which concentrations increase over progressive trophic levels, is measured a t Levels II and 111. An additional category of tests screens for potential accumulation in humans by measuring uptake, accumulation, and metabolism in rats. Toxicity has long been a concern associated with the production and use of chemical substances. Consequently, research on test methods and mechanisms of toxic action is more advanced than in any other area of hazard assessment. Where once an acute oral dosage test with rats (LD5o) was considered an adequate indicator of a substance's toxicity, today a wide range of methods and test organisms are available to define toxicity. In this area of hazard assessment, perhaps more than any other, tiered testing is required. It now appears likely that the premanufacture notification testing requirements in most toxic substances regulations will include one or more acute toxicity tests. The European Communities are including in their proposed Base Set (Level 0) tests for acute toxicity, subchronic toxicity, skin, irritation, eye irritation, sensitization, and acute aquatic toxicity (fish and
Daphnia). The primary factors determining which tests to perform a t Level I a r e exposure patterns, results from Level
FIGURE 1
Preliminary exposure assessment Exposure
Physical/chemical .properties
Production
Amount Environmental transport & fate predictions
I
.
Dispersion
Amount
,A
Temporal
Spatial
I
Disposal (function of production & use)
Dispersion
', Temporal
Spatial
Monitoring confirmation
FIGURE 2
Hazard testing process
0 tests, and structural similarity to known toxicants. Tests with aquatic and terrestrial organisms range from simple assays with microorganisms to long-term feeding studies. Genetic effects In this testing category, individual tests for mutagenicity, carcinogenicity, and teratogenicity a r e grouped according to the type of genetic defect
they can detect: point mutations, DNA repair, chromosome aberrations, cultured-cell mutagenesis, in vivo mutagenesis, cell transformation, and teratogenicity (Table 3). Within these groups, a combination of battery testing and the tiered-testing approach described for the other categories appears to be most efficient. By using the battery approach, a chemical is subjected to a number of tests simulVolume 13, Number 10, October 1979 1205
cell transformatton
In vivo and in vitro transformation
point mutation Sperm morphology Histocompat-
111
Viral transformation
Mammalian teratogenicity
Embryo toxicity
;it each ':est level. T h e test b:ttter> is nccess;ir\ because in the riiiit,ifenicit!!'c.arc:inogenicit) area. no iJnc indiLidual te:,t is sufficienti) :ICciir:ite. or broad criough in the tjpes of d c l ' c c t \ that i t c:ln detect to st:ind
t:ineciu\l!
;I
I o n i: .
During the pa',i decade the sornatic- cc: I I ni u t :I t i o n t heor > of c;i rc i nogcric\,is has gainec: 11 idespread accepta!ici:. I'hi5 theorb s t a t e s t h a t alter; i t i o n of gcnctic inaterial in a \omatic ccll u p o n cxpt?\ure to an cyternal agent o r \ h t ; i i i c c ma! result in change\ i n the ccll'4 g r o n t h p a t t e r n t h a t t r a n s 1'or.iii i t t o the iiiaiignant state of u n cor:~r.c!licd fro\\ t -I. This theor) has gairicd credence :is iiiost chemical c:irciiogens ;ilso ioiv positive results i n i i i u tagenici t y t c \ t \ . , \ I I.c\.cl 0. st;iritlard plate and liq11id - c u I I u rc sc rce r i i ng t e:,t ;I re s uggeltetl. I l i c s ~shou ' Id be supported M ith ho\t--iicdi:itcd a + , i > s :it Levei I to de( c c i coi iipou nds req ui ring metabolic ~ i c ! ! ~ ~ i i i(indirect oii mutagens). or \ t i t h b i ! i i i ! , i r test\ using additional test \ I' I cicl I .,hould :iIso include j>l,ltc t e s t \ using rcpair deficient bactcricii \ : r a i n \ ,end micronucleus tests. ; I I ; i p i d !hougii !e!,\ s c n i t i v e indicator 01 chroinosom,tl aberrLitions than the I I baric m a rrow test. I i i c i I I tchts iri,lude ;I c r( 1 L iii u t a s c n i ci t iiicit!, !c\t c:itegclries. A t this level. \ o i i i c oi' the icbts. for instance those i i i i o i i ing cell culture techniques. lire L:
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Environmental Science & Technology
extrcmelq time consuming a n d difficult to perform, 1% tiile others-the Drosophila sex-linked recessive lethai test. for example---are less rigorous and serve to confirm finding?, a t L.erels I and 1 I . Long-term carcinogenicity testing v.as purposely ignored i n this test skstcm since its use wouid be to confirm Level 1-1 I 1 findings, and its high costs place it i n a unique class. Tcratogcnicit! tests a t Level I l l include studies ot' both live-born progen!. and decreased fertilit). There has been soiiie controversy as to LI hether teratogeniciL1 testing should be required a t a lowel- level. T h e decision to place it at Level I I I came from the feeling that some of t h e specific iiiutagcnicit> tests. for example the ' * > t 21 nd a rd-do m i n;i n t I et ha I assij' i n rats using subchronic Iciposure," which is ;i Level I I test, ;ictually provide v ;I I u;i b IC teratogen ic i nfor m a t ion ;I s well a s data on mutagenicit!. The remLiining Level 111 tests are either high11 complex procedures or difficult to interpret with regard to hunian health effects. X variet! of mutagenicity. carcin og e n i c i t y , a n d t e r ;!. t oge n i c i t > tests that use different organism:, and testing s p t e n i s provide comparabie re5 u l i s . T h e tiered-testing system should be sufficiuntl) flexible to accept results from a n y of the cornpdrable test systenis. Similarly. in this test category. decisions m u 5 t be based on the results or '1 biitterq of tests. not those of a n ~
individual test, as false positives and false negatives a r e known to occur in i n d ivid u a I test systems. In summary T h e tiered- tes t i ng approach suggested here is not perfect, but i f used sagaciously it has the potential to pro\,ide those data required to assess the potential health and/or environmental hazard of a new chemical substance. A s shown in Figure 1 . this decision may be to allow production, to regulate, to withdraw a chemical voluntarily from the market. or to require Lidditional testing. Ever! decision should reflect the results achiwed from all tests in applicable areas; i t is the overall pattern of the results which must be considered a s results, of a single test could be misleading. Intcrmediary decisions a r e also possible because a chemical could be permitted for only certain contained uses, or production could be limited t c ' a ceiling poundage. Yo matter what the structure of the decision may be. tiered testing seems to provide the greatest guidance to the chemical hazard assessor. Much work remains to be done to define the decision criteria based on the results of internationally recommended test methods. With these definitions of decision criteria and selection of specific test methods. tiered teating will become a tool that the chcmica! haz'essor will find indispensable.
Additional reading Japan, 1974, “Order Prescribing the Items of the Test Relating to the New Chemical Substances, Order of the Prime Minister, the Minister of Health and Welfare and the Minister of International Trade and Industry, July 13, 1974,” applied to the Law Concerning the Examination and Regulation of Manufacture, etc. o f Chemical Substances, 1973. Howard, P. H., Saxena, J., Sikka, H., “Determining the Fate of Chemicals,” Enciron. Sci. Technol., 12, 398-407
(1978). Kimerle, R. A,, Gledhill, W. E., Levinskas. G . J., “Environmental Safety Assessment of New Materials,” in Estimating Hazard
of Chetnical Substances t o Aquatic Life,
ASTM, STP 657, 1978, pp 132-146. Sanders, W. M., 111, “‘Exposure Assessment, A Key Issue in Aquatic Toxicology,” in Aquatic Toxicology, ASTM, STP 667, 1979, pp 271-283. Duthie, J . R., “The Importance of Sequential Assessment in ‘Test Programs for Estimating Hazard to Aquatic Life,” in Aquatic Toxicology and Hazard Eualuation, ASTM, STP 634, 1977, pp 17-35. Stern, A . M., Walker, C. R., “Hazard
Assessment of Toxic Substances, Environmental Fate Testin,g and Ecological Effects Testing,” in Estimating Hazard of Chemical Substances to Aquatic Life, ASTM, S T P 657, 1978, pp 8 I - 13 I ,
r
Water Quality Area Training Center Proposal Solicitation The Columbus Laboratories of Battelle Memorial Institute, in cooperation with US. EPA’s National Training and Operational Technology Center, is soliciting proposals for the establishment and operation of a pilot Area Training Center (ATC). The Center, scheduled to be in operation by January 1980, will offer professional educational and training programs in water quality to personnel in federal, state, and local water pollution control agencies, and other private sector groups in EPA Federal Standard Regions I and II (Connecticut, Maine, Massachusetts, New Hampshire, Rhode Island, Vermont, New Jersey, and New York). Eligible to respond to this proposal solicitation are twoand four-year colleges and universities located within those Federal Standard Regions. Proposals will be due by November 15, 1979. The ATC is to serve as a functional model. Its performance will be evaluated, and the results used to recommend a national ATC implementation procedure. Battelle has been designated by EPA as the administrative grantee and evaluator, and is responsible for preparing the request for proposal (RFP), reviewing proposals, selecting an educational institution and helping in the implementation of the pilot ATC. The educational institution selected will be subcontractor to Battelle. For further information, contact the project director:
Lawrence G. Welling
Battelle Memorial Institute 505 King - Avenue, Columbus, Ohio 43201 6 14-424-7 172
f
D~~SSELDORF WEST OERMAIY FEBRUARY 11-15,lS80
THE WORLD’S LARQEST
ENVIRONMENTAL TECHNOLOGY TRADE FAIR & CONGRESS Judith M. Hushon ( t o p ) is a group leader in the Eticironmental Division at the M I T R E Corp. S h e has serced as project Ircirler f o r a number of projects investigtititig health and encironmental effects of to.yic sub.c.tance.r. Robert J. Clerman (1.) is an encironmental .rj’stems scientist at the M I T R E Corp. He has been incolced i n the reciew and derrloptwnt of’testing systems f o r chemical hti-urd assessment .
Burkhard 0. Wagner ( r . ) recently joined the OECD as principal administrator in t h e Chemical Dicision of the Environment Directorate. Preciously, he serced with the F‘cderal Encironmental Agency of the Federal Republic of Germany, and participated i n discussions on the principles of’tiered testing. Coordinated by LRE
ENVITEC 80 will be the most comprehensive exhibition on environmental technology ever presented Every modern nation in the world will participate bringing to one location the latest developments solutions and operating technology for today s industrial and community needs covering
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Volume 13, Number 10, October 1979
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