DRAFT I.R.LG. GUIDELINES
FOR
SELECTED ACUTE TOXICITY TESTS
Testing Standards ft Guidelines Work Group
INTERAGEIMCY REGULATORY LIAISON GROUP
      1979

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FOR FURTHER INFORMATION CONTACT:




INDUSTRY ASSISTANCE OFFICE  (TS-799)




U. S. ENVIRONMENTAL PROTECTION AGENCY




401 M Street, S.W.




Washington, B.C.  20460




TELEPHONE TOLL-FREE 800-424-9065




OR IN WASHINGTON 554-1404

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                PREFACE

        This package of "Draft I.R.L.G. Guidelines for Selected
Acute Toxicity Tests" represents the work of the members of the
Testing Standards and Guidelines Work Group of the Interagency
Regulatory Liaison Group  (I.R.L.G.).  It reflects the views of
staff members of the I.R.L.G. agencies who reviewed earlier drafts
and is being released to obtain public comment before final drafts
are prepared for submission to the I.R.L.G. agencies.  The tests in
this package are the first in a series on toxicity testing which
will include other acute as well as chronic effects tests.

        On August 21, 1979, the I.R.L.G. published a Federal Re-
gister notice announcing the public availability of these five
draft guidelines and a meeting for public participation in dis-
cussing them.  That meeting will be held on October 30 at 9:00 A.M.
in the Auditorium, Main Floor, of the Hubert H. Humphrey Building,
200 Independence Avenue, S.W., Washington, D.C., 20201.  Work
Group members will discuss their philosophy, comparisons of these
with other similar guidelines, the relationship of I.R.L.G. guide-
lines to other testing requirements, and future activities of the
Work Group.  The public will be invited to participate in this
discussion.  In addition, comment by attendees will "be requested on
the scientific issues raised at the beginning of each guideline.

        The Work Group has asked that written comments be submitted
by October 19, 1979, to allow some discussion of the'm at the public
meeting.  However, the public is encouraged to submit comments after
this suggested deadline as input to the Group's on-geing work.  They
should be addressed to Dr. Victor Morgen Roth III, HFF-185, Food
and Drug Administration, Bureau of Foods, Division of Toxicology,
200 "C" Street, S.W., Washington, D.C., 20204.  Comments may be
examined in the I.R.L.G. office, Room 509, 1111 18th Street, N.W.,
Washington, D.C., 20207, 9:00 A.M. to 4:00 P.M., Monday through
Friday.

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             Testing Standards and Guidelines Work Group
                Interagency Regulatory Liaison Group
 Chairman:   Dr.  Victor Morgenroth,  III,  Food and Drug Administration
 Surrogate Liaison:   Dr.  Allen H.  Heim,  Food and Drug Administration
Members:   Health Effects  Group
Dr. James R.  Beall



Dr. William D'Aguanno



Dr. Robert  Hehir



Dr. Zdenka  Horokova



Dr. Patricia  Marlow



Dr. Joseph  McLaughlin



Dr. Robert  E. Osterberg



Dr. Norbert Page



Dr. Orville E. Paynter



Ms. Peggy Perry



Dr. John A. Quest



Mr. Van Seabaugh



Dr. Frode Ulvedal
Occupational Safety  Health Admin.



Food and Drug Administration



Consumer Product Safety Commission



Food Safety  Quality Service



Occupational Safety  Health Admin.



Consumer Product Safety Commission



Food and Drug Administration



Environmental Protection Agency



Department of Commerce



Food and Drug Administration



Food and Drug Administration



Consumer Product Safety Commission



Environmental Protection Agency
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                 TABLE OF CONTENTS
Preamble	    1

Draft I.R.L.G. Guideline for
Acuate Eye Irritation Tests   	    9

Draft I.R.L.G. Guideline for
Acuate Oral Toxicity Studies  in Rodents   ....  39

Draft I.R.L.G. Guideline for
Acute Dermal Toxicity Tests	61

Draft I.R.L.G. Guideline for
Acute Inhalation Tests in Rats	87

Draft I.R.L.G. Guideline for
Teratogenicity Testing in Rat, Mouse
and Rabbit	113
                     ill

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IV

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                             PREAMBLE







I.  Background







    Four regulatory agencies, the Consumer Product Safety



Conmission  (CPSC), the Environmental Protection Agency  (EPA), the



Food and Drug Administration (FDA), and the Occupational Safety



and Health Administration (QSHA), agreed to work together to re-



form the regulatory process and to improve protection of workers,



public health, and the environment (42 FR 54856, 11 October 1977).



They formed the Interagency Regulatory Liaison Group (IRLG) to



implement their agreement. In January 1979, the Food Safety and



Quality Service (FSQS), Department of Agriculture, joined the



IRLG.







    These agencies recognized that although they often  regulate



the same chemicals, toxicity testing guidelines used by each



agency are not always uniform.  Among currently required tests,



differences exist primarily in details of methodology and not in



fundamental toxicological principles.  The Testing Standards and



Guidelines Work Group was established for the purpose of develop-



ing guidelines which would resolve existing differences and be



used by all of the IRLG agencies for testing chemicals  for health

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or environmental effects or both. The plan ,was to review current



tests and procedures in use or under development and prepare a



single set of toxicity guidelines that could serve the IRLG



agencies. This effort would be coordinated with the development of



other guidelines.  On December 17, 1977, the Work Group held a



public neeting to explain  its purpose and goals, and to answer



questions about  its activities  (42 FR 59106, 15 November 1977).







II.  Philosophy







     The Work Group agreed upon the  following  tenets:








      A.  Guidelines must  be sufficiently  comprehensive  to provide



a sound, scientific method for  gathering data  necessary  for char-



acterization of  the test substance.







      B.  Requirements of  the guidelines must  be  feasible.







      C.  Guidelines must  provide adequate guidance to investiga-




tors.







      D.  Guidelines must  be flexible, allowing the investigator



latitude for scientific judgment.

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      E.  Each guideline  should  be  complete in itself and be able



to stand alone.







      F.  Guidelines  should  avoid irrelevant or marginally useful



procedures, while retaining  the  value  of  the test.   Each recom-



mended procedure should result  in data essential for characteriza-



tion of the test substance and useful  for regulatory decisions.







      G.  Costs of conducting the tests must be considered and



kept to a minimum without jeopardizing the validity of the test.







      H.  Guidelines  should  be reviewed annually, and opportunity



must be provided for  modifications  which  improve the test and



incorporate advances  in the  state of the  art.







      I.  Guidelines  should  be constructed so that  they can be



harmonized with those under  development nationally  and



internationally.







      J.  Welfare of  the  test anijnals  must be considered.







      K.  Decisions of the Work  Group  are made by consensus.



Guidelines recommended by the work  group  are acceptable to every



member.

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III.  Development







      IRIJG guidelines  are  based on  those  currently used or  under



development  in the agencies,  in international  organizations,  and



in  industry.  Early drafts of the IRD3 guidelines were circulated



through each agency for  review, and comments from that review are



included  in  this proposed  guideline.   In  addition, drafts of  the



EPA Office of Pesticides Programs (OPP) proposed guidelines (43 FR



37336, 22 August 1978) under  the  Federal  Insecticide,  Fungicide



and Rodenticide Act  (FIFRA),  and  public comments to those guide-



lines were used extensively in an effort  to assure compatibility



and to benefit from the  appreciable ,time  and effort the OPP staff



put in to their development.   A second major source of information



was Principles and Procedures for Evaluating the Tbxicity of



Household Substances  (NAS  Pub. No.  1138), prepared for CPSC by the



National  Academy of Sciences. Still others heavily relied  on were



the Pharmaceutical Manufacturers' Association  Guidelines  for  the



Assessment of Drug and Medical Device Safety in Animals  (February



1977)the  FDA, Bureau of  Foods Direct Additive  Cyclic Review Draft



Guidelines; the Appraisal  of  the  Safety of Chemicals in Foods,



Drugs and Cosmetics, Association  of Food  and Drug  Officials of  the



U. S; and protocols submitted by  several  industrial  organizations

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in response to 43 FR 1987, 13 January 78.  To the extent possible,



IRLG guidelines are being coordinated with development of guide-



lines by the Organization for Economic Cooperation and Development



(OECD).  The Work Group acknowledges the contributions from each



of these information sources and takes this opportunity to express



its appreciation to the scientists who developed them and made



then available.







IV.  Relationship to Other Guidelines







     A.  Previously Issued Guidelines and Tests In Progress







         To assure that issuance of the IRD3 guidelines will not



cause confusion because other guidelines are being developed to



meet specific agency needs and will not negate tests already in



progress, the IRLG agencies published the following notice (42 FR



1528, 10 January 1978):  "To the extent permitted by each agency's



legislation, parts of published standards, regulation, and/or



guidelines may be amended to agree with uniform testing standards

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and guidelines developed by  the  IRLG.   In  that event, data result-
ing from use of pre-existing guidelines will  be  accepted by  the
agency requiring them so long as these  data are  generated from
studies begun before the IRLG guidelines are  promulgated  and the
data are valid and scientifically sound."

     B.  Use and Modification of IRLG Guidelines

         Use of this guideline will provide  test methods  for
acquisition of data acceptable  to all of  the IRLG agencies.   Under
most circumstances, the data obtained fron this  test should  be
sufficient to meet the requirements for a  specific aspect of the
toxicological characterization of the test substance.   Under cer-
tain circumstances, however, the data nay  show a need for further
study; or it may be recognized at the outset  this guideline  may
have to be modified by an  agency.  If the  modification requires
information in addition to that  required by  this guideline,  the
data will be acceptable to all  the IRLG agencies.  If the modifi-
cation requires less than  this guideline,  the data from the  test
may not be acceptable to all IRLG agencies.   It  is important that
modifications be agreed upon between the investigator and the
agency requiring than.

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         Each  agency will decide how  it will use  IRLG  guidelines.

and has made a commitment to adopt  them to  the  fullest  extent

possible consistent with its regulatory responsibilities.   For

example, in its proposed guidelines (43 FR  37337,  22 August 1978),

the EPA Office of Pesticide Programs stated that,  "At such  time  as

these conmittees complete their work,  EPA will  review the IRLG
                                    /
documents and revise its PIFRA guidelines if appropriate."




V.  Agency Responsibilities




    Each agency has the responsibility to decide:




      A.  what substance, and what  form of  the  substance, it

requires to be tested;




      B.  which tests  it will require;




      C.  how it will  use the data  derived  from the tests;  and




      D.  how it will  ijnplement use of IRLG guidelines.

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DRAFT I.R.L.G. GUIDELINE FOR




ACUTE EYE IRRITATION TESTS
Testing Standards  Guidelines Work Group



INTERAGENCY REGULATORY LIAISON GROUP








May 30, 1979

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                             PREFACE








     This guideline delineates test procedures to evaluate the



toxicity of liquids, solids, aerosols, and liquids propelled under



pressure, to ocular tissues of laboratory animals.  The test



should demonstrate the potential of a substance to produce injury



to the human eye.  Evaluation of gases for eye irritation requires



special techniques which are not specified in this guideline.
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      {Scientific Issues for Public Ctanment







     During the development of  this  proposed guideline,  many scien-



tific issues were discussed by  the Work Group.   These issues were



raised by members of the Work Group, by public  cornnents  to the EPA



proposed pesticide guidelines,  and by  the conments  of interagency



reviewers.  Consideration of these comments,  discussed below,  is



reflected in this proposed guideline;  and the public  is  invited to



conment further on these issues or any other aspect of this proposed



guideline.







     A.  Either males or females may be used for this test.   Although



a review of available data indicates that eye irritation is not a sex



dependent response, sane carmentors  have suggested  that  equal  numbers



of both sexes should be used.   Information about which sex,  if



either, is more appropriate for eye  irritation  studies and data show-



ing sex differences, or lack of differences,  in response to eye irri-



tants would be helpful.







     B.  The Work Group chose the albino rabbit as  the preferred



animal for this test, although  the rabbit's  lesser  predictive
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ability for human ocular  irritancy with  respect to other  species,



such as monkeys  is recognized.  Hie Work Group solicits comments



regarding the  use of ocular  irritancy data obtained from  other



species and whether they  should take  precedence over rabbit data.



Discussion of  the use  of  other animals  should include difficulty of



obtaining them,  cost,  ease of handling,  and structural differences of



ocular tissues.







     C.   The  Work Group  realizes that  the classical method of



instillation of  the test  substance  in eye  irritation studies is into



the cul-de-sac.  Sane  canmentors  raised the issue of approximating



human responses  and decreasing exaggerated rabbit responses by admin-



istration of the test  substance directly onto the cornea, rather than



into the conjunctival  sac.   The Work  Group would like information



concerning the predictive nature  of each procedure in approximating



the human response and which procedure  produces responses that take



into account the more  susceptible members  of the population in terms



of potential irritancy.







     D.   The  Work Group  reviewed comments on the OPP guidelines  re-



garding the amount of  test substance  that  would be expected to con-



tact human eyes  in accidental situations and its relevance  to the



volume applied to rabbit  eyes in  the  eye irritation test.   The Work



Group realizes that the use  of several volumes of test material (0.01



ml to 0.1 ml)  applied  to  rabits better delineates the dose-response



characteristics  of an  irritant. The purpose of this guideline,  how-



ever, is to detect irritance; and therefore the use of a  single,



large volume
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has been recommended.  The 0.1 ml volume closely approximates  2



drops, a realistic volume of exposure  that  a human  eye might



receive.







     E.   "Hie Work Group recognizes  the possibility of a traumatic



response from instillation of a  test substance  into the  eyes from an



aerosol spray. Coranents are solicited  on use of an  alternate method



whereby the aerosolized material is  collected  in a  chilled  container



and tested identically as with the other liquids not propelled under



pressure.







     F.   Several comments to the OPP  proposed  guidelines were



received concerning the examination  of eyes 24  hours prior  to



instillation of the test material.   Since eye damage could  occur



within the 24 hour period making the animal unacceptable for testing,



a shorter time interval, such as 1 or  4 hours,  was  suggested in  order



to minimize this potential.  The Work  Group decided that the phrase



"within 24 hours" would accomodate this concern, while allowing  the



investigators to use  their own judgement.







     G.   Another topic at issue is  the requirement to hold rabbits



beyond 72 hours even  though no responses have occurred or those  that



have, have disappeared.  The Work Group is  unaware  of chemicals  that
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that produce delayed type of ocular irritation  following  one  expos-



ure.  Comments on the need for observations beyond  72  hours are



solicited.







     G.   Properly used, fluorescein or other stains highlight



changes in tissue in eye irritation studies.  Improperly  admin-



istered, such stains can contaminate the eyes with  bacteria or irri-



tating materials.  In this guideline, the use of such  stains  is



proposed to be optional in the examination of the eye.







     H.   Anesthetics may obscure a pain response to the  irritant,



but rarely interfere with the response of an eye to an irritant;



therefore, the Work Group proposes that for humane  reasons  anes-



thetics should be used when the substance being tested is likely  to



cause extreme pain.  Information about the effects, including  effects



on healing time, of one instillation of a local anesthetic  in



altering responses of the tissues to eye irritants  is  solicited.







     I.  Terata have been induced in offspring  following  the  instil-



lation of glucocorticoids to the eyes of pregnant rabbits.  This and



other evidence indicate that substances can be absorbed following



ocular instillation; however, systemic toxicity is  usually  not



evaluated in acute eye irritation tests.  The Work  Group  invites



views on whether to require the evaluation of systemic effects in the



acute eye irritation test.
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evidence indicate that substances can be absorbed  following  ocular



instillation; however, systemic  toxicity is  usualy not evaluated in a



cute eye irritation tests.







     J.  The issue of the most appropriate scoring system was



discussed.  The Work Group  felt  that the widely  used  scoring system



utilized in this guideline  permits  a relatively  realistic classifica-



tion of degrees of hazard,  is less  subject to  the  distortion that



occurs using a weighted  system,  and is more  sensitive to subtle



ocular effects.







     K.  It was suggested that substances be tested in diluted  form



and also that substances be washed  from the  eyes soon after  admin-



istration in order to evaluate the  potential of  a  substance  to  cause



irritation under conditions of normal use.   Data obtained from  eyes



washed following instillation of the substance were considered  to be



indicative more of the value of  possible first aid treatment than of



the potential of the substance to cause eye  irritation.   The Work



Group also suggests that additional studies  may  be appropriate  for



shampoos or other substances which, in normal  use, might enter  the



eye in diluted form or might be  washed out immediately.   The Work



Group thinks that such studies could be useful,  but should be done in



addition to an initial eye  irritation test of  the  neat substance.
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                      TABLE OF CONTENTS







  I.   General Considerations







      A.   Good Laboratory Practices



      B.   Personnel



      C.   Test Substance



      D.   Animals



      E.   Dead Animals/ Necropsy, and Histopathology



      F.   Equipment



      G.   Documentation







 II.   Specific Considerations







      A.   Test Preparation



      B.   Test Procedure







III.   Data Reporting







      A.   Identification



      B.   Body of Report







          1.  Summary and Conclusions



          2.  Materials



          3.  Methods



          4.  Results



          5.  References






 IV.   Suggested Reading




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I.   General Considerations







     A.   Good Laboratory Practices







          Basic standards presented here  relating  to good  labora-



tory practices are to serve as general guidance for the conduct of



the study, but are not intended  to be all inclusive.  This guide1-



line does not set forth the managerial aspects of  science  or good



laboratory practices.  Studies should be  conducted according to



"Nonclinical Laboratory Studies, Good Laboratory Practice



Regulations," (43 FR 59986, 22 December 1978).







     B.   Personnel







          All testing and evaluation must be done  under the direc-



tion of personnel who have the education,  training, and experience



to perform the testing and evaluation in  accordance with sound



scientific experimental procedures.  The  agency, commission, or



department may require resumes of personnel who have performed,



supervised, reviewed, or evaluated the testing.  To the extent



possible, the same person or persons should perform all observa-



tions and necropsies in a single test in  order to  insure consis-



tency of evaluation.  When a histopathological examination is
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done, similar considerations should apply.







     C.   Test Substance  (materials or mixtures of substances or



materials)







          1.   As far as  is practical, composition of the test



substance must be known,  including the name  and quantities of



known contaminants and  impurities.  Unknown  materials,  if any,



must be quantified to account  for 100% of the  test sample.  The



specific substance to be  tested will  be determined in consultation



with each agency.







          2.   The lot  of the  substance tested should be the same



throughout  the study.   The test sample should  be stored under



conditions  that maintain  its stability, strength,  quality, and



purity from the date of its production until the tests  are



complete.







          3.   Safe handling and disposition of the test substance



is essential.







     D.   Animals
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          1.   Animals used for testing should not have been



subjected to any previous experimental procedures.







          2.   The test animal shall be characterized as to



species, strain, sex, weight and/or age.  Each animal must be



assigned an appropriate identification nunber.







          3.   Recommendations contained  in DHEW pub. no.  (NIH)



74-23, entitled "Guide for the Care and Use of Laboratory



Animals," should be followed for  the care, maintenance, and



housing of animals.







          4.   Animals may not be group-caged for this test.







          5.   Healthy animals must be used. Animals must be



assigned to groups in such a manner as to minimize bias and assure



comparability of pertinent variables.
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          6.   Each animal must be observed as necessary  to  insure



that animals are not lost due to autolysis of tissues, misplace-



ment, or similar management problems.







     E.   Dead Animals, Necropsy, and Histopathology







          When an animal is discovered dead, it must be refriger-



ated at temperatures low enough to minimize autolysis if  necropsy



cannot be performed immediately.  Necropsy must be performed with-



in 16 hours of death.  When animals are killed for examination,



the necropsy should be performed as soon after death as possible.



If histopathological examination is to be conducted, all  tissue



specimens should be placed in appropriate fixative when they are



taken from the animal.







     F.   Equipment







          All equipment used in conducting the test, including
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equipment used to prepare and administer the test substance and



equipment used to maintain environmental conditions, must be of



appropriate design and adequate capacity.  Equipment should be



inspected, cleaned, and maintained regularly.  The equipment must



be properly calibrated at the time of its use.







     G.   Documentation







          Color photograpic documentation to verify gross and



microscopic findings or to clarify conflicting data is a desirable



aspect of ocular toxicity studies.   If photographs are taken, the



equipment and film must be of sufficient quality to permit con-



trolled, close-up color photography  of the eye to yield clear,



sharp-focus images that literally fill the camera field.
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II.  Specific Considerations







     A.   Test Preparation







          1.   Testing  shall be performed  on  either male or female



albino rabbits weighing between 2.0  and  3.0 kilograms.  Other



species may also be tested  for oomparative purposes.







          2.   The nunber of animals to  be tested  for each  test



substance must be adequate  for analysis.   At  least 6 rabbits must



survive the test for each test substance.  If additional testing



is necessary for estimating dose response  or  for further evalua-



tion, more animals will be  required.







          3.   Animal facilities should  be so designed  and  main-



tained as to exclude sawdust, wood chips,  or  other extraneous



materials that might produce eye irritation.  In addition,  animals



under test should not be exposed for long  periods  to intense and



direct light, as it may damage the retina.
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     B.   Test Procedure







          1.   Both eyes of each animal  in  the  test groups must  be



examined (by use of optical instruments,  fluorescein,  ultraviolet



light, or other appropriate means) within 24 hours before subs-



tance administration.  Animals with eye defects or irritation must



be excluded.








          2.   For most purposes, anesthetics should not be  used;



however, if the test substance is likely  to cause extreme pain,



local anesthetics may be used for humane  reasons. In such cases,



anesthetics should be used only once,  just  prior to instillation



of the test substance; the eye used as the  control in  each rabbit



should also be anesthetized.  Proparacaine  0.5% and butacaine



sulfate 2% are acceptable anesthetics.







               For substance administration, the animal is held



firmly but gently until it appears to  be  quiet. The test sub-



stance is placed in one eye of each animal  by gently pulling the



lower lid away from the eyeball  (conjunctival cul-de-sac) to form



a cup into which the test substance is dropped. The lids are then



gently held together for one second and  the animal is  released.



The other eye, remaining untreated, serves  as a control.  Vehicle



controls are not included.  If a vehicle  is suspected  of causing
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irritation, additional studies should be conducted,  using  the



vehicle as the test substance.







          3.   For testing liquids, 0.1 milliliter  is  used.  For



solids or pastes, 100 miligrams of the test substance  is used.



For participate substances (flake, granule, powder,  or other



particulate form), the amount used must have a volume  of 0.1



milliliter weighing not more than 100 mg. The measure  should be



taken after gently compacting the particulates by tapping  the



measuring container in a way that will not alter their individual



form.  The weight of the 0.1 milliliter test dose must be



recorded.







          4.   For aerosol products, the substance should  be



administered as a single, short burst of about one second  at a



distance of about 4 inches directly in front of the  eye (held



open), provided that the distance insures that the velocity  of the



ejected material does not traumatize the eye.  The dose should be



approximated by weighing the aerosol can before and  after  each



treatment.  For other liquids propelled under pressure, such as



substances delivered by pump sprays, an aliquot of 0.1 ml  should



be collected and instilled in the eye as for liquids.







               The eyes are not washed following instillation of



the test substance, except as noted for fluorescein  staining.
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After the 24-hour examination, the eyes may be washed,  if desired.



Tap water or isotonic solution of sodium chloride  (U.S.P.) should



be used for all washings.







               For some  substances (such as shampoos) shown  to  be



irritating by this test, additional  tests  using rabbits with eyes



washed soon after instillation of the  substance may be  needed.   In



these cases, it is recommended that  6  rabbits be used.  Pour



seconds after instillation of the test substance,  the eyes of 3



rabbits are washed, and  at 30 seconds  after instillation, the eyes



of the other 3 are washed.   For both groups, the eyes are washed



for five minutes using a volume and  velocity of flow that are not



traumatizing.







     C.   Observations







          1.   The eyes  should be examined at 1, 24, 48, and 72



hours, and 7 days after  treatment. In  addition to  the required



observations of the cornea,  iris, conjunctivae, serious lesions



such as pannus, phlyctena, and rupture of  the globe should be



reported.  The grades of ocular reaction (Table I) must be



recorded at each examination. If the cornea, iris, or conjunctivae



has not healed completely by the seventh day, the  unhealed animals



should be retained and re-examined on  the  14th day, and again at



the 21st day if injury persists.  Evaluation of reactions can be



facilitated by use of a  binocular loupe, hand slit-lamp, or  other



expert means.
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          2.   After the recording  of  observations at 24 hours,



the eyes of any or  all  rabbits may  be  further examined after



applying flourescein stain.   For  this  optional examination, one



drop of flourescein sodium ophthalmic  solution (U.S.P) is dropped



directly on the cornea.  After flushing  out the excess flourescein



with tap water or isotonic solution of sodium chloride (U.S.P.),



injured areas  of the cornea  appear  yellow.   These changes are best



seen under ultraviolet  illumination in a darkened room.







          3.   A record of the discharge from treated eyes is not



required; however,  any  exudate above normal can be recorded as



additional information.







          4.   An animal has exhibited a positive reaction if the



test substance has  produced  at any  observation one or more of the



following signs:







               (a)  ulceration of the  cornea (other than a fine



stippling)







               (b)  opacity  of the  cornea (other than a slight



dulling of the normal luster),
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                (c)   inflammation of  the  iris  (other  than a  slight
deepening of the rugae or a  light hyperemia of  the circumcorneal
blood vessels), or

                (d)   an obvious  swelling  in the  conjunctivae
(excluding the  cornea and iris) with partial  eversion of the
eyelids and a diffuse crimson color  with individual  vessels not
easily discernible.

          5.    Table I - Grades for  Ocular Lesions

                The grading of ocular responses  is subject to vari-
able interpretations.  To promote standardization and to assist
in interpreting the  observations in  accordance  with  this guide-
line, a training film, entitled "Laboratory Procedures  for  Testing
Eye Irritation," (Digest No. 10237)  and  an "Illustrated Guide  for
Grading Eye Irritations" (Digest No. 10239) have been prepared.   A
limited number  of copies of  the guide are available  from the
Consumer Product Safety Commission Directorate  for Engineering and
Science, Washington, D. C. 20207.  The film is  available on loan
from Modern Talking  Pictures, Inc.,  2000 "L"  Street, N.W.,
Washington, D.  C.  20036.  Copies of the film (Identification  No.
CPSC M51361) can be  purchased from Movielabs, Inc.,  Movielabs
Building, 619 West 54th Street, New  York City 10019.

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                             TABLE I

                    Grades for Ocular Lesions

                              CORNEA

Opacity:  degree of density (area most dense taken  for  reading)

No ulceration or opacity	,          	0
Scattered or diffuse areas of opacity (other than slight dulling
  of normal luster/ details of iris clearly visible       	1
Easily discernible translucent areas/ details of
  iris slightly obscured                                   	2
Nacreous areas, no details of iris visible, size of pupil
  barely discernible	,	      	3
Opaque cornea, iris not discernible through the opacity	4

                                 IRIS

Normal	                                                   n
Markedly deepened rugae, congestion, swelling, moderate
  circumcorneal hyperemia, or injection, any of these
  or combination any thereof/ iris still reacting to
  light (sluggish reaction is positive)	       ,	    1
No reaction to light, hemorrhage, gross destruction (any
  or all of these)	                     ">.

                             CONJUNCTTVAF;

Redness (refers to palpebral ad bulbar conjunctivae excluding
         cornea and iris)

Blood vessels normal	, ,   , ,  ,                  ,,,..,.... 0
Some blood vessels definitely hyperemic (injected)    	I
Diffuse, crimson color, individual vessels not easily discernible. ..2
Diffuse beefy read                                          	3

Chemosis:  lids and/or nictitating membranes

No swelling	    	0
Any swelling above normal (includes nictitating membranes)	.1
Obvious swelling with partial eversion of lids           	7.
Swelling with lids about half closed	      ,, .3
Swelling with lids more than half closed	        	4
                               28

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      D.   Classification of Test Substances
           Classification*
       fteaction
           Non-irritant
No positive reactions
(opacity, iritis or conjunctiv-
itis on more than 1 out of 6
test animals at 1-3 days and
all eyes normal at 7th day.
           Irritant
Opacity grades 1.0 to 2.0 at
any observation up to 7 days.
All corneas cleared at 14
days.*
                                         Iritis 1.0 at 1-7 days, but  all
                                         iritis cleared by 14th day.*

                                         Conjunctivitis:
                                           Redness grade 2.0 at 1-7 days
                                           Chemosis grades greater than
                                            2.0 at 1-7 days.
*  If not cleared at 14 days, substance  is considered a  severe
 irritant.
                                 29

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               Severe
                 Irritant or
                 Corrosive**
Opacity greater than 2.0.
Injury persists 14-21 days.

Corneal perforation or
necrosis at any observation
period.

Pannus or phlyctenular
reactions.

Iritis grade greater than 1.0
at 1-7 days, all eyes not
clear at 14 or 21 days.
                              	       Conjunctivitis:
                                         Badness grades greater than
                                           2.0 at 1-7 days.
                                         Chemosis grades greater than
                                           2.0 at 1-7 days.
** Opacity grades 2 to 4 and/or perforation of the cornea are
considered to be corrosive effects when opacities persist to 21 days.
                              30

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III. Data Reporting







     A.   Identification







          Each test report must be  signed by  the person



responsible for the test and  identify:







          1.   The laboratory where the  test  was performed by



name and address;







          2.   The inclusive  dates  of  the test; and







          3.   Each person primarily responsible for  separate



ccraponents of the test and the component for  which the person  is



reponsible including  (a) the  conduct of  the test,  (b) analysis of



the data, (c) the writing of  the  report, and  (3) any  written or



other matter contained in the report.







     B.   Body of Report







          The test report must include all  information necessary



to provide a complete and accurate  description and evaluation  of
                              31

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     E.   Conclusions







          1.   The test shall be considered positive if four or more of



the animals in either of the test groups  (rabbits with  eyes washed,  or



with eyes unwashed) exhibit a positive reaction.  If only one animal



exhibits a positive reaction, the test shall be regarded as negative.



If two or three animals exhibit a positive reaction,  the toxioologist



in charge of the test may designate the substance to be an irritant.



If he/she does not, the test shall be repeated using a  different group



of six animals.  The second test shall be considered  positive if three



or more of the animals exhibit a positive reaction.







          2.   If only one or two animals in the second test exhibit a



positive reaction, the test should be repeated with  a different group



of six animals.  When a third test is needed, the substance will be



regarded as an irritant if any animal exhibits a positive response.
                              32

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the  test procedures  and  results.   Each  report must  include the



following  sections:







           1.    Summary and Conclusions.   This section of the test



report  should  contain  a  tabular summary of  the  data,  an  analysis



of the  data, and  a statement of the conclusions drawn from the



analysis.  The summary must highlight all positive  data  and



observations and  any other indications  of toxic effects.







           2.    Materials.   This section of  the  test report shall



include, but not  be  limited to/ the following information:







                (a)   Identification of the test  substance,



including:







                i.    chemical name,  molecular structure,  and a



qualitative and quantitative determination  of its chemical



composition, including names and quantities of  known  contaminants



and  impurities, so far as  is practical; the determinations shall



also include a listing of materials as  unknowns, if any,  so that



100% of the test sample  is  accounted for;
                               33

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               ii.   manufacturer  and  lot  nunber of the substance



tested, and such  information  as physical  state, pH,  stability, and



purity; and







              iii.   exact  identification  of  diluents,  suspending



agents, emulsifiers, or other materials used in administering the



test substance.







                (b)  Animal data,  including:







                  i. species and strain used  and rationale  for



selection of  the  strain if other  than a common  laboratory  strain;







                ii. source of supply  of the  animals;







                iii. description of  any pre-test conditioning,



including diet;







                iv. description of  the method used in



randomization  of  animals to test  groups;  and







                  v. nunbers of animals of each  sex in  each test



group.
                               34

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                (c)  Data on facilities should  include description



of the caging conditions including bedding material, ambient



temperature, and humidity.







          3.   Methods








                (a)  Deviation  from guidelines  - This section



shall indicate all ways in which the test procedure deviates from



this guideline and shall state the rationale for such deviation.







                (b)  Specification of test methods - This section



shall include a full description of the experimental design and



procedure, the length of the study, and the dates on which the



study began and ended.







               (c)  Statistical analysis - All statistical methods



used should be fully described or identified by reference.







               (d)  Data on dosage administration, including:







                 i. all dose levels administered;
                            35

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                ii. method and frequency of administration; and







               iii. total volume of substance (i.e., test



substance plus vehicle) contained in individual dosages.







               (e)  Data on observation methods, including:







                 i. duration; and







                ii. method and frequency of observation of the



animals.







          4.   Results







               The tabulation of data and individual results must



accompany each report in sufficient detail to permit independent



evaluation of results, including summaries and tables that show



the relationship of effects to time of dosing, sex, etc.
                               36

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          5.   References







               This section of  the  test report  shall  include  the



following information:







               (a)  Availability of original data,  specimens  and



samples of the test substance.  The location of all original  data,



specimens, and samples of the test  substances vrfiich are  retained



in accordance with the testing  requirement.







               (b)  Literature  or references, including, where



appropriate, those references for (1)  test procedures,  (2)



statistical and other methods used  to  analyze the data,  (3)



compilation and evaluation of results, and  (4)  the  basis upon



which conclusions were reached.







IV.  Suggested Reading







     1.   Draize, J. H., 1959.  Dermal Toxicity. In;  Association



of Pood and Drug Officials of the U. S.  Austin, Texas.  Appraisal



of the Safety of Chemicals in Foods, Drugs, and Cosmetics,  pp.



46-59.
                              37

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     2.   Green, W. R., J. B. Sullivan, R. M. Hehir, and L. F.



Scharpf.  A systematic comparison of chenically-induced eye injury



in the albino rabbit and rhesus monkey   In; The Soap and Deter-



gent Association.  Submission to the National Academy of Sciences



by the Soap and Detergent Association on toxicity test procedures



with Appendices A-P. Appendix C.







     3.   National Academy of Sciences - National Research



Council, 1977.  In;  Principles and Procedures for Evaluating the



Toxicity of Household Substances, Report No. 1138, prepared for



the Consumer Product Safety Commission.  Eye Irritation, pp.



62-91.







     4.   Prince, J. H., 1964.  The Rabbit in Eye Research.



Springfield, 111.  Charles C. Thomas.
                              38

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DRAFT I.R.L.G. GUIDELINE FOR



ACUTE ORAL TOXICITY STUDIES IN RODENTS
Testing Standards  Guidelines Work Group



INTERAGENCY REGULATORY LIAISON GROUP








May 30, 1979
               39-

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                             PREFACE
The LDjQ value is the nost frequently determined  index of



toxicity and is required by some Federal legislation.







Although several accepted methods for determining the LD5Q



values have been developed, many important determinants of tox-



icity are not represented either by these values  or slopes of



dose-response curves for lethality.  These determinants are  inte-



gral to an evaluation of acute toxicity and should be observed



during the course of an acute toxicity study.  Site and mechanisn



of action, early or delayed death, and recovery rate may be  better



indices of toxicity and hazard than LE^g values per se.  Mor-



bidity and/or pathogenesis may have more toxicological signifi-



cance than mortality.








This guideline is designed for use in acute ingestion tests  using



rodents, but is adaptable by example to other species.
                           40

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      Scientific Issues for Public Conttient

     During the development of this proposed guideline, many scientific
issues were discussed  by the Work Group.  These  issues were raised by
members of the Work Group,  by the public  comments  to the  EPA proposed
pesticide guidelines and by the comments, of interagency reviewers.
This proposed guideline reflects  the Work Group's  consideration of
these conments.  Consideration of these comments,  discussed below, is
reflected in this proposed  guideline; and the public is invited to
comment further on these issues or any other aspect  of this proposed
guideline.

     A.  Several commentors suggested at  least 5 to  6 dose levels in
order to achieve reliable mortality and obtain appropriate confidence
limits of the LD50 value.   The Work Group feels  that four dose
levels, when properly  spaced, should provide sufficient data for
     estimations.
     B.  Many commentors felt  that  restricting  the 95%  confidence
limits of the LD$Q to plus or  minus 20% or  less may be  too
restrictive.  Comments are solicited concerning the limitation of  the
confidence limits to 20%.
                              41

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     C.  The Work Group recognizes the difficulty of selecting dose



levels which will produce mortality rates between 10 and 90% and



requests comments concerning methods of dose selection  for  acute oral



toxicity studies.







     D.  The Work Group thinks that fasting of animals  is necessary  to



obtain more uniform absorption of the test substance in the acute oral



study, but the period of fasting should not be so long  as to induce



significant stress (metabolic or otherwise) in the test animals.



Comments are requested concerning the effect of fasting on  acute oral



toxicity and appropriate fasting periods for various species.







     E.  Many comments were received concerning the frequency of



clinical (visual) observations of the test animals.  The Work Group



thinks that observation at least twice a day following  the  day of



substance administration is necessary in order to determine the acute



toxicity profile of the test substance.  Comments on the frequency of



clinical observations are requested.
                              42

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                      TABLE OF CONTENTS

  I.  General Considerations

      A.  Good Laboratory Practices
      B.  Personnel
      C.  Test Substance
      D.  Animals
      E.  Dead Anijnals, Necropsy, and Histopathology
      F.  Equipment

 II.  Specific Considerations

      A.  Test Preparation
      B.  Test Procedure

III.  Data Reporting

      A.  Identification
      B.  Body of Report

          1.  Summary and Conclusions
          2.  Materials
          3.  Methods
          4.  Results
          5.  References

 IV.  Suggested Reading

                          43

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I.   General Considerations

     A.   Good Laboratory Practices

          Basic standards presented here relating to good  labora-
tory practices are to serve as general guidance for the conduct of
the study, but are not intended to be all inclusive.  This guide-
line does not set forth the managerial aspects of science  or good
laboratory practices.  Studies should be conducted according to
"Nonclinical Laboratory Studies/ Good Laboratory Practice
Regulations," (43 FR 59986, 22 December 1978).

     B.   Personnel

          All testing and evaluation must be done under the direc-
tion of personnel who have the education, training, and experience
to perform the testing and evaluation in accordance with sound
scientific experimental procedures.  The agency, commission, or
department may require resumes of personnel who have performed,
supervised, reviewed, or evaluated the testing.  To the extent
possible, the same person or persons should perform all observa-
tions and necropsies in a single test in order to insure consis-
tency of evaluation.  When a histopathological examination is
                              44

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done, similar considerations should apply.







     C.   Test Substance  (materials or mixtures of substances or



materials)







          1.   As far as  is practical, composition of  the  test



substance must be known,  including the name and quantities of



known contaminants and  impurities.  Unknown materials,  if  any,



must be quantified to account for 100% of the  test sample.  The



specific substance to be  tested will be determined in  consultation



with each agency.







          2.   The lot  of the substance tested should  be the same



throughout the study.   The test sample should  be  stored under



conditions that maintain  its stability, strength, quality, and



purity from the date of its production until the  tests are



complete.







          3.   Safe handling and disposition of the  test substance



is essential.







     D.   Animals
                                  45

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          1.   Animals used for testing  should  not have been



subjected to any previous experimental procedures.







          2.   The test animal shall be  characterized  as to



species, strain, sex, weight and/or age.  Each  animal  must  be



assigned an appropriate identification number.







          3.   Recommendations contained in DHEW pub.  no. (NIH)



74-23, entitled "Guide for the Care and  Use of  Laboratory



Animals," should be followed for the care, maintenance,  and



housing of animals.







          4.   Animals may be group-caged for this  test unless the



pharmacological action of the test substance dictates  otherwise.



However, the number of animals per cage  should  not  prevent



continued and clear observation of each  animal.  When  signs of



morbidity or excitability are observed in group-caged  animals



during the test, such animals should be moved to separate cages.








          5.   Healthy animals must be used. Animals must be



assigned to groups in such a manner as to minimize  bias and assure



comparability of pertinent variables.
                               46

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          6.   Each animal must be observed as  necessary  to insure



that animals are not lost due to cannibalism, autolysis of



tissues, misplacement, or similar management problems.







          7.   When control animals are used, they must be  housed,



fed, and handled exactly like the test animals; and  they must be



caged to minimize airborne or other contamination by the  test



substance.








     E.   Dead Animals, Necropsy, and Histopathology







          When an animal is discovered dead, it must be refriger-



ated at temperatures low enough to minimize autolysis if  necropsy



cannot be performed immediately.  Necropsy must be performed with-



in 16 hours of death.  When animals are killed  for examination,



the necropsy should be performed as soon after  death as possible.



If histopathological examination is to be  conducted, all  tissue



specimens should be placed in appropriate  fixative when they are



taken from the animal.








     F.   Equipment







          All equipment used in conducting the  test, including
                                47

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equipment used to prepare and administer the test substance and



equipment used to maintain environmental conditions, must be of



appropriate design and adequate capacity.  Equipment should be



inspected, cleaned, and maintained regularly.  The equipment must



be properly calibrated at the time of its use.
                                48

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II.  Specific Considerations







     A.   Test Preparation








          1.  Animals:  Laboratory strains of rats  (125-250 g each)



and/or mice (20-30 g each) should be used.  When attempting to estimate



hazards to young humans, additional studies designed to consider the



developmental stage of the test animal  in relation  to anticipated human



exposure should be performed.








          2.   Number and sex:  At least 10 animals, 5 per sex, random-



ly assigned should be used at each dose level.  The females should be



nonpregnant.







          3.   Controls:  Untreated controls are generally not re-



quired, since dose reponse during an LDjg may serve as an internal



control.  A negative or vehicle control group is not required; however,



if a vehicle or solvent of uncharacterized toxic potential is used, an



acute oral toxicity test should be done on the solvent.







          4.   Dose levels:  At least four dose levels should be used,



spaced appropriately to produce test groups ideally with mortality



rates between 10% and 90% to permit the calculation of the U)$Q



value for males and females with a 95% confidence limit.  Where



possible, the 95% confidence limit should not exceed approximately plus



or minus 20% of the ID   value.
                              49

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          5.   Fasting:  Animals  should  be fasted prior to admin-



istration of test substance.   Pood  should  be  withheld from rats



overnight; from mice  for 6  to  8 hours.







     B.   Test Procedure







          1.   Dosage:  For an acute  study, one  or more doses of



the  test substance may be administered within a  24-hour period.



Ideally the substance should be administered  in  a single dose.



The  determination of  Ll^g values  of insoluble solids can be



difficult because of  the nature of  the suspension that may have to be



administered.  Such limitations may be circumvented, when necessary, by



the  administration of the test substance in divided doses over a period



of several hours.  An adequate estimate  of acute hazard is obtained for



most purposes if data based on this test is submitted showing that the



     value of 5gA9-
          2.   Route of administration:  The dose should be admin-



istered by gavage, not in the food.  The dose  is  administered via



soft rubber or polyethylene tubing or a large  ball-tip needle.



The maximum volume of liquid that can be given depends on the



rodent's size and should not exceed 2 ml/lOOg  body weight.   When
                              50

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possible/ variability in test volume should be minimized, with



concentrations being adjusted accordingly.








          3.   Observation period:  The observation period should be at



least 14 days.  Although a 14-day observation period  is sufficient for



most compounds, animals demonstrating visible signs of toxicity after



14 days could be held longer.








          4.   Recording of clinical observations:  Observations should



be recorded systematically as they are made.  The animals should be



observed frequently during the first day and twice a day thereafter at



least 4 hours apart (once each morning and late afternoon).  Individual



records should be maintained for each animal.  Visual observations



should include, but not be limited to, changes in skin and fur, eyes,



mucous membranes and respiratory, cardiovascular, autonomic and central



nervous sytems, and somato-motor activities.  Particular attention



should be directed to observations for the presence of tremors, convul-



sions, salivation, hyperactivity, diarrhea, lethargy, sleep, coma,



blanching, cyanosis, and vasodilation.  The time at which signs of



toxicity appear and the time of death must be recorded.
                              51

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          5.   Weight change:  Individual weights of animals must
be determined on the day the test substance is administered,
weekly thereafter, and prior to sacrifice.

          6.   Necropsy:  A complete gross necropsy should be per-
formed on all animals that die during the course of the  test and all
remaining animals at termination of the test.  Gross pathological
changes of the intestinal tract and the major organs such as liver,
kidney, heart, brain, and spleen should be noted.  Liver, kidney, and
organs showing evidence of gross pathology of all animals surviving 12
or more hours should be preserved for possible future microscopic
examination.
                                52

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III. Data Reporting







     A.   Identification







          Each test report must be signed by the person



responsible for the test and identify:







          1.   The laboratory where the test was performed by



name and address;







          2.   The inclusive dates of the test; and







          3.   Each person primarily responsible for separate



components of the test and the component for which the person  is



reponsible including  (a) the conduct of the test, (b) analysis of



the data/ (c) the writing of the report, and (3) any written or



other matter contained in the report.







     B.   Body of Report







          The test report must include all  information necessary



to provide a complete and accurate description and evaluation  of
                               53

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the test procedures and results.  Each report must include the



following sections:







          1.   Summary and Conclusions.  This section of  the  test



report should contain a tabular summary of the data, an analysis



of the data, and a statement of the conclusions drawn from the



analysis.  "Hie summary must highlight all positive data or



observations and any deviations from control data which may be



indicative of toxic effects.







          2.   Materials.  This section of the test report shall



include, but not be limited to, the following information:







               (a)  Identification of the test substance,



including:








               i.   chemical name, molecular structure, and a



qualitative and quantitative determination of its chemical



composition, including names and quantities of known contaminants



and impurities, so far as is practical; the determinations shall



also include a listing of materials as unknowns, if any,  so that



100% of the test sample is acccounted for:
                                  54

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              ii.   manufacturer and  lot  number of the  substance



tested, and such information as physical  state, pH,  stability,  and



purity; and







             iii.   exact  identification  of diluents, suspending



agents, emulsifiers, or other materials used  in administering the



test substance.







                (b)  Animal data, including:







                 i. species and strain used and rationale  for



selection of the strain if other than a common laboratory  strain;







                ii. source of supply  of the animals;







                iii. description of  any pre-test conditioning,



including diet;







                iv. description of  the method used in



randomization of animals to test or control groups;  and







                 v. numbers of animals of each sex in each test



and control group.
                                    55

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                (c)  Data on  facilities  should  include description



of the caging conditions including number of animals  per cage,



bedding material, ambient temperature,  and  humidity.








          3.    Methods







                (a)  Deviation  from guidelines  - This  section



shall indicate  all ways in which the test procedure deviates from



these guidelines and shall state the rationale for such



deviation.







                (b)  Specification of test methods - This section



shall include a full description of the experimental  design  and



procedure, the  length of the study, and the dates on  which the



study began and ended.








                (c)  Statistical analysis -  All statistical methods



used should be  fully described or identified by reference.








                (d)  Data on  dosage administration, including:








                 i. all dose levels administered, expressed  as



mg/kg of body weight;
                                   56

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                ii. method and frequency of administration; and







               iii. total volume of substance  (i.e., test



substance plus vehicle) contained in  individual dosages.







               (e)  Data on obsevation methods, including:







                 i. duration; and







                ii. method and frequency of observation of the



animals.







          4.   Results







               The tabulation of data and individual results must



accompany each report in sufficient detail to permit independent



evaluation of results.







               (a)  Tabulation of the response data (i.e., nunber



of animals dying; number of animals showing signs of toxicity;



nunber of animals exposed) at each exposure level by sex, and time



of death after dosing;
                               57

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               (b)  11)50 values for each test substance
calculated at the end of the observation period, with method of
caluclation specified;
               (c)  95% confidence interval for the
values;
               (d)  Slope of the dose-mortality curve for each
substance tested; and

               (e)  Findings from all clinical observations,
necropsy, and histopathological examinations  (when made).

          5 .   References

               This section of the test report shall include  the
following information:

               (a)  Availability of original  data, specimens  and
samples of the test substance.  The location  of all original  data,
specimens, and samples of the test substances which are  retained
in accordance with the testing requirement.
                                  58

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               (b)  Literature or references, including, where
appropriate, those references for (1) test procedures,  (2)
statistical and other methods used to analyze the data,  (3)
compilation and evaluation of results, and (4) the basis upon
which conclusions were reached.
                                59

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IV.  Suggested Reading







     1.   Balazs, T. 1970.  Measurement of Acute Toxicity.  In;



Methods in Toxicology.  G. E. Paget, ed.  F. A. Davis Co.,



Philadelphia, Pa.







     2.   Hagan, E. C. 1959.  Acute Toxicity.  Jrp  Appraisal of the



Safety of Chemicals in Foods, Drugs and Cosmetics.  Association of Food



and Drug Officials of the United States.







     3.   Bliss, C. I.  1938.  The determination of the dosage mortal-



ity curve from small numbers.  Quarterly Journal Pharm. Pharmacol.



11:192-216.







     4.   Litchfield, J. T., Jr. and F. Wilcoxon.  1949.  A simplified



method of evaluating dose-effect experiments.  J. Pharmacol. Exp.



Therap. 96:99-115.







     5.   Thompson, W. R.  1947.,  Using of moving averages and inter-



polation to estimate median effective dose.  Bacteriological Rav.



11:115-145.
                              60

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DRAFT I.R.L.G. GUIDELINE FOR



ACUTE DERMAL TOXICITY TESTS
Testing Standards  Guidelines Work Group



INTERAGENCY REGULATORY LIAISON GROUP








May 15, 1979
                61

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                             PREFACE







A test for acute dermal toxicity should evaluate the potential for



systemic and local toxic effects of chemicals expected to come in



contact with the skin.  The acute dermal test refers to one period



of topical application of up to 24 hours (the exposure period) and



an observation period of at least 14 days.
                               62

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      Scientific Issues  for Public Gommsnt







     During the development of  this proposed  guideline, many scientific



issues ware discussed by the work group.  These  issues were  raised  by



members of the  Work Group,  by the public  comments  to the  EPA proposed



pesticide guidelines and by the comments  of interagency reviewers.



Consideration of these  comments/  discussed below,  is reflected  in this



proposed guideline; and the public is  invited to comment  further on



these issues or any other  aspect  of this  proposed  guideline.







    A.  Several commentors stated that dermal toxicity studies  should



be conducted on the product as  it will be encountered in  actual use.



Specifically, granular  and pelleted formulations will not come  into



contact with the skin as a paste  as would occur  using the IRD3



guidelines.







        The Work Group  requests comment on the form in which the test



substance should be applied to  the skin.







     B. Because of the  various  methods used to remove fur from  the



dermal test areas or the times  at which test  areas are prepared prior



to application  of the test substance,  there are  potential problems  in



the reproducibility of  the test responses.







        Information regarding the most appropriate method of test  site



preparation is  requested.
                               63

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    C.  Although the use of abraded skin may be apropriate for testing
drugs and cosmetics intended for use on damaged or diseased skin,
abraision techniques have not been standardized;  and  responses obtained
from substances applied to abraded sites may be variable.

        Information is requested concerning the validity of performing
acute dermal toxicity tests using abraded skin and the  use of abraded
skin in trial test mortality determinations with  2 gAg or 2 ltd/kg
application of the test substance.

    D.  Several oommentors suggested that dermal  exposure  should not be
limited to 24 hours, but should be varied to simulate actual use condi-
tions.

        The Work Group requests comments on this  issue.

    E.  Another issue is the frequency of clinical observation of  the
test animals.  The Work Group feels that at least twice a  day observa-
tions is necessary in order to determine the time of  onset,  duration
any acute effects.

        Comments on this topic are invited.
                              64

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                        TABLE OF CONTENTS
  I.  General Considerations







      A.  Good Laboratory Practices



      B.  Personnel



      C.  Test Substance



      D.  Animals



      E.  Dead Animals, Necropsy, and Histopathology



      F.  Equipment







 II.  Specific Considerations







      A.  Test Preparation



      B.  Test Procedure







III.  Data Reporting







      A.  Identification



      B.  Body of Report
                         65

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         1.  Summary and Conclusions



         2.  Materials



         3.  Methods



         4.  Results



         5.  References
IV.  Suggested Reading
                      66

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I.   General Considerations

     A.   Good Laboratory Practices

          Basic standards presented here  relating  to good labora-
tory practices are to serve as general guidance for the conduct of
the study, but are not intended to be all inclusive.  This guide-
line does not set forth the managerial aspects of  science or good
laboratory practices.  Studies should be  conducted according to
"Nonclinical Laboratory Studies, Good Laboratory Practice
Regulations," (43 PR 59986, 22 December 1978).

     B.   Personnel

          All testing and evaluation must be done  under the direc-
tion of personnel who have the education,  training, and experience
to perform the testing and evaluation in  accordance with sound
scientific experimental procedures.  The  agency, commission, or
department may require resumes of personnel who have performed,
supervised, reviewed, or evaluated the testing.  Tto the extent
possible, the same person or persons should perform all observa-
tions and necropsies in a single test in  order to  insure consis-
tency of evaluation.  When a histppathological examination is
                               67

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done, similar considerations should apply.

     C.   Test Substance  (materials or mixtures  of  substances or
materials)

          1.   As far as  is practical, composition  of the test
substance must be known,  including the name and  quantities of
known contaminants and impurities.  Unknown materials,  if any,
must be quantified to account for 100% of the test  sample.   The
specific substance to be  tested will be determined  in consultation
with each agency*

          2.   The lot of the substance tested should be  the same
throughout the study.  The test sample should be stored under
conditions that maintain  its stability, strength, quality,  and
purity from the date of its production until the tests are
complete.

          3.   Safe handling and disposition of  the test  substance
is essential.

     D.   Animals
                             68

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          1.   Animals used for testing should not have been sub-



jected to any previous experimental procedures.







          2.   The test animal shall be characterized as  to



species/ strain, sexf weight and/or age.  Each animal must be



assigned an appropriate identification number.







          3.   Reccmnendations contained  in DHEW pub. no. (NIH)



74-23, entitled "Guide for the Care and Use of Laboratory



Animals," should be followed for the care, maintenance, and



housing of animals.







          4.   Because it is necessary to prevent oral ingestion



of the test substance, animals may not be group-caged for this



test.







          5.   Healthy animals must be used. Animals must be



assigned to groups in such a manner as to minimize bias and assure



comparability of pertinent variables.
                              69

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          6.   Each animal must be observed as necessary to  insure
that animals are not lost due to cannibalism, autolysis of
tissues, misplacement, or similar management problems.

          7.   When control animals are used, they must be housed,
fed, and handled exactly like the test animals; and they must be
caged to minimize airborne or other contamination by the test
substance.

     E.   Dead Animals, Necropsy, and Histopathology

          When an animal is discovered dead, it must be refriger-
ated at temperatures low enough to minimize autolysis if necropsy
cannot be performed immediately.  Necropsy must be performed with-
in 16 hours of death.  When animals are killed for examination,
the necropsy should be performed as soon after death as possible.
If histopathological examination is to be conducted, all tissue
specimens should be placed in appropriate fixative when they are
taken from the animal.

     F.   Equipment

          All equipment used in conducting the test, including
                              70

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equipment used to prepare and administer the test substance and



equipment used to maintain environmental conditions, must be of



appropriate design and adequate capacity.  Equipment should be



inspected, cleaned, and maintained regularly.  The equipment must



be properly calibrated at the time of its use.
                                71

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II.  Specific Considerations








     A.   Test Preparation







          1.   Animals:  The young, adult, albino rabbit  weighing



2.0 to 3.0 kg is the preferred species because of its  size,  ease



of handling and restraint, and skin permeability.  Selection of



other species may be acceptable but must be justified.







          2.   Number and sex:  Bgual numbers of animals  of  each



sex with intact skin are required for each dose level.  The  number



of animals per dose depends on the level of statistical confidence



desired.  All methods for estimating 11)50 values require  that



the test animals be randomly assigned to dose groups.  TVo rabbits



per sex per dose are recommended in most cases.  If a  toxicolog-



ical effect occurs with a marginally significant incidence,  data



from further testing with larger numbers of animals may be



required.








          3.   Females:  The females should be nonpregnant since



pregnancy may modify response.








          4.   Dose levels:  To establish a dose regimen, a  trial
                               72

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test is recommended.  It should  include one dose level  higher  than
the expected ID$Q and at least one dose level  below the
expected 11)50 .  J^ a ^se f 2g/kg (or 2ml/kg) or more, placed
on the abraded (within two hours prior to application)  skin of at
least 2 animals per sex, produces no mortality, no  further testing
at other dose levels is necessary.  However, if mortality occurs,
at least three dose levels should be used to estimate the
    ' using rabbits with intact  skin.
          5.   Preparation of skin:  Twenty-four hours before
testing, fur from the trunk of animals must  be  clipped so  that  no
less than 10%  (about 240 cm-*) of  the dorsal  body surface area is
available for application of material.  The  abraded  area is
prepared by making four epidermal incisions  with a clean needle
through the stratum corneun, but  not deep enough to  disturb  the
derma or produce bleeding.

     B.   Test Procedure

          1.   Test substance:  When testing solids, the test
substance should be moistened sufficiently with normal saline or
tap water to make a paste that will insure good contact with the
skin.  For some applications, it  may be appropriate  or necessary
                               73

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to use other vehicles.  If a carrier or diluent  is used,  it  should
be non-irritating and of known low toxicity.  When such vehicles
are used, consideration should be given to the effects of those
vehicles on absorption of the test substance.

          2.   Dosage:  When technically feasible, the maximum
quantity of substance plus vehicle to be applied  is 2 gAg body
weight.  The test substance should be applied uniformly over at
least 10% of the dorsal surface area.  When possible, at  least 3
levels of exposure should be tested to permit development of a
dose-response trend.

          3.   Administration (application):  The test substance
must remain in contact with the skin throughout the exposure
period of 24 hours.  Liquid or solid substances should be held in
contact with the skin with a porous gauze dressing and non-
irritating tape.  The test site should be covered in a semi-
occlusive fashion with an impermeable material such as plastic
film or rubberized cloth.

               Routine use of occlusive dressings is not  recom-
mended.  Occlusive skin dressings may enhance penetration of the
test substance and should be used only when testing for effects
                                 74

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that may occur under similar conditions  in humans.








               During exposure, animals  should  be prevented  from



ingesting or inhaling the  test substance.   Restrainers,  such as



Elizabethan collars, that  permit animals to move about their cages



should be used for this purpose.   Immobilization is  not  a



recommended method.








               At the end  of the exposure  period, all residual



material should be removed by washing, using an appropriate



solvent.  About one half hour later, and once again  at 72 hours,



the exposed area should be examined, and all lesions noted and



graded (Table I).








          4.   Observation period:  The  observation  period must  be



at least 14 days.  However, duration of  observation  should not be



fixed; rather, it should be determined by  the toxic  reactions,



rate of onset, and length  of recovery period.   Although  a 14-day



observation period is sufficient for most  compounds, animals



demonstrating visible signs of toxicity  after 14 days could  be



held longer.
                                75

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          5.   Recording of clinical observations:  The animals



should be observed frequently during the first day and twice a day



thereafter at least 4 hours apart (once each morning and late



afternoon).  Observations should be recorded systematically as



they are made, and individual records should be maintained for



each animal.  Observations should include, but not be limited to,



grossly visible changes in skin and fur, eyes, mucous membranes



and respiratory, cardiovascular, autonomic and central nervous



systems, and somatomotor activities.  Particular attention should



be directed to observations for the presence of tremors, convul-



sions, salivation, diarrhea, lethargy, sleep, and coma.  The time



at which toxicity signs appear and the time of death must be



recorded.







          6.   Weight change:  Individual weights of animals must



be determined on the day the test substance is administered,



weekly thereafter, and at death or sacrifice.








          6.   Necropsy:  A complete gross necropsy should be



performed on all animals that die during the course of the test



and on all remaining animals at termination of the test.  Gross



pathological changes of the intestinal tract and the major organs



such as liver, kidney, heart, brain, and spleen should be noted.
                               76

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Liver, skin/ kidney/ and organs showing evidence of gross path-



ology of all animals surviving 12 or more hours should be



preserved for possible future microscopic examination.
                               77

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                             TABLE  I








                   EVALUATION OF SKIN REACTION







                                                         Value



Erythema and Eschar Formation







No erythema	                                  0



Very slight erythema  (barely perceptthl.e)	I.



Well-defined erythema	,                          7.



Moderate to sever erythem	,    	,3



Severe erythema  (beet readness) to  slight



  eschar formation (injuries in depth)            	4



Edema Formation



No edema	            ,	             0



Very slight edema (barely perceptible)	,,	..,,,,I



Slight edema (edges of area well defined by



  definite raising	.2



Moderate edema (raised approximately 1 milliliter)	3



Severe edema (raised more than 1 millimeter and



  extending beyond the area of exposure)	4



Severe eschar and/or corrosion	Note



                                                        occurence
                               78

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III. Data Reporting








     A.   Identification








          Each test report must be signed by the person



responsible for the test and identify:








          1.   The laboratory where the test was performed by



name and address;







          2.   The inclusive dates of the test; and








          3.   Each person primarily responsible for separate



components of the test and the component for which the person  is



reponsible including  (a) the conduct of the test, (b) analysis of



the data/ (c) the writing of the report, and (3) any written or



other matter contained in the report.








     B.   Body of Report








          The test report must include all information necessary



to provide a complete and accurate description and evaluation  of



the test procedures and results.  Each report must include the
                                79

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following sections:







          1.   Summary and Conclusions.  This section of the  test



report should contain a tabular summary of the data, an analysis



of the data, and a statement of the conclusions drawn from the



analysis.  The summary must highlight all positive data or



observations and any deviations from control data which may be



indicative of toxic effects.







          2.   Materials.  This section of the test report shall



include, but not be limited to, the following information:







               (a)  Identification of the test substance,



including:








               i.   chemical name, molecular structure, and a



qualitative and quantitative determination of its chemical



composition, including names and quantities of known contaminants



and impurities, so far as is practical; the determinations shall



also include a listing of materials as unknowns, if any, so that



100% of the test sample is acccounted for:







              ii.   manufacturer and lot number of the substance
                               80

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tested, and such information as physical state, pH,  stability,  and
purity; and

             iii.   exact identification of diluents, suspending
agents, emulsifiers, or other materials used  in administering the
test substance.

               (b)  Animal data, including:

                 i. species and strain used and rationale for
selection of the strain if other than a common laboratory strain;

                ii. source of supply of the animals, diet (lot
number, composition, etc.), and water;

               iii. description of any pre-test conditioning;

                iv. description of the method used in
randomization of animals to test or control groups;  and

                 v. numbers of animals of each sex in each  test
and control group.
                                81

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               (c)  Data on facilities  should  include description



of the caging conditions including number of animals per cage/



bedding material, ambient temperature,  and humidity.








          3.   Methods







               (a)  Deviation from guidelines  - This section



shall indicate all ways in which the test procedure deviates from



these guidelines and shall state the rationale for such



deviation.







               (b)  Specification of test methods - This section



shall include a full description of the experimental design  and



procedure, the length of the study, and the dates on which the



study began and ended.








               (c)  Statistical analysis - All statistical methods



used should be fully described or identified by reference.








               (d)  Data on dosage administration, including:







                 i. all dose levels administered, expressed  as



mg/kg of body weight;
                                82

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                ii. method and frequency of administration;  and







               iii. total volume of substance  (i.e./ test



substance plus vehicle) contained  in  individual dosages.







               (e)  Data on obsevation methods/ including:







                 i. duration; and







                ii. method and frequency of observation of the



animals.







          4.   Results







               The tabulation of data and individual results must



accompany each report in sufficient detail to permit independent



evaluation of results.







               (a)  Tabulation of  the response data (i.e./ number



of animals dying; number of animals showing signs of toxicity;



number of animals exposed) at each exposure level by sex/ and time



of death after dosing;
                               83

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               (b)  ID$Q values for each test substance



calculated at the end of the observation period, with method of



caluclation specified;
               (c)  95% confidence interval for the



values;
               (d)  Slope of the dose-mortality curve for each



substance tested; and







               (e)  Findings from all clinical observations,



necropsy, and histopathological examinations (when made).







          5.   References







               This section of the test report shall include the



following information:







               (a)  Availability of original data, specimens and



samples of the test substance.  The location of all original data,



specimens, and samples of the test substances which are retained



in accordance with the testing requirement.
                                84

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               (b)  Literature or references, including/ where



appropriate, those references for (1) test procedures,  (2)



statistical and other methods used to analyze the data,  (3)



compilation and evaluation of results, and (4) the basis upon



which conclusions were reached.
                             85

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IV.  Suggested Reading








     1.   Draize, J. H.  1959. Dermal toxicity.  In;  Appraisal of



the Safety of Chemicals in Poods, Drugs, and Cosmetics. Austin,



Texas.  Association of Pood and Drug Officials of the U. S.  pp.




46-59.








     2.   National Academy of Sciences - National Research



Council, 1977.  Dermal and eye toxicity tests.  In; Principles and



Procedures for Evaluating the Toxicity of Household Substances,



Report No. 1138, prepared for the Consumer Product Safety



Commission,  pp. 23-28.








     3.   McCreesh, A. H. and M. Steinberg,  1977.  Dermato-



Toxicology and Pharmacology, Hemisphere Publishing Corp.,



Washington, D. C.








     4.   Mailbach, H. I. and P. N. Marzulli. 1975.  Animal Models



in Dermatology. Churchill and Livingston, Edinburgh.
                             86

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DRAFT I.R.L.G. GUIDELINE FOR
ACUTE INHALATION TESTS IN RATS
Testing Standards  Guidelines Work Group



INTERAGENCY REGULATORY LIAISON GROUP








June 6, 1979
                87

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                             PREFACE








The purpose of this test is to characterize the toxicity of a  sub-



stance administered acutely to test animals.  This characterization



goes beyond simply counting dead animals or calculating LCso



values and includes clinical observation, identification of target



organs, etc.







Careful consideration was given to inhalation tests and techniques



which assess acute injuries to the lungs and systemic effects.  This



guideline is for acute inhalation studies using rats in dynamic



airflow chambers, and it may be used, with minor changes, for other



species of rodents.
                            88

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      Scientific Issues for Public Comment







     During the development of  this proposed  guideline/ many scientific



issues were discussed by the Work Group.  These  issues were  raised  by



members of the Work Group, by the public  comments  to the  EPA proposed



pesticide guidelines and by the comments  of interagency reviewers.



Consideration of these comments, discussed below/  is reflected  in this



proposed guideline; and the public is  invited to comment  further on



these issues or any other aspect of this  proposed  guideline.







     A.  Although commentors have suggested that inhalation  effects may



not be sex dependent and that either sex  may  be  used or if there is a



sex difference in the response  to the  test substance, the difference



will appear in the acute oral test, the Vtork  Group thinks that  both



males and females should be used in the acute inhalation  test.  Any



comments or information about which sex/  if either,  is more  appropriate



for an acute inhalation study and data showing sex differences  or lack



of differences would be helpful to the Work Group.
     B.   This guideline requires no  further acute  inhalation  testing



if an exposure to 5 mg/1 of the test  substance  for  four hours  produces



no mortality.  Some commmentors pointed out the problems associated
                              89

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with achieving an exposure concentration of  5 mg/1.   The Work Group



recognizes this problem and  in  this guideline has  allowed for the



physical, chemical properties to be a determining  factor in setting the



maximum dose.







     C.   While there are various opinions on the  need  for controls



(vehicle, sham, negative)in  the acute inhalation study,  the Work Group



thinks control groups are unnecessary in this test.   When a solvent of



uncharacterized toxicity is  used, however, an acute  study should be



done on the solvent.







     D.   Temperature and humidity ranges for acute  inhalation



toxicity testing were another issue.  Lower  or  higher temperature



ranges were suggested, as well  as no temperature limits.  The Work Group



decided that temperature and relative humidity  measurements were



necessary and that a temperature of 22C + 2C, with a  relative



humidity of 30% to 50% would be appropriate  for the  conduct of this



test.  One consideration in  this decision was the  fact  that scientific



literature indicates that both  these variables  should be maintained



within relatively narrow ranges, since changes  in  either direction can



alter the toxic responses of the test substance.
                             90

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     D.   In view of the primary  importance of the lungs  in an
                                                            
inhalation study, special, specific treatment of lungs prepratory  to

histopathological examination was recommended. The Work Group

acknowledges the importance of special treatment of the lungs in an

acute inhalation toxicology study, but thinks there are several

acceptable procedures for preserving lung tissue, and decided to allow

for individual investigator judgment on this matter.
                               91

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                      TABLE OF CONTENTS
  I.  General Considerations







      A.  Good Laboratory Practices



      B.  Personnel



      C.  Test Substance



      D.  Animals



      E.  Dead Animals, Necropsy, and Histopathology



      F.  Equipment








 II.  Specific Considerations







      A.  Test Preparation



      B.  Test Procedure







III.  Data Reporting








      A.  Identification



      B.  Body of Report
                        92

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         1.  Summary and Conclusions



         2.  Materials



         3.  Methods



         4.  Results



         5.  References
IV.  Suggested Reading
                        93

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I.   General Considerations







     A.   Good Laboratory Practices







          Basic standards presented here relating  to good  laboratory



practices are to serve as general guidance for  the conduct of  the



study, but are not  intended to be all  inclusive.   This guideline  does



not set forth the managerial aspects of science or good laboratory



practices.  Studies should be conducted according  to "Nonclinical



Laboratory Studies, Good Laboratory Practice Regulations,"  (43 FR



59986, 22 December  1978).







     B.   Personnel







          All testing and evaluation must be done  under the direc-



tion of personnel who have the education, training, and experience



to perform the testing and evaluation  in accordance with sound



scientific experimental procedures.  The agency, commission, or



department may require resumes of personnel who have performed,



supervised, reviewed, or evaluated the testing.  Tb the extent



possible, the same person or persons should perform all observa-



tions and necropsies in a single test  in order to  insure consis-
                             94

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tency of evaluation.  When a histopathological examination  is



done, similar considerations should apply.







     C.   Test Substance  (materials or mixtures of  substances or



materials)







          1.   As far as  is practical/ composition  of  the test



substance must be known,  including the name and quantities of



known contaminants and  impurities.  Unknown materials,  if any,



must be quantified to account for 100% of the test  sample.  The



specific substance to be  tested will be determined  in  consultation



with each agency.







          2.   The lot  of the substance tested should  be the same



throughout the study.   The test sample should be stored under



conditions that maintain  its stability, strength, quality, and



purity from the date of its production until the tests are



complete.







          3.   Safe handling and disposition of the test substance



is essential.







     D.   Animals
                              95

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          1.   Animals used for testing should not have been sub-



jected to any previous experimental procedures.







          2.   The test animal shall be characterized as to species,



strain, sex, weight and/or age.  Each animal must be assigned  an



appropriate identification number.







          3.   Recommendations contained in DHEW pub. no.  (NIH) 74-23,



entitled "Guide for the Care and Use of Laboratory Animals," should be



followed for the care, maintenance, and housing of animals.







          4.   Animals may be group-caged for this test unless pharma-



cological action of the test substance dictates otherwise.  However,



the number of animals per cage should not prevent continued and clear



observation of each animal.  When signs of morbidity or excitability



are observed in group-caged animals during the test, such  animals



should be moved to separate cages.







          5.   Healthy animals must be used. Animals must  be



assigned to groups in such a manner as to minimize bias and assure



comparability of pertinent variables.
                             96

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          6.   Each animal must be observed as necessary  to  insure



that animals are not lost due to cannibalism, autolysis of tissues,



misplacement, and similar management problems.








     E.   Dead Animals, Necropsy, and Histopathology








          When an animal is discovered dead, it must be refriger-



ated at temperatures low enough to minimize autolysis  if  necropsy



cannot be performed immediately.  Necropsy must be performed with-



in 16 hours of death.  When animals are killed for examination,



the necropsy should be performed as soon after death as possible.



If histopathological examination is to be conducted, all  tissue



specimens should be placed in appropriate fixative when they are



taken from the animal.








    F.    Equipment








          All equipment used in conducting the test, including equip-



ment used to prepare and administer the test substance and equipment



used to maintain environmental conditions, must be of appropriate



design and adequate capacity.  Equipment should be inspected, cleaned,



and maintained regularly.  The equipment must be properly calibrated at



the time of its use.
                             97

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II.  Specific Considerations







     A.   Test Preparation







          1.   Animals:  Standard laboratory strains of healthy young,



adult rats weighing between 125 and 250 g should be used, but other



species or younger animals may be required for specific purposes.   For



example, immature animals must be used when attempting to estimate



LCso values that may apply to infants; and these studies should be



performed in addition to the studies on mature animals.







          2.   Number and sex:  The number of animals must be adequate



for analysis.  At least 10 animals, 5 per sex, should be used at each



concentration level.  Also, if sex differences are seen in LC50



values, the study should be repeated using 10 of each sex.   If  a



toxicological effect occurs with a marginally significant incidence,



data from further testing with larger numbers of animals may be



required.








          3.   Females:  Since estrus and pregnancy may modify  female



responses,  the females should be nulliparous and nonpregnant.
                              98

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          4.   Dosage concentration:  To establish a  regimen,  a  trial



test is recommended.  It should  include at  least one  concentration



level higher and one concentration lower than  the expected  LC$Q.



No further testing is necessary  if an exposure of 5 mg/1  (or a maximum



concentration as permitted by the physical/chemical properties of  the



test substance) for the prescribed 4 hour duration administered  to 5



male and 5 female test animals produces no  mortality.   If mortality



occurs, however, an additional test using at least 4  concentration



levels and a negative control group should  be  done.   The doses should



be spaced appropriately to produce test groups with mortality  rates of



1-20%, about50%, and 70-99% permiting the calculation of the IC$Q



value with a 95% confidence interval of +_ 20%  or less.








          5.   Use of solvent:   If necessary to help  generate  an



appropriate concentration of the substance  in  the atmosphere,  a solvent



may be added to the test substance.  If the product's labeling instruc-



tions specify use of a particular solvent,  that solvent is  recommended.



If no solvent is specified in the product's labeling  instructions, the



solvent(s) in the product formulation should be used  if possible.   If  a



vehicle or solvent of uncharacterized toxicity is used  in generating



the exposure atmosphere, an acute inhalation test should be done on the



solvent.
                               99

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          6.   Selection of equipnent:   The  animals should be tested



using  inhalation equipnent designed  to  sustain a dynamic airflow and an



evenly distributed exposure atmosphere.   If  a  chamber is used, its



design should minimize crowding of the  test  animals and maximize their



exposure to the test substance.  References  to examples of accceptable



experimental designs appears  in Section IV,  "Suggested Reading."








     B.   Test Procedure







          1.   The chamber should be maintained at  22C +_ 2, and



the relative humidity should be 30%  to  50%.







          2.   Air flow should be adjusted to  insure that the oxygen



content of exposure atmosphere is at least 19% and  concentrations of



the test substance in the chamber at the outlet and the inlet are



essentially the same.








          3.   Monitoring or measurements shall be  made of:







               (a)  the rate of airflow,  continuously;







               (b)  the actual concentration of the test substance by



sampling chamber air as near as practical to the animals'  breathing
                               100

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zone as frequently as necessary to obtain an average,  integrated



external exposure which is representative of the entire exposure



period.  Concentration and particle size distributions of  the  test



substance in the chamber should be controlled.  During the development



of the generating system, particle size analysis should be carried out



as frequently as necessary to  insure proper stability of aeresol



particles.  During exposure, analysis  should be made as often  as



necessary to determine the consistency of particle distribution (at



least 20% of the particles should be 10 microns or less in diameter).







                (c)  the temperature and humidity, continuously.







          4.   The exposure period  (duration of compound administra-



tion) shall be 4 hours.







          5.   The observation period  must be  at least 14  days.   Dura-



tion of observation should not be fixed; it should be determined  by  the



toxic reactions, rate of onset, and length of  recovery period. Although



a 14-day observation period is sufficient for  most compounds,  animals



demonstrating visible signs of toxicity after  14 days  could  be held



longer.
                                101

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          6.   Recording of clinical observations:  The  animals  should



be observed frequently during the first day and  twice  a  day thereafter



at least 4 hours apart (once each morning and late afternoon).   Obser-



vations should be recorded systematically as they are  made,  and  indi-



vidual records should be maintained for each animal.   Visual observa-



tions should  include, but not be limited to, changes in  skin and fur,



eyes, mucous  membranes and respiratory, cardiovascular,  autonomic and



central nervous sytems, and somato-motor activities.   Particular



attention should be directed to observations for the presence of



tremors, convulsions, salivation, hyperactivity, diarrhea,  lethargy,



sleep, coma,  blanching, cyanosis, and vasodilation.  The time at which



toxicity signs appear and the time of death must be recorded.







          7.   Weight change:  Individual weights of animals must



be determined on the day the test substance is administered,



weekly thereafter, and prior to sacrifice.








          8.   Necropsy:  A complete gross necropsy should  be per-



formed on all animals that die during the course of the  test and all



remaining animals at termination of the test.  Gross pathological



examination should include nasal passage, trachea, bronchi,  lungs,



major organs of detoxification such as liver and kidneys, and any other



tissues known to be affected by the test substance.  All abnormalities
                              102

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must be recorded.   Lungs, liver, kidney, and organs showing evidence



of gross pathology of all animals surviving 12 or more hours should be



preserved for possible future microscopic examination.
                             103

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III. Data Reporting







     A.   Identification







          Each test report must be signed by the person



responsible for the test and identify:







          1.   The laboratory where the test was performed by



name and address;







          2.   The inclusive dates of the test; and







          3.   Each person primarily responsible for separate



components of the test and the component for which the person  is



reponsible including (a) the conduct of the test, (b) analysis of



the data, (c) the writing of the report, and (3) any written or



other matter contained in the report.







     B.   Body of Report,








          The test report must include all information necessary



to provide a complete and accurate description and evaluation  of
                            104

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the test procedures and results.  Each report must include the



following sections:








          1.   Summary and Conclusions.  This section of the test



report should contain a tabular summary of the data, an analysis



of the data, and a statement of the conclusions drawn from the



analysis.  The summary must highlight all positive data or



observations and any other indications of toxic effects.







          2.   Materials.  This section of the test report shall



include, but not be limited to, the following information:







               (a)  Identification of the test substance,



including:







               i.   chemical name, molecular structure, and a



qualitative and quantitative determination of its chemical



composition, including names and quantities of known contaminants



and impurities, so far as is practical; the determinations shall



also include a listing of materials as unknowns, if any, so that



100% of the test sample is acccounted for:
                               105

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               ii.   manufacturer  and  lot  number of the substance



tested, and such  information  as physical  state, pH,  stability, and



purity; and







              iii.   exact  identification  of diluents,  suspending



agents, emulsifiers, or other materials used  in administering the



test substance.







                (b)  Animal data,  including:







                  i. species and strain used and rationale  for



selection of  the  strain if other  than a common  laboratory  strain;








                ii. source of supply  of the animals;







                iii. description of any pre-test conditioning,



including diet;








                iv. description of the method used in



randomization of animals;  and








                 v. numbers of animals of each  sex in  each test



group.
                            106

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                (c)  Data an facilities should include description



of the caging conditions including number of animals per cage,



inhalation chambers, bedding material, ambient temperature,



lighting conditions, and humidity.








          3.    Methods








                (a)  Deviation  from guidelines - This section



shall indicate  all ways in which the test procedure deviates from



these guidelines and shall state the rationale for such



deviation.







                (b)  Specification of test methods - This section



shall include a full description of the experimental design and



procedure, the  length of the study, and the dates on which the



study began and ended.







                (c)  Statistical analysis - All statistical methods



used should be  fully described or identified by reference.







                (d)  Data on dosage administration, including:
                          107

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                 i. all dose levels administered, including all



concentrations of substances expressed as milligrams/liter or
                ii. method and frequency of administration; and








               (e)  Data on observation methods, including:








                 i. duration; and








                ii. method and frequency of observation of the




animals.








               (f)  Data on equipment, including:








                 i. a description of the exposure chamber used



with justification for deviation from the design suggested in this



guideline;








                ii. the rate of airflow through the chamber



(liters per minute);
                          108

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               iii . gas measurements and analysis for the test
compound; and

                iv. the method used in determining particulate
size and the results of the analysis.

          4.   Results

               The tabulation of data and individual results must
accompany each report in sufficient detail to permit independent
evaluation of results.

               (a)  Tabulation of the response data (i.e., number
of animals dying; number of animals showing signs of toxicity;
number of animals exposed) at each exposure level by sex, and time
of death after dosing;

               (b)  1X50 values for each test substance
calculated at the end of the observation period, with method of
caluclation specified;
               (c)  95% confidence  interval  for  the
values;
                          109

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                (d)  Slope of the dose-mortality  curve  for each



substance tested; and







                (e)  Findings from all clinical observations,



necropsy, and histopathological examinations  (when made).








          5.   References







               This section of the test report shall include  the



following information:







                (a)  Availability of original data, spec linens  and



samples of the test substance.  The location of all original  data,



specimens, and samples of the test substances which are retained



in accordance with the testing requirement.







                (b)  Literature or references, including, where



appropriate, those references for (1) test procedures, (2)



statistical and other methods used to analyze the data, (3)



compilation and evaluation of results, and (4) the basis  upon



which conclusions were reached.
                              110

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IV.  Suggested Reading








     1.   Drew, R. R., and S. Laskin.  1973.  Environment inhalation



chambers.  In;  Methods of Animal Experimentation.  W. I. Gay, ed.



Academic Press, New York.  Vol. 4, pp. 1-41.








     2.   Fraser, D. C., R. E. Bales, M. Lippmann, and H. E.



Stockinger.  1959.  Exposure chamber in research in animal inhalation.



Public Health Monograph No. 57, U. S. Public Health Service, Department



of Health, Education and Welfare.







     3.   National Academy of Sciences - National Research Council.



1977.  Inhalation exposure.  In;  Principles and Procedures for



Evaluating the Toxicity of Household Substances, Report No. 1138.



Washington, D. C.  4:60.
                               Ill

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112

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DRAFT I.R.L.G. GUIDELINE FOR




TERATOGENICITY TESTING IN RAT,



MOUSE AND RABBIT
Testing Standards  Guidelines Work Group



INTERAGENCY REGULATORY LIAISON GROUP








May 16, 1979
              113

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                             PREFACE







This guideline is for use with substances given orally to the rat,



mouse, or rabbit.







The purpose of this test is to yield data to help determine



whether a test substance is potentially embryotoxic and/or terato-



genic.  Treatment must be started early enough and continued long



enough to include the period of organogenesis for the particular



species used.
                                  114

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     Scientific Issues for Public Comment








     During the development of this proposed guideline, many  scientific



issues were discussed by the Work Group.  These  issues were raised  by



members of the Work Group, by the public comments  to  the EPA  proposed



pesticide guidelines and by the comments of interagency reviewers.



Consideration of these comments, discussed below,  is  reflected  in this



proposed guideline; and the public is  invited to comment further on



these issues or any other aspect of this proposed  guideline.








    A.  In this proposed guideline, dosing begins  after implantation



and continues through organogenesis up to one day  prior to term, which



could be Day 6 through 19 in the rat,  or Day 7 through 29 in  the rabbit



(depending upon the strain used).  Since the purpose  of this  test is to



determine the teratogenicity of a substance, the Work Group believes



that implantation of the embryo should occur before dosing begins in



order to assure that the dosing will not interfere with implantation.



Also, this guideline  provides that dosing will  occur through most  of



the period of gestation, which will in most species,  go beyond  the



period of organogenesis.








        Comment on the duration of dosing relative to the gestation




period is encouraged.
                                115

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    B.  Some ooramentors suggested that doses should be adjusted peri-
odically throughout pregnancy rather than basing the dosage level on
the dam's weight on the first day of compound administration (Day 6 of
pregnancy in the rat and mouse, and Day 7 in the rabbit).  Because of
the lack of evidence of the transplacental movement of the test
substance, and the uncertainty of escalating the maternal concentra-
tion relative to each dose level, the work group believes that dosage
should be based on the maternal weight just after implantation.

        Additional information regarding the setting of dosage levels
would be helpful.

    C.  Another issue is the proper selection of dosages to be tested.
The highest dose suggested was one that either causes overt maternal
toxicity or affects fetal development.  Because excessive maternal
intoxication may indirectly prevent normal fetal development, care
must be taken in choosing this dose.  Hie main purpose of a teratology
study is to evaluate the potential of a chemical to affect fetal
development, and to produce anomalies in the offspring.  Therefore,
the highest dose should not cause so many fetal deaths that the
assessment of its teratogenic potential is not possible.  Some
commentors have suggested that the maximum dose should be limited to
10,000 times the expected human dose or somehow otherwise limited when
                              116

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testing relatively inert substances.  The Work Group  recognizes  that



human exposure may not be known, constant, or measurable.  Also,  the



Work Group is unaware of a method for defining the maximum amount to



be given, except by either observing toxicity or by the  limit of  the



physical/chemical properties of the substances.







        The public is encouraged to comment on the proper selection  of



dose levels for teratology testing.







    D.  The appropriate number of pregnant animals per group is



another issue.  Government agencies have traditionally requested  that



at least 20 pregnant rats or mice and at least 10 pregnant rabbits be



used in each group.  Recently other governments have  asked for at



least 20 pregnant rabbits.  The National Academy of Science recently



suggested that at least 20 pregnant animals, regardless of specie, be



used in each group.  Although the Work Group supports the use of  20



pregnant rodents per group, it does not endorse the use of 20 pregnant



rabbits per group because of limited supplies of healthy rabbits  and



the costs involved.  The Work Group questions whether the additional



animals produce sufficient data to warrant the difficulties and



suggests the use of 15 pregnant rabbits per group as  a compromise.







        The public is encouraged to comment on the proper number  of
                             117

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pregnant animals per group in teratology studies.  Of particular  value
would be comments on statistical or scientific rationale for determin-
ing the proper number of pregnant animals per group.

    E.  The necessity for positive control groups in a teratology
study has been questioned.  This guideline recommends positive
controls to assure that the strain and species being used  is sensitive
to known teratogens and that those conducting the studies  are thor-
oughly familiar with identification of terata.  Some ccranentors have
suggested that historical data may be sufficient for these purposes.
Others have suggested that positive controls should be used to char-
acterize the strain or species being used and that positive control
studies are necessary only when a laboratory selects a new or
different species or strain for use.

        The Work Group would appreciate receiving comments on ways to
assure that the species being used are characterized as to their
ability to respond to known teratogens and to assure that  individuals
examining offspring have had experience detecting a wide variety  of
terata.
                            118

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                        TABLE OF CONTENTS
  I.  General Considerations







      A.  Good Laboratory Practices



      B.  Personnel



      C.  Test Substance



      D.  Animals



      E.  Dead Animals, Necropsy, and Histopathology



      F.  Equipment







 II.  Specific Considerations







      A.  Test Preparation



      B.  Test Procedure







III.  Data Reporting







      A.  Identification



      B.  Body of Report
                          119

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         1.  Suiroary and Conclusions



         2.  Materials



         3.  Methods



         4.  Results



         5.  References
IV.  Suggested Reading
                         120

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I.   General Considerations







     A.   Good Laboratory Practices







          Basic standards presented here relating  to good  labora-



tory practices are to serve as general guidance  for  the conduct of



the study, but are not  intended  to be all inclusive.  This guide-



line does not set forth the managerial aspects of  science  or good



laboratory practices.   Studies should be conducted according to



"Nonclinical Laboratory Studies, Good Laboratory Practice



Regulations," (43 PR 59986, 22 December  1978).







     B.   Personnel







          All testing and evaluation must be done  under the direc-



tion of personnel who have the education,  training, and experience



to perform the testing  and evaluation in accordance with sound



scientific experimental procedures.  Bie agency, commission, or



department may require  resumes of  personnel who  have  performed,



supervised, reviewed, or  evaluated the testing.  To  the extent



possible, the same person or persons should perform  all observa-



tions and necropsies in a single test in order to  insure consis-



tency of evaluation.  When a histopathological examination is
                               121

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done, similar considerations should apply.

     C.   Test Substance (materials or mixtures of substances or
materials)

          1.   As far as is practical/ composition of the test
substance must be known/ including the name and quantities of
known contaminants and impurities.  Unknown materials/ if any,
must be quantified to account for 100% of the test sample.  The
specific substance to be tested will be determined in consultation
with each agency.

          2.   The lot of the substance tested should be the same
throughout the study.  The test sample should be stored under
conditions that maintain its stability/ strength/ quality/ and
purity from the date of its production until the tests are
complete.

          3.   Safe handling and disposition of the test substance
is essential.

     D.   Animals
                               122

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          1.   Animals  used  for  testing  should  not have been sub-
jected to any previous  experimental  procedures.

          2.   The test animal shall be  characterized  as to
species, strain, sex, weight and/or  age.   Each  animal  must  be
assigned an appropriate identification number.

          3.   Recommendations contained  in DHEW pub.  no. (NIH)
74-23, entitled "Guide  for the Care  and Use of  Laboratory
Animals," should be  followed for the care, maintenance,  and
housing of animals.

          4.   Animals  may be group-caged unless the pharmacolog-
ical action of the test substance dictates otherwise.  However,
the number of animals per cage should not prevent  continued and
clear observation of each animal.  When  signs of morbidity  or
excitability are observed in group-caged  animals during  the test,
such animals should  be  moved to  separate  cages.

          5.   Healthy  animals must  be used. Animals must be
assigned to groups in such a manner  as to minimize bias and assure
comparability of pertinent variables.
                                123

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          6.   Bach animal must be observed as necessary to insure



that animals are not lost due to cannibalism, autolysis of



tissues, misplacement, or similar management problems.







          7.   When control animals are used, they must be housed,



fed, and handled exactly like the test animals; and they must be



caged to minimize airborne or other contamination by the test



substance.







     E.   Dead Animals, Necropsy, and Histopathology







          When an animal is discovered dead, it must be refriger-



ated at temperatures low enough to minimize autolysis if necropsy



cannot be performed immediately.  Necropsy must be performed with-



in 16 hours of death.  When animals are killed for examination,



the necropsy should be performed as soon after death as possible.



If histopatnological examination is to be conducted, all tissue



specimens should be placed in appropriate fixative when they are



taken from the animal.







     F.   Equipment








          All equipment used in conducting the test, including
                                 124

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equipment used to prepare and administer  the  test  substance and



equipment used to maintain environmental  conditions, must be of



appropriate design and adequate capacity.  Equipment should be



inspected, cleaned, and maintained  regularly.  The equipment must



be properly calibrated at the time  of  its  use.
II.  Specific Considerations







     A.   Test Preparation







          1.   Animals:  Strains with low fecundity should not be



used. All test and control animals must be young, mature, pregnant



females of uniform age, size, and parity.  Untreated males of



proven fertility should be used to produce the pregnancies.







          2.   Test groups:  At least three  test groups and one



vehicle control group must be used.  When the test substance is



administered in a vehicle, the vehicle only  should be  administered



to the controls.  If no vehicle is used, then the controls should



be sham treated.  If there are insufficient  data on the toxic



properties of the vehicle used in administering the test  sub-



stance, a sham control group should  also be  included.  In all



other respects, the controls must be handled and maintained  in  a



manner identical to that used with the groups given the test
                                  125

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substance.  For quality assurance, either a positive control group
of at least 5 pregnant animals should be included in every  study
or a positive control group of 20 pregnant animals should be
included in a study at least once a year and when the species or
strain of animals being studied is changed.  Any known teratogen
may be used as the positive control.  Examples include aspirin or
Vitamin A for rats, 6 Amino nicotinamide for rabbits, and
corticosteroids for mice.

          3.   Number of animals:  Sufficient numbers of anijnals
must be bred to assure that each test group and the vehicle
control group will consist of at least 20 pregnant rats or  mice,
or at least 15 pregnant rabbits.  These are the minimum numbers of
pregnant animals at or near term.  The objective is to assure that
sufficient pups are produced to permit evaluation of the terato-
genic potential of the substance.  As mentioned above, the  posi-
tive control groups should routinely consist of at least 5
pregnant animals.

     B.   Test Procedure

          1.   Duration of test and time of delivery:  Day  0 is
defined as the day a vaginal plug and/or sperm are found.   The test
substance should be administered daily beginning soon after
implantation (Day 6 for rats or mice, Day 7 for rabbits) and con-
tinuing through most of the gestation period until about one day

                              126

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prior to term.  Thus,  the  treatment period for the rat would be
Day 6 through Day 19 of pregnancy;  for the rabbit it would be Day
7 through 29 of pregnancy;  and  in the mouse,  the period would be
Day 6 until one day before expected delivery.  In the mouse, the
period of gestation varies with the strain used.

               For  substances that cause enzyme induction,  or are
highly toxic, shorter  dosage periods may be appropriate.

               In all  cases, fetuses shall be delivered by
hysterotony about one  day  prior to term.

          2.   Dosage:  At least three dosage levels must be
tested in addition  to  the  controls.   Unless limited  by the
physical/chemical nature,  or biological effects of the compound,
the highest dosage  level should induce overt  maternal toxicity or
affect fetal development.   Maternal toxicity  should  not be  so
great as to compromise the  integrity of tlie study or obscure
meaning of the malformations.   The intermediate dose(s)  should
induce some observable fetal effects attributable to the test
substance.  The low dosage level should not induce observable
adverse effects attributable to the test substance.   The dosage
administered should be based on the individual animal's body
weight on the first day of  substance administration.
                               127

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          3.   Route of Administration:  The test substance or



vehicle should be administered by oral intubation unless the



chemical or physical characteristics, or pattern of human exposure



to the test substance suggest a more appropriate route of admin-



istration.  The test substance should be administered at approxi-



mately the same time each day.







          4.   Animal care:  Pood and water should be provided ad



libitum.  Pregnant females may be provided nesting materials,



although it is not considered necessary.







          5.   Observation:  Throughout the test period, each



animal must be observed at least once daily, by an appropriately



trained observer.  Pertinent behavioral changes, and all signs of



toxicity, including mortality, must be recorded.  Any female



showing signs of abortion or premature delivery must be sacrificed



on the data such signs are observed.  These observations should be



reported individually.  Females should be weighed at the start of



substance administration (Day 6 or 7), at the time of sacrifice,



and at least weekly between these times.








          6.   Necropsy:  Immediately after a female is sacri-



ficed, the uterus should be excised and examined for embryonic or
                           128

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fetal deaths and the lumber of live fetuses.  When possible,  the



tune of death in utero should be established.  The fetuses  should



be examined externally, weighed individually, and the weights



recorded.  The sex of each fetus should  be determined if possible.



In rodents about one half of each  litter should be eviscerated,



prepared, and examined for skeletal anomalies using  the method of



Staples  (11) or equivalent.  The remaining one half  of each litter



should be prepared and examined for soft tissue anomalies,  using



the method of Wilson (15) or equivalent. In rabbits, all fetuses



should be examined by gross dissection for soft tissue anomalies



and subsequently processed  for skeletal examinations.







          7.   Statistical Analysis:  Values from the control and



test groups should be compared statistically.  Any of several



methods  are acceptable.  The following are suggested:  Anomalies



may be compared by chi-square methods or the binomial expansion



method.  Maternal body weight gains and  weight of fetuses may be



compared to those of controls by F-test  and Student's t-test.



Petal survival and incidence of abnormalities per litter may  be



compared by nonparametric, rank-order methods.  Other statistical



methods may be substituted.
                            129

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III. Test Report







     A.   Identification







          Each test report must identify:







          1.   The laboratory where the test was performed by



name and address;







          2.   The inclusive dates of the test; and







          3.   Each person primarily responsible for separate



components of the test and the component for which the person is



reponsible including (a) the conduct of the test, (b) analysis of



the data, (c) the writing of the report, and (3) any written or



other matter contained in the report.







     B.   Body of Report







          The test report must include all information necessary



to provide a complete and accurate description and evaluation of



the test procedures and results.  Each report must include the



following sections:







          1.   Summary and Conclusions.  This section of the test
                           130

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report should contain a tabular  summary of the data,  an analysis



of the data, and a statement of  the conclusions drawn from  the



analysis.  The summary must highlight  all  positive data or



observations and any deviations  from control data  which may be



indicative of toxic effects.







          2.   Materials.  This  section of the test report  shall



include, but not be limited to,  the following  information:







               (a)  Identification of  the  test substance,



including:







               i.   chemical name, molecular structure,  and  a



qualitative and quantitative determination of  its  chemical



composition, including names and quantities of known  contaminants



and impurities, so far as is practical; the determinations shall



also include quantitites of unknown materials, if  any,  so that



100% of the test sample is accounted for:







              ii.   manufacturer and lot number of the  substance



tested, and such information as  physical state, pH, stability, and



purity; and
                            131

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             iii.   exact identification of diluents, suspending
agents, emulsifiers, or other materials used in administering the
test substance.

               (b)  Animal data, including:

                 i. species and strain used and rationale for
selection of the strain if other than a common laboratory strain;

                ii. source of supply of the animals;

               iii. description of any pre-test conditioning,
including diet;

                iv. description of the method used in
randomization of animals to test or control groups; and

                 v. numbers of animals of each sex in each test
and control group.

                vi. parity

               (c)  Data on facilities should include description
                         132

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of the caging conditions  including  number of  animals  per cage,
bedding material, ambient temperature,  humidity,  and  lighting
conditions.

           3.   Methods

               (a)   Deviation  from  guidelines - This  section
shall indicate all ways in which  the  test procedure deviates  from
these guidelines and shall state  the  rationale for such
deviation.

               (b)   Specification of  test methods - This section
shall include a full description  of the experimental  design and
procedure, the length of  the study, and the dates on  which the
study began and ended.

               (c)   Statistical analysis  - All statistical methods
used should be fully described or identified  by reference.

               (d)   Data  on dosage  administration, including:

                 i.  all dose levels administered, expressed as
mg/kg of body weight;
                               133

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                ii. method and frequency of administration; and

               iii. total volume of substance (i.e., test
substance plus vehicle) contained in individual dosages.

               (e)  Data on obsevation methods, including:

                 i. duration; and

                ii. method and frequency of observation of the
animals.

          4.   Results

               The tabulation of data and individual results must
accompany each report in sufficient detail to permit independent
evaluation of results.

               (a)  Data on dose levels including the number of
animals initially on study, number and percentage that were
pregnant, number and percentage that died, and the average*
maternal body weights and all weight changes.
*  All averages should be accompanied by an appropriate measure  of
   variability.
                               134

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               (b)  Maternal data  for each animal  should  include
the following information arranged by group.

                    i.  clinical signs of toxicity:  a description
of all observed signs of toxicity  accompanied by each animal's
identification number, test group  and (i.e., day of pregnancy) of
observation,

                    ii.  age (or weight) at the start of the
test,

                    iii.  body weights on the first day of admin-
istration, at sacrifice, and at least once near mid-gestation; the
body weight change  based on the carcass weight, i.e., body less
the uterus and its  contents; and

                    iv.  signs of  resorptions, abortion, or
premature delivery.

                    (c)  Fetal data:  The following information
arranged by test group should be supplied.

                    i.   cumulative data, showing  mean and
                             135

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variability for each dose level:  number of litters examined,



number of implantations per litter, average number of live pups



per litter, total of dead fetuses per litter, total number of



fetuses, number and percent of pups with anomalies, skeletal vs.



visceral anomalies, number and percent of litters containing



anomalous pups, and number and percent of abnormal pups par



litter.







                   ii.   numerical data for each litter including:



identification number of each dam and/or its litter; number of



implantations; weight, number and percent of dead fetuses; number



and percent of live pups; average weight of live pups per litter;



when determined, number of each sex and percent of male pups;



number and percent of pups with any abnormality.








                  iii.   anomaly data for each litter including:



identification number of the litter; number of pups examined;



number of pups having anomalies; and number of pups having



visceral anomalies.  When an anomaly is difficult to describe,
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color photographs of anomalies may be submitted.  If photographs
are taken, the equipment and film must be of sufficient quality to
permit controlled, close-up color photography of the anomaly to
yield clear, sharp-focus images that literally fill the camera
field.

                    (d)  Evaluation of the results should
include:

                    i.   an evaluation of the relationship, if
any, between exposure to the test substance and the anomalies,
and

                   ii.   an indication of the dosage level at
which no toxic effects attributable to the test substance
appeared.

           5.   References

               This section of the test report shall include the
following information:

               (a)  Availability of original data, specimens and
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samples of the test substance.  The location of all original data/



specimens, and samples of the test substances which are retained



in accordance with the testing requirement.







               (b)  Literature or references, including, where



appropriate, those references for (1) test procedures, (2)



statistical and other methods used to analyze the data, (3)



compilation and evaluation of results, and (4) the basis upon



which conclusions were reached.







IV.  Suggested Reading







     1.   Asling, C. W.  1969.  Nutrition and teratogenesis.



Irj: Methods for Teratological Studies in Experimental Animals and



Man.  H. Nishimara, J.R. Miller, and M. Vasuda, eds.  Igaku-Shoin



Ltd.:  Tokyo,  pp. 76-92.








     2.   Collins, T. F. X. and E. V. Collins.  1976.  Current



methodology in teratogenicity research.  In; Advances in Modern



Toxicology. M. A. Mehlman (NIH), Vol. 1, Part 1, New Concepts  in



Safety Evaluation, M. A. Mehlman, R. Shapiro, and H. Blumenthal.



Chapt. 6.  Hemisphere Pub. Corp., Washington, D. C.  pp. 144-174.








     3.   Perm. V. H.  1967.  The use of the golden hamster  in
                                 138

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experimental teratology.  Lab. Animal Care, 17:452.







     4.   Health Protection Branch.  1977.  The Testing of



Chemicals for Carcinogenicity, Mutagenicity and Teratogenicity.



Ministry of Health and Welfare:  Canada.







     5.   Oser, B. L.  1971.  Toxicology of pesticides to



establish proof of safety. In;  Pesticides in the Environment.



Vol. 1, Part II, pp. 411-456.  R.  White-Stevens, ed.   Marcel



Dekker, Inc.:  New York.







     6.   Palludan, B.  1966.  Swine in teratological studies.



In;  Swine in Biochemical Research.  L. K. Bustad and R. 0.



McClellan, eds.  Battelle Mem. Inst.:  Richland, Washington,  pp.



51-68.







     7.   Palmer, A. K.  1974.  Problems associated with the



screening of drugs for possible teratogenic activity.  Exp.



Embryol. and Teratol. 1:16-33.







     8.   Robson, J. M.  1970.  Testing drugs for teratogenicity



and their effects on fertility.  Brig. Med. Bull. 26:  212-216.
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     9.   Bassoon, H.  1976.  Survey and Evaluation of Techniques



Used in Testing Chemical Substances for Teratogaiic Effects.  EPA



Report No. 560-5-77-007, NTIS Pub.  PB-273-195.  National



Technical Information Service:  Springfield, Va.







    10.   Shepard, T. H., J. R. Miller and M. Morris.  1975.



Methods for Detecting of Environmental Agents that Produce



Congenital Defects.  Proceedings of the Gaudeloupe Conference



Sponsored by 1'Institut de la Vie.  North-Holland Publishing Col:



Amsterdam-Oxford:  American Elsevier.







    11.   Staples, R. E. and Schnell, V. L.  1964.  Refinements  in



Rapid Clearing Technic in the KOH-Alizarin Red S Methods for Petal



Bone.  Stain Tech. 29:61-63.







    12.   Tachmann Deplessis, G.  1972.  Teratogenic drug



screening.  Present procedures and requirements.  Teratology.



5:271-285.







    13.   Weil, C. S.  1970.  Selection of the valid number of



sampling units and a consideration of their combination in



toxicological studies involving reproduction, teratogenesis or



carcinogenesis.  Food Cosmet. Toxicol.  8:177-182.
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    14.   Wilson, J. G.  1971.  Use of rhesus monkeys in



teratological studies.  Pad. Proc.  Am. Soc. Exp. Biol.



30:104-109.







    15.   Wilson, J. G.  1973.  Environmental Birth Defects.



Academic Press Inc.  New York.







    16.   World Health Organization.   1967.  Principles for the



testing of drugs for teratogenicity.   WHO Tech. Rep. Ser. No. 364.



Geneva.
                                    U.S. GOVERNMENT PRINTING OFFICE ! 1979 0-300-043/6416
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