Pesticide Assessment Guidelines, Subdivision F
        Revised Policy for Acute Toxicity Testing
        (U.S.)  Environmental  Protection Agency,  Washington,  DC
        22  Sep  88

       •nd Subtitle
       ;ticide Assessment Guidelines,  Subdivision  F, Revised
      licy for Acute -Toxicity Testing
  7. AutlwxU)

  9. Performlnf Orfanlratloo Name and Address
    OPP/HED (H7509C)
    401 M St., S.W.
    WASHINGTON, D.C.   20460
  12.' Sponsoring Organization "*me and Address

    Same  as  #9

  IS. Supplementary Notes

    For more information, refer to Subdivision F:   Series 81-1, 81-2  and 81-3
    Addendum 6 PB89-124077
                (. Report Dale

                (. Performing OrcanUetlon Rept. No.
                10. Prolect/Task/Work Unit No.
                11. Contract(C) or Cr«nl(G) No.
                13. Type of Report & Period Covered
 16. Abstract (Limit: ?00 words)
    In response to the need  to improve  scientific and regulatory decisions and to
    provide additional guidance for evaluating the acute toxicity of chemical  exposures
    under  the Federal Insecticide, Fungicide and  Rodenticide Act and the Toxic Substances
    £t>ntrol Act.  This action builds upon a previous revision of the acute toxicity
     esting strategy tc reduce the use  of experimental animals whii'e providing adequate
      formation about chemical safety.

    The Environmental Protection Agency is disseminating this notice to industry,
    governmental bodies., scientific societies, animal welfare groups and interested
    parties to apprise them  of our new position.   The Agency's acute toxicity
    testing guidelines are being revised to reflect the positions articulated in this
 17. Document Analysis a. Descriptors
    b. Id«ntifi«rs/Op*n-Cnd«d
   c. COSAtl ricld/G'Oun

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                                                        orrici e*
                                                 ritTICIDU AND TOXIC iU««TA~CI
      22  1988
 Re:  Revised  Policy  for Acute  Toxicity Testing

     Appended  is  a  revised  policy  for evaluating  the  acute
 toxicity of  chemical  exposures under the  Federal  Insecticide.
 Fungicide and Rodeziticide Act and  the Toxic  Substances  Control
 Act.  This action builds upon a previous  revision of  the  acute
 tox?city testing  strategy to  reduce the use  of  experimental
 animals while providing adequate  information about chemical

     The Environmental Protection  Agency  is  disseminating this
 notice to industry, governmental bodies,  scientific societies,
 animal welfare groups and interested parties to apprise them of
 our new position.   The Agency's acute toxicity  testing  guidelines
 are being revised to  reflect  the positions, articulated  in this
                         Victor J. Aimm
                         Acting i^sistant Administrator
                           for Pesticides
                           and Toxic  Substances

        Alternative Methodology for Acute Toxicity Testing
      The Environmental  Protection Agency announces a revision to
 its  approach to acute toxicity testing in fulfillment of actions
 under the Federal  Insecticide, Fungicide and Rodenticide Act
 (FIFRA)  and the Toxic Substances Control Act (TSCA).  This
 revision reflects  the Agency's concern about animal welfare and
 its  continued efforts to  reduce the impacts on animals of EPA's
 testing  requirements.   While maintaining the tiered approach
 adopted  in 1984, the Agency now recommends  (when appropriate) the
 use  of abbreviated test methods and consideration of using only
 one  sex,  as a means of  reducing the numbers of animals in
 deriving important information on acute toxicity.

      EPA  considers  the  evaluation of toxicity following short-
 term  exposure  to  a  chemical  (i.e., acute toxicity) to be a
 limited but  integral  step  in  the assessment of the toxic
 potential of a chemical substance under the regulatory framework
 of  its pesticide  and  toxic substances programs.  The Agency also
 supports  measures dedicated to reduce the use of animals in
 toxicity  testing  and  conducts research on test methods which can
 lead  to further reduction  or  elimination of animal usage and
 suffering.   Through the careful selection of test methodology and
 maximization of the data obtained from acute studies, EPA strives
 to  achieve a balance  between  the welfare of animals and the need
 to  utilize animals  in evaluating chemical safety.
          The  approach  to  acute toxicity testing previously given
 in  EPA's  Test  Guidelines (U.S. Env. Prot. Agency, 1978; 1979)
 emphasized the  determination  of the median lethal dose  (LD50)
with a 95% confidence interval.  A 1984 update of the guidelines,

published  in  1985  (U.S. Env. Prot. Agency,  1985)  stated  that  the
Agency discouraged the uses of the "classical"  LD50 test
employing  large numbers of animals for determination of  lethality
only.  Instead, the Agency emphasized the use of  a tiered
approach to obtain acute toxicit.' data which reduced the number
of animals used, but maximized the amount of relevant
information that could be obtained from such testing. That
approach included the following:

a.   Using Data From Structurally Related Chemicals,  The  Agency
     encourages the review of existing acute toxicity information
     on chemical substances that are structurally related  to the
     agent under investigation.  Using this approach, one  may be
     able  to  compile enough information from these surrogate
     chemicals to make preliminary safety evaluations that
     reduce the need for further animal testing or which
     indicate the type of testing to be pursued.

b.   "Limit" Test.  When information on structural analogs is
     inadequate, one should consider the "limit"  test.  The
     relative toxicity of a chemical is determined by
     professional judgement; for chemicals judged to be
     relatively non-toxic,  a single group of animals is  given a
     large  dose of the agent.   If no lethality is demonstrated,
     no  further testing for this information IB pursued.

 c.    Multifaceted  Testing.  A three-dose multiple endpoint
      evaluation  may  be  important for those substances judged to
      be  relatively toxic or which demonstrate lethality in the
      limit  test.   Using this procedure, animals are evaluated as
      to  the onset, duration, intensity, and reversibility of
      behavioral  effects, body weight changes and lethality; all
      animals are submitted to gross necropsy.  Histopatholcay and
      certain follow-up studies may be warranted where t^re are
      gross  indications of target organ toxicity.

      EPA has reevaluated its data needs on acute toxicity and
 continues to espouse the tiered approach that was developed in
 the  1984 update.   Thus, the first consideration for a chemical
 for  which there  is no acute toxicity data, should be a review of
 structurally related compounds, followed by the limit test when
 appropriate.   In those cases where testing beyond the limit test
 is indicated,  consideration should be given to well-designed
 abbreviated test schemes which employ minimal numbers of animals,
 «s discussed below.  In most cases, it is expected that these
 tests can be structured to give enough information on acute
 toxicity to obviate the need for further acute  studies  (e.g.,  the
 three-dose  multi-faceted testing approach).  We continue to
 stress the  need for collecting information on behavioral
 effects, gross pathology and lethality  (as developed in "c"

     While more complete animal testing may be necessary in some
cases  (based on scientific evidence from the abbreviated test,
e.g., delayed toxicity, unusual central nervous system effects,
irreversible effects),  the Agency generally supports limiting
such tests to those using the lowest feasible number of animals.
     Several abbreviated methods to investigate acute toxicity
have been developed over the years.  Some of them have rather
extensive data bases and have been validated against more
traditional test methods which estimate median lethal dose.
Their merit lies in the fact that they allow for the evaluation
of the full spectrum of acute responses; numerical calculations
can be made; and fewer animals may be employed in the generation
of the information than with most other approaches.  For some
methods, statistical calculations are simple or are aided by


      EPA has investigated four methodologies that might be used.

 These include (1)  the approximate  lethal dose method1 of

 Deichmann and Le Blanc (1943);  (2) the moving averages method2 of

 Thompson (1947); (3)  the  up-and-down method3 of Dixon and Mood

 (1948)  and Dixon (1965);  and  (4) the cumulant method of Reed-

 Muench4 (1938) .

      The methods vary as  to the  assumptions that are made, the

 number of groups of animals and  number of animals per group.

 Toxicologists Should be familiar with these differences before

 employing a given method.  For  instance, the up-and-down method

 is especially difficult to apply when chemicals induce delayed

 toxic effects.   Therefore,  other methodologies may be more

 appropriate.   When an alternative  method for acute toxicity

 testing is selected,  a rationale for such a selection should

 accompany the submission.  The Agency solicits discussions with

 data  generators  on still  other methods that may be employed.
      1The  approximate  lethal  dose  method  was  further  refined by
 Deichmann  and Mergard  (1948) ;  these  authors performed eighty-
 seven  determinations  (calculated by  the methods of  Behrens
 (1929)  and Bliss  (1938)).   The approximate lethal dose method was
 also used  by  Kennedy et  al.  (1986).

     2The  moving  averages  method was refined  by Weil  (1952, 1983)
 and Gad and Weil  (1982), and  was used by  Smythe and Carpenter
 (1944,  1948)  and  Smythe  et al.  (1949,  1951, 1954, 1962).
          up-and-down method was  recently  used and  refined  by
Bruce  (1985,  1987)  (calculated by the method  of Bliss  (1938)).
The up-and-down method was  also used by  Brownlee et al.  (1953),
Oixon and Hassey  (1957), Klassen  ana Plaa  (1967) and Hsi  (1969).

     4The Department of Defense has had  considerable experience
using the Reed-Muench method with a large  number of chemicals  (F.
Vocci, personal communication); it has also been used  by  Lorenz
and Bogel (1973), Bhan (1974), Aubert and  Amdral  (1979),  and
Thakur and Fezio  (1981).

      The Agency  emphasizes  that parallel assays on ir.ale ar.J
 female  animals to  determine an approximate estimate of acute
 toxicity need not  be  routinely determined, since male and female
 animals of  the same strain  generally show only slight and
 insignificant differences in susceptibility to toxic agents.
 However,  for some  chemicals, one sex may be somewhat more
 sensitive than-*.he other  (Muller and Kley (1982); Schutz and
 Fuchs  (1982);  (Bruce  (1985)).  Cassarett and Doull (1980)
 indicate that the  class of  compound is important in specific sex
 differences.  De Pass et al. (1984) shoved that for 91 chemicals
 tested  for  oral  toxicity in rats,  females were slightly more
 sensitive than males  (p<.001).  Muller and Kley (1982) performed
 152 parallel studies on male and female animals for which 129
 showed  no significant differences.  However, when statistically
 significant differences were observed  (23 compounds), 17 were
more toxic  to females.  Therefore, consideration should be given
to limiting studies to the  more sensitive sex.  Previous history
on the  class of chemical being evaluated would be helpful in
making  this determination.   For confirmation, a few animals of
the other sex should also be tested.

      In summary,  EPA has  modified  its approach to acute toxicity
 testing,  recognizing that appropriate information for safety
 evaluation can be developed  using  fewer animals than had been
 recommended in the past.   We strongly urge industry to use these
 abbreviated test  methodologies,  whenever appropriate, as
 replacements for  the three-dose  multifaceted method EPA
 previously had recommended.   Four  such methodologies which might
 be  used have been identified;  other methods may also be employed,
 if  adequate rationale can be provided.  It IB expected that
 studies will still include behavioral observations, gross
 necropsy  and ancillary observations, as before.

      EPA  urges industry to begin submitting data obtained with
 alternate methods which use  fewer  animals on a routine basis;
 the Agency is planning to revise its testing guidelines to
 incorporate the above guidance.  We plan to accept only newly
 generated industry data that conforms with our revised guidance
 unless  an adequate rationale (e.g., data generated in accordance
 with  regulatory requirements other than those of EPA) accompanies
 the submission; data without a rationale may be returned to  the

      The  Agency encourages the public to comment on  this
position  and  provide information on still other alternate
methodologies  which  have  progressed to a stage of validation
which would be acceptable to the scientific community.


 Aubert  M.F.A.  and  Ajndral  L.  (1979).   Potency Testing of
 Veterinary Vaccines  Containing Inactivated Virus.  Coir.p. Inununol
 Microbiol.  Infect. Dis.,  1,  341-349.

 Behrens B.  (1929).   The Assay of Digitalis on Frogs.  Arch.
 Exptl.  Path.  Pharmakol.,  140. 237-256.

 Bhan  A.K.  (1974).  Computing LD50 More Efficiently and Accurately
 - A Modification of  Reed-Muench Method.  Indian Soc. Nucl.
 Agricul.  Biol., l, 5-6.

 Bliss C.I.  (1938).   Determination of  the Small Dosage Mortality
 Curve from SmaM Numbers.  Quart. J.  Year Book Pharro., 11. 192-

 Brownlee  K.A., Hodges J.L. and Rosenblatt M.  (1953).  The Up-and-
 Down  Method With Small Samples.  Amer. Statist. Assoc. J., 48.

 Bruce R.D.  (1985).   An Up-and-Dovn Procedure  for Acute Toxicity
 Testing.   Fundam.  Appl. Toxicol., 5_,  151-157.

 Bruce R.D.  (1987).   A Confirmatory Study of the Up-and-Dovn
 Method  for Acute Oral Toxicity Testing.  Fundam. Appl. Toxicol.,
 8, 97-100.

 Casarett  L.J.  and  Doull J.  (1980).  Factors Influencing
 Toxicology.   In: Toxicology. The Basic Science of Poisops. J.
 Doull,  C.D. Klaasen  and M.O. Amdur, eds., 77-88.  New York:
 Kacmillan Publishing Co.,  Inc.

 Deichmann W.B. and LeBlanc T.L.  (1943).  Determination  of  the
 Approximate Lethal Dose With About Six Animals.  J.  Indust.  Kyg.
 Toxicol.,  21,  415-417.

 Deichmann W.B. and Mergard E.G.  (1948).  Comparative Evaluation
 of Methods  Employed  to Express the Degree of  Toxicity of  a
 Compound.   J.  Indust. Hyg. Toxicol.,  30. 373-378.

 De Pass L.R.,  Myers  R.C.,  Weaver E.V. and Weil C.S.  (1984).  An
Assessment  of  the  Importance of Number of Dosage Levels,  Number
of Animals  per Dosage Level, Sex and  Method of LD50  and  Slope
Calculation in Acute Toxicity Studies.   In: Acute Toxicity
Testing;  Alternative Approaches. A.M. Goldberg,  «d.  (Alternative
Methods in  Toxicology, Vol.  2), 142-153.  New York:  Mary  A.
Liebert,  Inc., Publisher.


                        References  (contd,)

 Dixon W.J. (1965).   The Up-and-Dovn  Method  for Small Samples.
 Arner. Statist. Assoc.  J.,  60,  967-978.

 Dixon W.J. and Massey  F.J.  Jr.  (1957).   Sensitivity Experiments,
 In:  Introduction to Statistical  Analysis. 318-327.  New York:

 Dixon W.J. and Mood A.M.  (1948).   A  Method  for Obtaining and
 Analyzing Sensitivity  Data.   Ainer. Statist. Assoc. J., 4 3. 109-

 Gad  S.C.  and  Weil C.S.  (1982).   Statistics  for Toxicologists.
 In:  Methods in Toxicology.  A.W.  Hays,  ed.,  273-309.  New York:
 Raven Press.   ^

 Hsi,  B.P. (1969).  The  Multiple  Sample Up-and-Down Method  in
 Bioassay.  Amer.  Statist.  Assoc. J.,  64.  147-162.

 Kennedy,  G.L.  Jr.,  Ferenz,  R.L.  and  Burgess,  B.A.  (1986).
 Estimation of Acute Oral  Toxicity  in Rats by  Determination of  the
 Approximate Lethal  Dose Rather Than  the LD50.  J. Appl. Toxicol.
 6,  145-148.

 Klassen,  C.D.  and Plaa, G.L.  (1967).   Relative Effects of  Various
 Chlorinated Hydrocarbons  on Liver  and Kidney  Function  in Dogs.
 Toxicol.  Appl. Pharmacol.,  10.  119-131.

 Lorenz R.J. and Bogel  K.  (1973).   Methods of  Calculation.  Method
 of Reed and Muench. Laboratory  Techniques  in Rabies.  WHO
 Monograph Series  21, 3rd  Ed.,  329-335.

 Muller H. and  Kley  H.P. (1982).  Retrospective Study on the
 Reliability of an "Approximate LD50"5 Determined  with  a Small
 Number of Animals.   Arch.  Toxicol.,  51, 189-196.

 Reed  L.J. and  Muench H. (1938).  A Simple Method  for Estimating
 Fifty Percent  Endpoints.   Amer.  J. Indust.  Hyg. Toxicol.,  30,

 Schutz  E.  and  Fuchs H.  (1?B2).   A  New Approach to Minimizing the
 Number  of Animals Used  in  Acute  Toxicity Testing  and Optimizing
 the Information of  Test Results.   Arch. Toxicol., 51.  197-220.

 Smythe  H.F. Jr. and Carpenter  C.P.  (1944).  Tlace of the Range-
 Finding Test in the Industrial Toxicology Laboratory.  J.  Indust.
Hyg.   Toxicol., ££,  269.
     5 "Approximate LD50"  in the  title  of  this  paper is  placed ir,
quotation marks eo as not  to confuse  it with  the  method  of
Deichman and Le Blanc (1943).


                        References  (contd.)

 Snythe H.F.  Jr.  and Carpenter  C.P.  (1948).  Further Experience
 with the Range-Finding  Test  in the  Industrial Toxicology
 Laboratory.   J.  Indust.  Hyg. Toxicol.,  30. 63.

 Smythe H.F.  Jr.,  Carpenter C.P. and Weil  C.S. (1949).  Range-
 Finding Toxicity Data:  List  III.  J.  Indust. Hyg. Toxicol., 3_J.,

 Sirythe H.F.  Jr.,  Carpenter C.P. and Weil  C.S. (1951).  Range-
 Finding Toxicity Data:  List  IV.  AKA Arch. Indust. Hyg. Occ.
 Med.,  4;  119.

 Smythe H.F.  Jr.,  Carpenter C.P., Weil C.S. and Pozzani U.C.
 (1954).   RangeeFinding  Toxicity Data:  List V.  AMA Arch. Indust.
 Hyg.  Occ.  Med. 10,  61.

 Smythe H.F.  Jr.,  Carpenter C.P., Weil C.S., Pozzani U.C.,  and
 Striegel  J.A.  (1962).   Range-Finding Toxicity Data: List VI.
 Amer.  Indust.  Hyg.  Assoc. J.,  23.  95.

 Thakur A.K.  and  Fezio W.L.  (1981).   A Computer Program for
 Estimating LD50  and It's Confidence Limits Using Modified
 Behrens-Reed-Kuench Cumulant Method.   Drug Chem. Toxicol.,  4.,

 Thompson,  W.R.  (1947).   Use  of Moving Averages and Interpolation
 to Estimate  Median  Effective Dose.   Bacteriol. Rev.  11.  115-145.

 U.S.  Environmental  Protection  Agency.  (1978).  Proposed
 Guidelines for Registering Pesticides in  the U.S.  Hazard
 Evaluation:  Humane  and  Domestic Animals.  Fed. Reg.  43,  37336-
 37345;  37351-37356.   (August 22).

 U.S.  Environmental  Protection  Agency.  (1979). Proposed Health
 Effects Test Standards  for Toxic Substances Control  Act  Test
 Rules  and  Proposed  Good  Laboratory  Practice Standards  for  Health
 Effects.   Fed. Reg.  44.  44054-44059;  44066-44067.   (July  26).

 U.S. Environmental  Protection  Agency.  (1985).  Health  Effects
 Testing Guidelines.  Fed. »eg.  5_0,  39397-39398.   (September 27)

 Vocci. F.  (1988).   Personal  Communication (Unpublished Data).

 Weil C.S.  (1952).   Tables for  Convenient  Calculation of  Median
 Effective  Dose (LD50 or  ED50)  *nd  Instructions on Their  Use.
 Biometrics 8, 249-263.

Weil C.S.  (1983).   Economical  LD50  and Slope  Determinations.
 Drug Chem. Toxicol. £,  595-603.