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i i ‘4 EPA 540/09—84-014 T014c $jttsncei----——- fA Pesticide Assessment Gud&ines Subdivision

F HAZARD EW\LUAT ION HUfN AND ANIMALS DOMESTIC fEPROOUCEO
BY U.S. DEPARTMENT
OF COMMERCE NATIONAL TECHNICAL INFOPMATION SERVICE SPRINGFIELD
VA. 22161r._ it DDUUENTAT1O4

I REOfi1 NO. JE

,... PAGE

j PBSG—108958

j 4. and IJO4M Pesticide Assesint ciide1ines, Sulxuivision

F Hazard Evaluation Iftmn

& Dcnstic Anirnis- Rcvised Edition 7. A4toc(s) BmceR. aecr,. Pr1a4-11kt( OrItWKttO4 Nryq and A.3dea’$ U. S. IDTAL PRYflX’rION AGI3CY opp/F (H75OC) 401 M St..

, S.W. WASHE’UION, D.C. 20460 OfSf1Z*IOfl

. Guidelines (Pevisions) 14. 1 Abstra-1 (limit 2 worcts) Pevisicri of acute oral, dermal and inhalation toxicity guidelines
of the original guidelines piblication (cctoxr 1q82). These revisions
to the acute toxicity guidelines phasiz. EPA’s philoso±iy effort. to riünirnize use of test anirals in deter.Lning huiran safety, ..tiile similarly supxrtinq research
in test nticxs ich hoxfuil will lead to further rcxuction
or elirination
of riLT1 sacrifice
or suffering. 17. Docum.nt AnaIys

• Descntnt b ld..itef..’i/Open Ended Tem C COSATI F.’dfC.tup ii. Avalabily Stat.”er’. Uncassfc. (S... ANSi-ZJ 11>

I S.•cv.-ft Class fTPi. R.r.orl) P0 S.cu’ty CI•ts (TP. P41 T’;—l S.. •.itrvc1Io’.i

o’ •‘•‘•‘e

21. No & : 167 22. P’c. OPTO4AL FO 7Z (—‘ (FOfI%4’Iy NTIS—)) D.p.f1m.e

O ,n. to)

•— 0.le Ncvcrber 1984 1. I. P.i 4 onium O•4Lr(Io .Q4. Po

• 54C/09-84-ON 10. PtfTe*IIW

N• 11. Cr.CtCC)

cc Gt•i*(G No (C) (C) Saiias#9 15. Supp4erner..ry No(es Supersede Subdivision

F PB83-153916

1).
o4 R.4
4. P.:nod Co.er,dEPA540/09—34—014.:..:•.,.PESTICIiZASSESSZ€N’IGUIDELINESSUBDIVSIO?1FRAZARDEVLLUATION:nmcANDDO!€STICANttSREVISEDEDITIONbyOfficeofPesticiaeProgramsGuidelinesProjectManagerBruceP.JaegerHazardEvaluationDivisionOfficeofPesticidePrograzs1,.s.EnvironmentalProtectionagencyOfficeofPesticideandToxicSubsta4icesWashington,D.C.20460.11/I’Foreword Toxicity data reLated
to hurtan and dcziiestic animal safety eValuation are ruired
to support pesticide registration under the Fera1 Insecticide, Fungicide, and Rodenticide Act (FIFRA). Sibdivision

F describes study protocoLs which may be used to develop these toxicity data. Public cment on Subdivision

F was accepted in a series of public uieetings, the last of ich was held in July 1982. ‘3ecause of the importance

of the issues raised in the Subdivision

F ccuiments, Sthd.iision

F ws redrafted witii the help of a reicw cm.ittee ccrnprisel
of representativ fr indusy, environmental oups and acadeaia. This guideline refers to ruiresnts established

in 40 CFR Part 158 so that this document can read as a cplete package ar so that the protocols ay be explained
in their proper context.—— /1

I’7BDIVISIQN

F

: HIZAJD EVALUATiON: HUMANS AND DOST IC AN IJ4ALS Ta.b,le of Contents pçusq I
. Organiza Lion aM I4iil csophy of Subdivision

F

1 tI. The Stamards for A.cceptable Tes tinq

3 111

• Discussion
of Individual Tea ts

12 k. Acute Oral, DeraI

, and Irthalat ion ‘oxici ty

12 B. Irri tation and Sens
i tiza tion Studi

.

13 C. Netrcotoxicity Evaluations

13 D. Subth.ronic Studies

‘

.

. .

.
14 z• Chronic and Oncogenici
ty Studies

15 F. Teratogenicity
and Reproduction Studies.

16 G. Mutaenicit! Studies

17 . Special Testing

18 GUIDELINES Series 80 Overview, Definition, and General Requiranents 80.-i Overview

20 80—2 Definitior

20 80—3 General Provisions

21 80-4 Reporting
of Data

28 805 C=hined Te.s ti ng

32 ries
81 acute Toxicity and Irritation Studi 81—1 Acute Oral Toxicity Study

34 81—2 Acute Derrnal Toxicity Study

39 81-3 Acute Inhalation Toxicity Study

44 81 -4 Primary Eye Irri tatio
n Study

51 81-5 PriliLary Dermal Irritation Study

55 816 Deratal Sensitization Study

59 81—7 Acute Delayed Neuerotoxici

ty of cnophc€phonis Substances

62 Eies 82 Subchronic Testing 82—1 Subthronic Oral Tox.ici ty

( Roden

: aid Non-rodeat): 90—Day Study

66 iTable of Contents (Continued) GUIDELINES 82—2 Repeati Dose Dexmal Toxicity: 21-Day Study

76 823 Subchronic Deruiai Toxici ty: 90-Day Study

83 82-4 Subdironic Irth&lation Th:jcity: 9ODay Study

92 82—5 Subchronic Neurotoxicity: 90—Day Study

103 Series 83 ironic ar3 Lor Term Studies 83—1 Chronic Toxicity Studies

107 83—2 Oncogenicity Study

117 83—3 Teratonicity Study

125 83—4 Reproductive and Fertility Effects

130 83—5 Combined Chronic Toxici ty/0nconici

cy Studies

137 ‘ Series 84 Mutagenicity 4

84—

1 Purpose and Generai Recanmenda tions fo r Mutagenici
ty : Testing

147 84-2 Mutagenicity Tests

, Series 85 Special Studies 85—1 Metabolism study.. 152 852 Dczrtestic Animal Safety Testing

156 85—3 Dermai. Absorption Studies of Pesticides (Reserved) ii

IEPA 540/09—84—014 varber 1984 ‘ Pesticide Assessment Guidelines . Subdivision

F t
REVISEO ID1TIc Hazard Evaluation: Human and Domestic Animals Prepared by Office of Pesticide Programs Guidelines Coordinator 13n]ce

R

. Jaeger Hazard Evaluation Division Office of Pesticide Pr.ograms U.S. Environmental Protection Agency Office of Pesticides and Toxic SuL’stan Washington, D.C. 2046016 of n—oiicogenic chronic effects (see

§ 83-1), The nori-roden, usually the dog .ay zneta.bolize the test substance differently fran the rodent, and therefore the two species, together, stay r?veal

a much broader range f toxic effects than if only one species were tusted. (See, gnera.11y, Goidenthal,
E., “Current Views on Safety Evaluation

of Dnigs,” FD1 Papers, 13 (May, 1968); Fit.zhui, O.G., 1965, “Appraisal
on the safey f chemicals in foods, drugs, ar cosmetics— chronic oral toxicity,” pp. ‘6, 38. As soc

• Food

& Drug Of fic

• of the U. S

. , pe, Kansas.] rp; believes that conducting oncogenic arid chronic feeding studies for preeterriiine long-term periods
is valuable fo several reasons

• EPA thinks that these tire periods are long enough t ‘.low turrrs and other chronic effects
to velop, and ye t sho rt enough to as sur e tha t a reaso nab lt. percencage
of the animals wil

1 s urvive to the

s th eduled te rmina tion point. A high level of survival would provide the cperimenter with

a larger data base on .th ith to perfo rm
s ta tis tical analys is

• In ad di t ion

, s ince geri
a tric diseases often make diagnosis
of neoplàstic cowth difficult, the sPudies would be desicied
to produce meaningful histology
by terminating the studies before suc* diseases noxmally becane

a significant problem.
By ‘ising

a relatively imiforta duration, EPA would also be better able to cpare the rults of different studies ar to cpare results with the historical data base. Finally, the principles establish

a fairly definite endpoint for thronic

S tudies wh ich would a]. low expe rimenters

to plan the ir
s tudies more efficien t.ly. F. Teratogenicity
and ReproductionStudies; 83-’3,.4. Two studies in the chronic/long-term series are desied

to evalte the effects of a pesticide
on the process of reproduction. Data trcrn

a Teratogenicity Study, 83—3, indicate whether expure

to a pesticide dur ing preq-ancy can cause fetotoxicity

or birth defects
in offspring. The Reproduction/Fertillty Study, 83-4, involves expcsure

to a pesticide and is desiqne
to assess mtre subtle effects on rroduction, such as decreased fertility, prematire delivery, and smaller offspring. Commenters have sugsted that, in adition

to the Teratogenicity Study in

§ 83—3, tests on behavioral and central nervous system (CNS) defects may be useful. Evaluation

of defects
in the CNS would have to be done in Utters during the postnatal period. The need for pcetnatal teratological evaluation arises mainly because the CNS does not mature fully in the human fetus and may still be liable to certain teratogenic influences

in late pregnancy. The studies would involve treating pregnant female animals during the latter third of gestation, observing the neonate throughout sexual isabirity, and perhaps perfonning objective tests on these animals, if appropriate. Cments are encouraged

on the ctharacteristics

of a suitable tt species, number of test animals, dce levels, duration
of neonate observation, and tests to be perforrn
on hese neonates. The princip

s for the reproduction study represent

a signficant depar— ture from traditional testing procedures. Instead of a three-generation 1617 study with two litters in each generation, E!’k proves

a two—generation study with only one litter in each genera’ion. The Agery considers that this methodology

is generally re sensitive thaxi the traditional test design.
In addition, this ethodo1oqy

is considLably le3s expensive than the traditional test design because fewer aninials are reçuired. A traditional reproduction study requires two litters per generation because the first litters produced by adolescent siothers often show

a great deal of variation. Secause of these variations, data baeed on the first litters are less useful than data from the sec,nd set of litters, which are more uniform n size and health. The current guidelines recocnsend only one litter, but the animals would not be bred until they had fully matured. Consequently, under the current principles, the first litter would be less likely to show the variability
of the first litters produced by juvenile mothez. in a traditions]. study. In adcdtion
to reducing the ni.ber of litters per generation, this guideline would reduce the number of generations. Part of the reason : reproduction studies traditionally have tested three generations

is to detect genetic abnormalities. Under the Subdivision

F guidelines, however, the potential
of a pesticide
to cause genetic damage uld be assessed
in a battery of znutagenicity tests which are collectively more sensitive than than
a three—generation reproduction study. (See also part III. C. of this Discussion.

) The other reason for extending

a reproduction study into the third generation

is to detect cumulative effects. The Agency, however,
is not aware of any toxic effect which first appeared
in the third generation of a reproduction study. While the current guidelines reduce the number of generations,

it would extend the perIod of dosing for each litter—producing animal. Accordingly, EPA believes this test design to be more sensitive for cumulative effects than the traditional three-generation study. G. Mutagenicitt4ies

84—1 and—2. Data from mutagencity tests are used for several purposes. Data are used directly
to determine
if a test substance

is genotoxic, that is, capable of interacting with or damaging genetic material (DNA) and/or mechanisms,

or mutaaenic, and hence capable of producing heritable genetic changes. Ti 1 Agency recognizes that

a mutagen causing

a heritable defect in humans has not yet been id: tified, however the desiqn and conduct of an epidemiologic study to veri.fy such

a relatively rare event is difficult. Since mutations
do occ in humans, the Agency feels that it needs to collect information

in experimental test systems in order to make reason— able decisions regarding risks associated with chemicals which nay be capable of inducing human genetir alteration. Mutagencity data nay also be used as an indication
of the oncogenic potential
of a pesticide. 17130 (ii) Species and st-rain; (ii) Time of death during the study or tiether aniEna1 sivived
to termination; (iii) Time of observation

of eth abnormal sign and its subseqnt course; (iv) Food ard body weight data; (v) Pregancy ar1 Utter data; and (vi) Fetal data (live/dead, sex, soft tissi. az skeletal de.fects, resorptions).

2 4 83—4 Reproductiveandferti1itffects. C a ) i en reqi ired

• Da ta on a tMo- nera tion
r e product ion

s tudy

e reiir& by 40 CFR Part 158 to supprt the rsistration

of each manufacturing—use product and form1ated product the t mets ei ther of the foflowii
on e.ria

: (1) The esticida1 use of a sticide roduct
is likely to resu.1t in S igi f icant human exp ure to the prthi, its act ive
i nedien
t( a)

, metabolite(s),

or degr&ation product(s);
or‘ ( 2
) The
u se r equi.r es a to lera nce for the çs t ic i de or an ezemp tic n fran the rouirerent

to obtain

a ,1erance,
or requires is8uance of a food additive requlation. (3) See, specificaI1,

40 CFR 158.50 and 1S8.135
to cterm1ne 4iet.her these data nust be subzitted. Section 11-A of this subdivision con ta ir
a n &:i di tio na]. di. scu
s s Lo n of the Fo i1a to ra

‘ Exemp

t So n” &nd who ozus t sulznit the requir

ii La as a ner a]. rule. (b) Purpose. iii guideline for tioneration reproduction teting is desicied
to praide cynera.1 inforn.ation concerning the effects of a tt substance
on gon&a1 fction, estrus cycles, mating behavior, conception, parturi io, La cta t So, an

i

, a rid th e owth

a nd developmen

t of the offsring. The study ttay also prc.’ide information abcxit the effects of the test suttance
on neonatal arbidity, rtality, and preliminary cta on teragenis
ar may serve as a guide for s’bseqnt tests. (C) Principle of_est nethod. Th test sutance

is ainistered to parental(P 1 ) ania1s xior to Lher mating, during the resultant ice— na.nci es

, a nd thr ou the we aning of the Sr F ef fs r ing

. Th e su± tance is then ainistered

to selected
F offsprirq during their growth into adult hood, tating, and production

of an F 2 generation,

up xitil the F 2 generation is 21 days old.

_*1 I

JI131Cd)Subtancetobetested.Testingshallbeperfortnedwithtedinica.1gradeofehactive£ngrientintheproduct.(e)Testprocedures:(1)Mi.rna1selection.(i)peciesandstrain.Theratorthemousearethepreferredspecies.Ifanothermammalianspeciesisused,thetestershouldprovidejustifcation/reaningforitsselection.Strairwithlowfecundityshouldnotbeused.(ii)Parental(P1)&nimalsshouldIabout8weeksoldattheStartofdoeing.SeecposurecorzlitiorinparagraphCc)(4)ofthissectionfordirregin.(iii)!.!•(A)Foran&equateassessmentoffertility,bothtalesandfalesmust*studied.(B)Thefemalesshouldmlliparousandnon—prnant.(iv)Eadtestarcontroloup.shouldcontainatleast20salesardasufficientnumberoffeaialestoyieldatleast20pregnantfaiia.lesatornearterm.(2)Controlrgs.(i)Aconcurrentcontrolisrecrnended.ThiSshouldanmtreatedorshamtreatedcoitrolouporifavehicleisusedineministeringthetestsubstance,a7ehiclecontrolgroip.(ii)Ifavehicleisusedin&ministerirgthetestsubstance,thecontroloupshouldreceivethevehicLeinthehighestvolunused.(Ui)Ifavehicleorotheradit1veisusedtofacilitatedcinq,itshouldnotinterferewithabrptioricithetestsu1tanceorproducetoxicaffeCts.(3)poselevelsanddosese1ect:on.(i)Atleastthreedelevelsaraconcurrentcontrolshouldbeused.(ii)Thehighestdoselevelshouldirxhicetx,cicitybutnotmortalityintheparental(P1)animals.(iii)Thelowestdoselevelshouldnotproduceanyevidenceoftoxi—city.Wherethereisusableestimatioiofhumanexpwethelowestdoseshouldexceedthis.(iv)Theintermediatedoselevel(s)s,ulaproduceminimalobservabletoriceffects.If,rethanoneintermediatedoseisused,doselevelsst’uldbesecedtoproduceaqriationoftoxiceffects.(v)Theincicnceoffatalitiesinlowandintermediatedosegroupsandinthecontrolsshouldlow,topermitmeanirgfuievaluationoftheresults.1)I j•1 132 (4) Extosure conditions. The animals are dced with the test substance ia11y on a seven-days per week basis using the testing sthedule presented in Table 3. ci) T&ble

4 contains the dirig, mating, delivery, and sacrifice B di edule for ania1s on test. (A) Daily dcing of the parental (P 1 ) males ar females should begin ien they are about

8 eks old. For both sexes, ding should be continued for a leaGt eight weeks before the eating period.

I (B) Dcir of P 1 mal should contin throu the thating period. At the erzi of the ma ting pe nod

, P males should be sac rific ed and exa.min. Dcsing of the i Dales saved for mating should continue from the time they are weaned throu the period they are mated with the F females (11 weeks for mice and 17 weeks for rats).
F males may be sacrificed after the F 1 mating period. (C) Daily dosing of the P females should continue through the three week ma ting pe

, pr &nancy, aM to the time of we aning thr ee weeks after delivery. Dcir of the F fales saved for mating should continue frc the time they are weanai

, thr ough the je nod they are ma ted wi th the i males (11 weeks for mice and 17 weeks for rats), thro4i jreancy, ar to the weaning of the F 2 offsring. (ii) All animals ar sacrificed

as follows: (A) All Pi males should be sacrificed immediately after delivery of the litter last sired or, in cases of infertility, after the coritioris
of (7)(i)(B) are rtt. (B) F 1 males selected for mating should be sacrificed immediately after livery of the last F 1 litter sired, or, in cases of infertility, after the conditions

of (7)(i)(B) are met. (C) F 1 males az females not selectel for mating should be sacrific1 en weane. CD) The 4renta.l females in all nerations should be sacrific&1 upn weani r of the ir las
t ii t ters. (E) F 2 offsing are sacrifice.i *en the offsing are 21 days of age. (5) Observationperiod. Duration
of obeervation should be for at least 28 weeks frc dcirq of P 1 animals
to sacrifice
of F 2 offspring
at weaning. (6) Adrdnistration of_the test substance. The oral. route of adutinis— t ra io n is LX .f e rr Ei.

1.36,.

jWeeks on Sudv

113 0 D4Deing of P 1 a1es and females begins 8—14
P 1 mating period 11-17 Dosing of P 1 males may er at Week 25; p 1 males are sacrificed

• 14-20 Ding of P 1 females end.. P 1 feaales are sacrif icel

Li born ai Utter sizes randcily aduste
to 8 pups e1 i dcaing of F 1 fa1es begins F 1 offspring not sel.cted for mating are sacrificed 1 mating; Dcir of 1 males may end at Week 40; ? males are sacrif iced’s Ra in 1.nc; F 1 females

F 2 born and are sacrificed litter sizes ra ndc.ly adjusted
to to 8 pups each F 2 offsring are sacrf iced ‘The lor rai.nber in the ranges is cormidered apropriate for mice, and the upper number is appropriate for studies using rats. Earliest breeding ac for mice is approximately

8 weeks and that for rats is 14 weekg. This scthedule
is for studi.es usir one mating per cneration. Numbers should bead j us ted by ap pr opr ia te numbers of eks to ac c te the se cond ma ti ng, gestation and weaning of offspring
as well as rest period tween matings. •‘Thjs is appropriate for studies with two matings r generation
or the time may be lonr in order that paragraph (7)(i)(A) can satisfied. TABLE

4 • APPROXD1AL’E DOSING AN D BREWING ScI!ULE P 1

Fl 22—34 25—37

:-• -—. UI

.1134 ci) en administer

by gavage or capsule, the doeage ministered to ead &nimal prior to iating should be based on the individual animals body ight and adjusted weekly. arin’j pregnancy the dcage should based on the body it at Day
0 and
6 of preancy. (ii) It is recantnended that the test substance
be aiinistere

in the diet or drinking water. (iii) If the test substance

is &thninistered

in the clrin.king water, or mixed in the diet, expceure
is continuous. (iv) For
a diet mixture, the highest concentraticn should not ccee five rcent with the exception
of nutrient. (7) Matingprocedure.

(i) Parental. (A) For each tztating, each fenale should be placed with
a single rarcxaly select1 male from the sane dosei.evel until pregnancy occurs or three weeks ?ave elapsed. Pair3 atinqs should be clearly identified and ix ttings with other males avoided.

( B) Those pairs that fail to ma te successful
ly shculd be evalua ted to termine the cause of the apparent infertility. This may involve such procedur
es as ad ii tio sal pz rtu.n

i ties to ma te wi th o the r a ir es o r dams

, histological examination

of the reproductive orçns

, and examination

of the trus
o r s e rma ge n ic cyc lea. (C) Each morning the female should
) am.ined for presence
of srm or vaginal plugs. Day
0 of regiancy
is defined as the day vaginal plugs or sxm are found. (ii) cross. (A) For mating the F 1 offsing, one nale &nd one faa.le are randauly selected frc each litter for crcs mating to produce the F 2 generation. Mating of siblings should be avoided. (B) F 1 males and females not selected for matir are sacrificed un weaning. (iii) Spcial housing. Near parturition, pregnant animals should be caged separately

in delivery or aiaternity cages and provided with nesting maqrials. (iv) Standardization

o.. _;.ttersizes.

(A) On Day
4 after birth, the size of each litter should be djusted by eliminating extra pups by randzn selection yield, as nearly
s x,ssible,
at least four males and four faale,s er litter. (B) enever the number of male or female pups ents having at leat four of each sex r Utter, partial adjustment (for exanpie, five males ar three females)
is permitted. Adust.ments are not appropriate for litters of less than eight pups.

I ij.:.- --
- —---- —---

- 135 (C ) Mus ments ,f the ?2 U tters

i a co riducted in the
s azne manner. (8) Observation

of &nimais. (i)
A careful camination for clinical signs of toxicity should be niac at Least once each day. Pertinent bebavi— a.z:al thazqes, ai.gns of difficult
or oioried partur

i tio

, food corisption and all signs of toxicity, £nclucling rta]J.ty, eboiild be recorded. These oera tiorts should be rorted for eh ixividuaJ. animal. (ii) The dura tion of gee tation should be calcula ted fr Day
0 of pr;nancy.

( iii) F:at:ti ii tte.r should be ecamii
as aoon as s sib 1. a! ter de Uv— •Z7 for the number of pups

, stillbirths, live birth, and the eeence ofgrs ancmialies

• De& pupa aria pups sacrific ed at ty 4 should be eserved and a tudied for ps ible da!ects and cse af &ath

• Live pu should be cczmted and litters we ighed, by w ighi eh individual pup (op tional

) at birth, or soon ther, atxl on Days 4, 7
( optional)

, I 4
, and 2 1 afterbirth. ‘iv) Physical or behavioral abnom&li ti oervea ui the dams or offsxring should be recorded. (v) P 1 males ai:1 females uld we ic1ied on the firs
t day of dc ing ai weekly thereafter

. i ‘- tt Should e we ighed

a t birth, or soon thereat ter

, and on Days
4
, 7
, ( optional

) 14, ai1
2 1 • Those P offs ingselected
to produce F 2 lit tes should be .igh ed at birth

, or soon there—I and on cys 4 7
i op Lioe.al

) , 14, az 2 1 after birth. !ndividu&l pup weiiits should he meas i ed at day
2 1. . (9) Gro9snecs.

(i) cplete grs camination should be doae on ail animals

, inc luding those i ich di e during the exer iinen

t. o r were killed in moribund coritioz

, to insure that sot more than 10% of theanimals in any tes t oup are

1 ct from the tes t di. to canniba, anal y is of tiss

, misplac en
t ar
s imilar manageme

n t r ohiems .“ ,- (ii

) Special attentio

n wiLl he directed
tn the o rgans of the reproduc tJ.ve eystn. (iii) Th following oraris axx tissts, or r,rentative samples thereof

, should be preserved
in a s ui tabi
e medium fo r ps ible future his to— pa t!zlcica

i aminai.on: C iii) The fo llcing orga. ar1 tisss

, or rr entative samples: (A) Vagina, (B) Uterus; (C) Ovaries; (D) Testes;

I 39136 (:E) Epididyzus; (p) Seminal sIc1es; (G) frtate; &nd (H) Targe
t organ(s) of al 1 a P 1 amizala selected for matirq. ( I 0 ) Histopatho1oy.

U) ‘tie Uoinq

h isa tixlogy Should be perfozmed: (A) Fuji histopat,1cxy

on the argar listed in paragraph (e) (9) Ciii) of this Section for all. high dozse aiz1 control P selected for mating. (B) Organs dezs ating pa t1ogy in these animals should theii be examined in aTiI1Al fri the other dose oups. (C) N:rcopic camination S1UId be made of all tissiz showing cs athoiogica1 cthanges. (f) Data and reportin.
(1) Treatwofresu1ts.

(.1.) Data shallbe

in tabular fora

, showizq

, for ear*i tee t oup: (A) The ber of animals at the start of the tee t; (B) The number of animals iant; (C). The tys of change (see sectioi 83—4(e)(8),(9),
and (10); am CD) The ercenta
of animals di.playing eadi type of thanc. ( 2 ) Evaluation ofs tudy results.
Afl 5ValU. tiOn Of tS t UItS, including the
a ta tis tical analysis

, bas ed on the
c linical findings

, the gra

s nropsy findings

, and the micrcsc cçic resiil ts
• shal
I be made and suppli. This should incluc an evaluation
of the relationship,

or lack eof, between the aiiiiaal’

s pos e to the t t subs tance arid the thci dence and seven ty of all absomali ties. ( 3 ) Tes t report

. In & di tion to the ixiforn.a tion reuir by
§ 80-4,the test reprt shall incluc the following infomtion: (i
) Toxic rxxe
ra ta by sex and d e , inclnding fertili
y indices a length of gee tation; (ii) Species arri
S am; C iii) Time of death during the

s tudy or wh e ther animals svived
to termination;

( iv) ‘Toxic or otbe.r ef fe cts on rroduct.ion

, offs ing

• or xs tna tal c:-=....__—-...- 137 Cv) Time of obervation

of each abnormal

s iqn a.nd its subsequent course;

C vi) Body igh

t da ta fo r P 1

, F 1 and F 2 animals

;- (vii) Necropsy findings; (viii) Detailed cscription

of all histopat}iological findings; arK (ix) Statistical treatment
of results kiere *ppropriate. S 83—5 Combined chronic toxiciy/oncogenicity studies. (a) Purpose. The obectve
of a cosibin1 chronic toxicity/carcino genicity £tudy is to cterniine the effects of a substance
ii a mMmm1ian species following prolonged ar repeated expe. The application

of these staiards should generate data on which to identify the majority of chron Ic a rr onc on ic ef fe cts
a nd to de te rriine

d os e-r xnse

r e la t it sh ips. Ideally, the design and coruct should allow for the tection

o neoplastic effects and
a tertnination

of onconic potential
as well as neral toxi— city, including narological

, physiological

, biothnical

, and hnatolc*ica.l effects arx expcsure—relatedtxrpholoical (pathology) e!fects. (b) Test procedures.
(1-) Animalselection.

U.) Species and strain. Preliminary studies providing data on acute

, subchronic

, arid toxicokine tic r r es shou

3. d h ave be en ca r r ii ai t to pe re i t an ap pr opr ia te th o ic e of animals (species and strain).
As discussed
in other guideilnes, the nouse and rat have been most widely used for assessment
of carcinogenic potential, kiile the rat ari dog have been mt often studied for chronic toxicity. The rat is the scies of choice for caibin3 d-ironic toxicity and carcino— genicity studies.
If other species are used, the tester should provide justification/reasoning

for their selecti,n. Where available, the strain selected should
) susceptible

to the carcinogenic

or toxic effect of the class of substances being test if )aown, and provided
it does not have

a sxntaneous background too hii for meaningful assessment. Canrrnly used l&boratory strains should be nployed. (ii) 3!• (A) Dosing of rats should begin as soon as ;sible after weaning, ideali.y before the rats are six, but in any case not rire than eight weeks old. (B) At ccinmencement

of the study, the weight variation
of animals should not exce€d
20 percent of the mean weight for eadi sex. (C) StudJ. using prenatal
or neonatal animals may be reccxnnended under special coritions.

1AI I ‘-‘i