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Subdivision

F

HAZARD EW\LUATIONHUfN AND

ANIMALS

DOMESTIC

fEPROOUCEO BY

U.S. DEPARTMENT OF COMMERCE NATIONAL TECHNICAL SPRINGFIELD INFOPMATION SERVICE VA. 22161
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

hoxfuil

huiran safety, ..tiile similarly supxrtinq research in test nticxs ich 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

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,d
, .

PESTICIiZ ASSESSZ€N’I GUIDELINES

SUBDIVSIO?1 F

RAZARD EVLLUATION :

nmc AND DO!€STIC AN ttS

REVISED EDITION

by

Office of Pesticiae Programs

Guidelines Project Manager

Bruce P. Jaeger Hazard Evaluation Division

Office of Pesticide Prograzs

1,.s. Environmental Protection agency

Office of Pesticide and Toxic Substa 4ices

Washington, D.C. 20460

.11/I’
y 

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’
pçusq

I . Organiza Lion aM I4iil csophy of

Subdivision

F 1 tI. The Stamards for A.cceptable Testinq 3 111 • Discussion of Individual Tea ts

12

k. Acute Oral, DeraI , and Irthalation ‘oxicity 12 B. Irritation and Sens itiza tion Studi . 13 C. Netrcotoxicity Evaluations

13 D. Subth.ronic Studies ‘ .

. . . 14 z• Chronic and Oncogenici ty Studies

15 F. Teratogenicity and Reproduction Studies . G. Mutaenicit! Studies 16

17

. Special Testing

18

GUIDELINES

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

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

90—Day ty ( Roden : aid Non-rodeat): Study

66

i
Series 83 ironic ar3 Lor Term Studies 83—1 Chronic Toxicity Studies 83—2 Oncogenicity Study 107 83—3 Teratonicity Study 117 83—4 Reproductive and Fertility 125 Effects 83—5 Combined Chronic Toxici 130 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

. 852 Dczrtestic Animal Safety . 152 Testing 156 85—3 Dermai. Absorption Studies of Pesticides (Reserved)

ii

I
. 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. 20460
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 assur 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 tistical analysis • In ad di t ion , since 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

16
A traditional

because reproduction study requires two litters per generation

deal of variation.

the first litters produced by adolescent siothers often show a great

litters are

Secause of these variations, data baeed on the first

more uniform

less

n

useful than data from the sec,nd set of litters, which are

litter, but

size and health. The current guidelines recocnsend only one

Consequently,

the animals would not be bred until they had fully matured. under the current principles, likely to show the first litter would be less

mothez.

the variability of the first litters produced by juvenile in a traditions]. study.

In adcdtion to reducing the ni.ber of litters per generation, guideline would this : reproduction reduce the number of generations. Part of the reason studies traditionally have tested three generations is to detect genetic abnormalities. Under the Subdivision F guidelines, however,

a

the

battery

potential

of znutagenicity

of a pesticide to cause genetic damage uld be assessed in tests which are collectively more sensitive than

Discussion. than a three—generation reproduction study. (See also part III. C. of this

third generation

) The other reason for extending a reproduction study into the is to detect cumulative effects. The Agency, however, not aware of any is

of a

toxic effect which first appeared in the third generation

generations,

reproduction study. While the current guidelines reduce the number of it would extend the perIod of dosing for each litter—producing animal.

for

Accordingly, EPA believes this test design to be more sensitive cumulative effects than the traditional three-generation study.

G. Mutagenicitt4ies 84—1 and—2.

used directly

Data from mutagencity tests are used for several purposes. Data are to determine if a test substance is genotoxic, capable of that is,

mechanisms,

interacting with or damaging genetic material (DNA) and/or

changes. Ti 1

or

Agency

mutaaenic, and hence capable of producing heritable genetic

in humans recognizes that a mutagen causing a heritable defect

an epidemiologic

has not yet been id: tified, however the desiqn and conduct of

Since mutations

study to veri.fy such a relatively rare event is difficult.

collect

do occ in humans, the Agency feels that it needs to information in experimental able decisions regarding test systems in order to make reason— capable of risks associated with chemicals which nay be

be used as

inducing human genetir alteration. Mutagencity data nay also an indication of the oncogenic potential of a pesticide.

17
(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 ) ien reqiired • Da ta on a tMo- neration r eproduction study e reiir& by 40 CFR Part 158 to supprt the rsistration of each manufacturing—use product and form1ated product the t mets either of the foflowii on e.ria :

(1) The esticida1 use of a sticide roduct is likely to resu.1t in S igif icant human expure to the prthi, its act ive inedient( a) , metabolite(s), or degr&ation product(s); or

‘

( 2 ) The u se r equi.r es a to lerance for the çstic 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

conta ir a n &:i di tio na]. di. scu ss Lo n of the Fo i1a to ra ‘ Exemp tSo 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 tSo, ani , 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

na.nci

to parental(P 1)

es

ania1s xior to Lher mating, during the resultant ice—

, a nd thr ou the we aning of the Sr F ef fsring . Th e su± tance is then ainistered to selected F offsprirq during their growth into adult hood, tating, and production of an F2 generation, up xitil the F2 generation is 21 days old.

_*1 I

J
(ii) Parental (P1) &nimals should I about 8 weeks old at the

S tart of doe ing . See cpos ur e corzli tior in paragraph C c) (4 ) of this

section for dir regin.

(iii) !.!• (A) For an &equate assessment of fertility, both tales

and fales must * studied.

( B) The females should ml liparous and non—prnant.(iv) Ead test ar control oup. should contain at least 20 sales

ard a sufficient number of feaiales to yield at least 20 pregnant faiia.les

at or near term.

(2) Controlrgs. (i) A concurrent control is recrnended.

Thi S should an m trea ted or sh am trea ted coi trol oup or if a

vehicle is used in eministering the test substance, a 7ehicle control

groip.

(ii) If a vehicle is used in &ministerirg the test substance, the

control oup should rece ive the vehic Le in the h ighes t volun used.

(Ui) If a vehicle or other adit1ve is used to facilitate dcinq, it

should not interfere with abrptiori ci the test su1tance or produce toxica f fe Cts.

(3) pose levels and dose se1ect:on. (i) At least three de levels

ar a concurrent control should be used.

(ii) The highest dose level should irxhice tx,cicity but not mortality

in the parental (P1) animals.

(iii) The lowest dose level should not produce any evidence of toxi—

city. Where there is usable estimatioi of human expwe the lowest dose

should exceed this.

(iv) The intermediate dose level(s) s,ula produce minimal observable

toric effects. If ,re than one intermediate dose is used, dose levels

st’uld be seced to produce a qriation of toxic effects.

(v) The incicnce of fatalities in low and intermediate dose groups and

in the controls should low, to permit meanirgfui evaluation of the results.

1)

I j
g

ore the eating period.

(B) Dcir of P1 mal I At should contin throu the thating period. 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 F1 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 F2 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) F1 males selected for mating should be sacrificed immediately after livery of the last F1 litter sired, or, in cases of infertility, after the conditions of (7)(i)(B) are met.

(C) F1 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 weanir of the ir las t iit ters.

(E) F2 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 P1 animals to sacrifice of F2 offspring at weaning.

tra io

(6)

n is

Adrdnistration of_the test substance. The oral. route of adutinis— LX .f e rrEi.

1.36

,

. j
0 D4Deing of P1 a1es

and females begins

8—14 P1 mating period

11-17 Dosing of P1 males

may er at Week 25;p1 males are

sacrificed •

14-20 Ding of P1

females end..

P1 feaales are

sacrif icel

Li born ai Utter

sizes randcily

aduste to 8 pups

e1

i dcaing of F1 fa1es begins

F1 offspring not

sel.cted for mating

are sacrificed

1 mating; Dcir of

1 males may end at Week 40; ? males are

sacrificed’s

Ra in 1.nc; F1 females F2 born and are sacrificed litter sizes

ra ndc.ly

adjusted to

to 8 pups each

F2 offsring

are sacrficed ‘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 be ad j us ted by ap pr opr ia te numbers of eks to ac c te the se cond ma ting, 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.

22—34

25—37

.

UI

.1
mixed

is &thninistered in the clrin.king water, or 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

dosei.evel

fenale should be placed with a single rarcxaly select1 male from the sane 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

histological

procedur es as ad ii tio sal pz rtu.n ities to ma te wi th o the r a ir es o r dams , examination of the reproductive orçns , and examination of the trus o r serma ge n ic cyc lea.

or vaginal

(C) Each

plugs.

morning the female should ) am.ined for presence of srm Day 0 of regiancy is defined as the day vaginal plugs or sxm are found.

(ii) cross. (A) For mating the F1 offsing, one nale &nd one faa.le are randauly selected frc each litter for crcs mating to produce the F2 generation. Mating of siblings should be avoided.

weaning.

(B) F1 males and females not selected for matir are sacrificed un

(iii) Spcial housing. Near parturition, pregnant animals should be

maqrials.

caged separately in delivery or aiaternity cages and provided with nesting

(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.:
imal.

(ii) The dura tion of gee tation should be calcula ted fr Day 0 of pr;nancy.

( iii) F:at:ti iitte.r should be ecamii as aoon as ssib 1. a! ter de Uv— •Z7 for the number of pups , stillbirths, live birth, and the eeence of grs ancmialies • De& pupa aria pups sacrificed at ty 4 should be eserved and a tudied for psible da!ects and cse af &ath • Live pu should be cczmted and litters we ighed, by wighi eh individual pup (op tional ) at birth, or soon ther, atxl on Days 4, 7 ( optional) , I 4 , and 2 1 after birth.

‘iv) Physical or behavioral abnom&li ti oervea ui the dams or offsxring should be recorded.

(v) P1 males ai:1 females uld we ic1ied on the first day of dc ing ai weekly thereafter. i ‘-tt Should e we ighed a t birth, or soon thereatter, and on Days 4 , 7 , ( optional ) 14, ai1 2 1 • Those P offsing selected to produce F2 littes 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 measied at day 2 1.

. (9) Gro9snecs. (i) cplete grs camination should be doae on ail animals , inc luding those iich die during the exeriinen t. o r were killed in moribund coritioz , to insure that sot more than 10% of the animals in any tes t oup are 1 ct from the test di. to canniba, anal

,-

y is of tiss

, misplac ent ar similar managemen t rohiems .“ (ii ) Special attention 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 psible future histo— pa t!zlcica i aminai.on:

C iii) The fo llcing orga. ar1 tisss, or rrentative samples: (A) Vagina,

(B) Uterus;

(C) Ovaries;

(D) Testes;

I 39
( 

0 ) Histopatho1oy. U) ‘tie Uoinq h isatixlogy 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 dezsating 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 shall be 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 ofstudy results. Afl 5ValU. tiOn Of tS t UItS, including the a ta tistical 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 pose to the tt substance arid the thci dence and seven ty of all absomali ties.

( 3 ) Test report. In & di tion to the ixiforn.a tion reuir by § 80-4, the test reprt shall incluc the following infomtion:

a

(i ) Toxic rxxe ra ta by sex and de , inclnding fertili y indices length of gee tation;

(ii) Species arri S am;

C iii) Time of death during the study or wh e ther animals svived to termination;

( iv) ‘Toxic or otbe.r effe cts on rroduct.ion, offsing • or xstna tal

c:
(viii) Detailed cscription of all histopat}iological findings; arK

(ix) Statistical treatment of results kiere *ppropriate.

S 83—5 Combined chronic toxiciy/oncogenicity studies.

genicity

(a)

£tudy

Purpose. The obectve of a cosibin1 chronic toxicity/carcino

species following

is to cterniine the effects of a substance ii a mMmm1ian

these staiards should prolonged ar repeated expe. The application of

chron Ic a rr onconic generate data on which to identify the majority of effe cts a nd to de te rriine d ose-rxnse r e la titsh ips. Ideally,

effects and

the

a

design

tertnination

and coruct should allow for the tection o neoplastic

city, including narologicalof, physiological onconic potential as well as neral toxi— , biothnical effects arx expcsure—relatedtxrpholoical , and hnatolc*ica.l (pathology) e!fects.

(b) Test procedures. (1-) Animalselection. Preliminary studies U.) Species and strain.

r res shou 3. d h aveproviding be en data on acute , subchronic , arid toxicokine tic ca rrii ait to pe rei t an ap pr opr ia te th o ic e of animals (species and strain). As discussed in other guideilnes, and rat have been most widely the nouse kiile the rat ari dog used for assessment of carcinogenic potential, The rat is have been mt often studied for chronic toxicity.

genicity studies.

the scies

If other

of choice

species

for caibin3 d-ironic toxicity and carcino—

justification/reasoning are used, the tester should provide for their selecti,n. Where available, selected should ) susceptible the strain class of substances being to the carcinogenic or toxic effect of the

sxntaneous background

test if )aown, and provided it does not have a

l&boratory strains should

too hii for meaningful assessment. Canrrnly used be nployed.

(ii)

weaning, ideali.y 3!• before (A) Dosing of rats should begin as soon as ;sible aftereight weeks old.

the rats are six, but in any case not rire than

should

(B)

not

At

exce€d

ccinmencement of the study, the weight variation of animals 20 percent of the mean weight for eadi sex.

under

(C)

special

StudJ. using prenatal or neonatal animals may be reccxnnended coritions.

1AI

I ‘-‘i