PIPERCNYL BOTOXIDE^
EECISICN DOCTMENT
Carol E. Iangley
Project Manager (S1RD)
Clayton Bushong
Daniel Byrd
Chris Qiaisson
Chao Chen
Ftoger Gardner
Linda Garczynski
Rranklin G&e
Bernard Ifaberman
Irving ffeuer
Gerald Marquardt
Ftobert ffcG&ughy
Barbara Mxre
Delores Williams
Biologist (EEB/HED)
Science Advisor (SPS/HED)
Biochemist (TCK/HED)
Statistician (CAG)
Ibxicologist (TCK/HED)
Writer/Editor (SFRD)
Etoduct Manager (RD)
Pathologist (CAG)
Qsneticist (TCK/HED)
ibxicologist (TOVHED)
Ibxicologist (CAG)
Typist (SPRD)
Typist (SPRD)
September 30, 1981
Cffice of Ffesticides and Tbxic Substances
Environmental Protection Agency
401 M Street, SW
Washington, D.C. 2D460
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- T&ble of Contents -
I. Introduction 2
II. Chemical Profile 6
A. Chemical Identity 6
B. Registered Products, Uses, and Tblerances 6
C. Exposure - 7
III. Piperonyl Butoxide [FB] as a Potential REAR Candidate 8
A. Introduction .. 8
B. Oncogenicity 8
1. Sources Originally Suggesting PB as an Chcogen 8
2. Other Oncogenic Studies . 9
a. The NCI Study 9
b. The Halter Iong-1£rm Study ......11
3. FOssible Oncogenic Gontaminants or impurities 12
C. Cbcarcinogenicity (Co-oncogenicity) 12
D. Mutagenicity 14
E. Chronic Tbxicity 16
1. Reproductive and Fetotoxic Effects 16
2. "teratogenic Effects . .................... 16
3. Metabolic Effects: Mixed Function Qcidase Inhibition...... 17
a. Inhibition Studies in Vitro ...18
b. Inhibition studies in Vivo ........ 18
c. Inhibition Studies with Wiowi Carcinogens
or Hi tag ens . .......19
d. Inhibition Studies in Hunans ...........................20
4. Other Chronic Effects 20
a. . Biological F&te of EB and its Metabolites 20
b. ethology 21
c. Mortality 22
F. Effects on Fish and Wildlife ....22
IV. Gbnclusicns and Recannendations ....................................24
A. Summary of Conclusions ..24
1. Oncogenicity 24
2. Rroduct Chemistry 24
3. Mutagenicity .......24
4. Reproductive Effects Tfesting 25
5. Teratogenic Ifesting 25
6. Metabolite Testing ....25
7. Chronic feeding Stu3y ..26
V. liable I: Cbcarcinogenicity of IB and Freons 27
VI. T&ble II: Statistical Significance of Epstein Study 28
VII. Bibliography
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Executive Summary
Piperonyl butoxide is an insecticide synergist principally employed to enhance
the activity of natural pyrethrun. It is contained in approximately 4,200
federally registered formulated products manufactered by over 900 registrants.
Although in recent ^ars there has been a trend toward the use of unsynergised
synthetic pyre thro id formulations, piperonyl butoxide, in combination with
pyrethrun, remains ujiumi in the marketplace. Piperonyl butoxide is formulated
into hand held aerosols, pressurized sprays, total release pressurized
products, intermittent aerosols, fogging concentrates, emulsions, dusts, and a
number of additional specialty products.
Piperonyl butoxide first came to the attention of the Agency because of its
appearance cn the teak list (1969) of chemicals. This list indicated chemicals
warranting additional study with regard to carcinogenicity. The Mrak
Commission based their asses anent upon a study by Innes et al. (1969)/ which
reported turners in one of the two mouse strains tested. Piperonyl butoxide
was, thus, referred to the Agency far scientific review in July of 1976. It
was identified as a ccnpound requiring intensive scientific review in July 1978
(43 FR 30613).
Subsequent to the initiation of the agency's review, -the National Cancer
institute (NCI) tested piperonyl butoxide for carcinogenicity in both 'rats and
mice. The NCI report.(1979) found increases in the incidence of lynphonas
in female rats and the incidence of lacrimal gland adenomas in male mice.
Although of statistical significance vfoen viewed from the perspective that the
incidence of lymphomas in the control rats ves low by chance-in ccnpariscn to
the historical spontaneous incidence of lymphomas in the rat strain tested, the
NCI suggested that had the experimental group been compared to the historical
controls there would have been no significant difference. NCI did not,
however, perform a statistical test to confirm their hypothesis.
With regard to the mouse study, the NCI stated that "...adenomas of the eye or
lacrimal gland occurred at incidences that were dose related, but in direct
comparison the incidences in the individual dosed groups were not significantly
higher than that in the control group..., thus, the occurrence of this turner in
male mice was not clearly related to the administration of the test chemical."
NCI later indicated that, historically, spontaneous adenomas of the eye or
lacrimal gland normally occur at a higher frequency than in the matched
controls in this study, and that, therefore, one could not call piperonyl
butoxide a carcinogen based on the dose related incidences-. NCI concluded that
under the conditions of their bioassay, piperonyl butoxide was not carcinogenic
in the strains of rats and mice tested.
The NCI study vas additionally reviewed by the Hazard Evaluation Division
(HED), of the Office of Pesticide Programs (OPP/EPA). The HED review found a
statistically significant increase in the occurrence of lymphomas in female
rats when compared to matched controls and a significant increase in the
occurrence of adenomas of the eye or lacrimal gland in male mice when compared
to matched, pooled or historical controls.
With the conflicting assessnent of piperonyl butoxide's carcinogenic potential,
HED referred the data to EPA's Carcinogen Assessment Qroup (CAG). HED
additionally referred four studies on possible mutagenic effects to EPA's
Reproductive Effects Assessment Group (REAG).
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CAG, upon review of the data, concluded that the Innes study provides one
positive result for reticulum oell sarcana in one strain of treated male mice
through one route of administration. Regarding the NCI stiriy, CAG disagreed
with the NCI. CAG stated that the lymphoma incidence in the female rats, when
evaluated by careful statistical analysis was significantly higher than that of
matched controls, pooled controls, historical controls and controls cited by
another auther (Goodman, 1979). The" CAG further provided, however, that the
male control rats in the NCI study had a very high incidence of lymphomas
(45%). This, they said, casts suspicion upon the whole stu3y and, thus, no
conclusions could be reached concerning piperonyl butoxide's carcinogenicity.
Regarding the lacrimal gland adenomas found in the male mice, CAG -stated that
the numbers of tuners were small in the NCI study, and that the findings
probably occurred by chance variation.
As a result of the multiple reviews, both CAG and HED have concluded that the
available evidence is not sufficient to mate a definitive judgsnent vis-a-vis
piperonyl butoxide's potential for carcinogenicity. Both CAG and HED have
agreed' that further testing is necessary.
A related issue, cocarcinogenicity, was additionally identified as a potential
cause far concern. Mrak (1969) reported that HB appeared capable of enhancing
the toxic effects of certain substances and that cne study (Epstein, 1967)
suggested that it may be a cocarcinogen with Fteon 112 and 113. Agency review
of the Epstein study found no conclusive evidence of cocarcinogenicity.
With regard to mutagenicity, KEAG could not mate a definitive statement
regarding PB because of inadequacies in all four studies reviewed. HED, as
well as KEAG, has concluded that additional testing must be undertaken.
Other data gaps have been identified by CAG and HED. These data gaps include
product chemistry, mannalian metabolisn, and reproductive effects. Both the
product chemistry and metabolisn data requirements are related, in part, to an
understanding of piperonyl butoxide's potential far oncogenicity. The product
chemistry data requirements are intended to identify synthesis processes and to
establish the presence or absence of potentially oncogenic manufacturing
impurities. The metabolism studies will be designed to facilitate
interpretation of those data derived frcm feeding studies vis-a-vis the
principal route of exposure, ie. inhalation. Reproductive effects data are
being sought due to both the high exposure potential of individuals of
reproductive age, and the current absence of valid reproductive effect*data.
As Agency review of all available scientific' literature has failed to
conclusively establish that piperonyl butoxide either meets car exceeds
established risk criteria, the Agency is returning piperonyl butoxide to the
registration process. A Notice will be sent to all registrants informing them
of the requirement to perform additional testing. These tests, the appropriate
protocols and time schedules will be described in detail within the Notice.
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I. Introduction
Section 3(a) of the Ffederal Insecticide Fungicide and Rxtenticide Act [FIFRA)
requires all pesticide products to be registered by the Administrator of EPA
before they may be sold or distributed. Section 6(b) of IJIFRA authorizes the'
Administrator to issue a notice of intent to cancel the registration of a
pesticide or to change its classification if it appears that the pesticide or
its labeling "does not comply with the provisions of [FIFRA] or, when used in
accordance with widespread and caomonly recognized practice, generally causes
unreasonable adverse effects on the environment." Thus the Administrtator may
cancel the registration of a pesticide whenever he or she determines that it no
longer satisfies the statutory standard for registration, which requires,
amcng other things, that the pesticide not cause "unreasonable adverse effects
on the envirorment" (Section 3(c)(5) of FIFRAJ. These "unreasonable adverse
effects" are defined in Section 2(bb) of FIFRA to include "any unreasonable
adverse effects to man or the environment, taking into account the economic,
social and environmental ¦ costs and benefits of the use of any pesticide."
Ihe Environmental Protection Agency, hereafter referred to as the Agency,
created the Rebuttable Presumption Against Registration [RPARJ process to
facilitate the identification of pesticide uses which may not satisfy the
statutory standard for registration and to provide a public, informal procedure
for the gathering and evaluation of information about the risks and benefits of
these uses. Ihe regulations governing the RFftR process are set forth in 40 CFR
162.11. In broad sunmary, these regulations set forth certain criteria of risk
and provide that an HPAR shall arise against a pesticide if the Agency
determines that the ingredient( s), metabolite!s), or degradation product(s) of
the pesticide in question meet or exceed any of these risk criteria.
In administering the RPAR process, the Agency adheres to the standard for
initiating the REAR process established by Section 3(c)(8), one of the 1978
ftnendments to FIFRA," which provides that the Agency may not start an RPAR
unless it has "a validated test or other significant evidence raising prudent
concerns of unreasonable adverse risk to man or the environment."
Vfcen the Agency publishes a notice indicating that an REAR has arisen, the
40 CFR 162.11 regulations require .that an opportunity then be provided for
registrants, applicants, and interested persons to submit evidence to rebut the
presuiption, or evidence relating to the economic, social, and environmental
benefits for any use of the pesticide. If the presumptions of risk are not
rebutted, the evidence on the benefits of the pesticide is evaluated and
considered along with the information on the risks. Ihe Agency then analyzes
various methods of reducing the amount of risk from the pesticide together with
their costs and determines whether the pesticide can be regulated so that the
benefits of continued use outweigh the risks. If measures short of
cancellation cannot reduce the risks associated with any given use of the
pesticide to a level which is outweighed by benefits, the use in question must
be cancelled.
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With regard to pipercnyi butoxide [IB), tvo published docunents, Innes et al.
(1969) and Mrak (1969), revealed a potential oncogenic hazard. Innes et al..
using a preliminary tunorigenic screening test, classified re as a compound
requiring additional study. D:. Mrak, Chairman of an advisory committee to
the Secretary of Health, Education, and Vfelfare (the Mrak.Gsnmission),
supported this conclusion in his report on pesticides. Piperonyl butoxide was,
thus, suspected by the U.S. Ehviromjental Protection Agency of meeting 40 CFR
162.11 risk criteria. Although potential oncogenicity was the primary basis of
concern, the Agency initiated a review of all available toxioological data.
The potential effects examined in this decision document are oncogenicity, co-
oncogenicity (co-carcinogenicity), mutagenicity, reproduction and
teratogenicity. An additional concern involves the metabolic fate of PB in
mammalian systems. Ihis latter concern is not directly involved with any
40 CFR 162.11 risk criteria, but rather with the availability of those data
necessary for an interpretation of chronic effects data.
Ihis docunent presents the review of scientific data gathered to determine
whether PB met or exceeded any of the risk criteria set out in 40 CFR 162.11.
The Agency found no valid evidence to indicate that IB met or exceeded any of
the risk criteria; however, there vets not sufficient information to determine
whether IB caused any reproductive, or mutagenic adverse effects. Results of
studies addressing these toxic effects did not indicate immediate cause for
concern. They did not, however, establish, with a reasonable degree of
certainty, that IB is either nomutagenic or nonteratogenic.
Those studies assessing carcinogenic effects were found to be inadequate. The
Agency, therefore,, can not reach a definitive conclusion concerning IB's
potential as a carcinogen. Lata gaps also exist in the areas of metabolism and
product chenistry. IB has not, however, been found to meet or exceed any risk
criteria. The Agency, therefore, will not initiate the RPAR process. Rather,
the Agency is recotmending that IB be returned to the registration process with
the stipulation that the registrants conduct appropriate tests to provide data
on carcinogenicity, mutagenicity, reproduction, metabolism, and product
chemistry.
Ihis decision docunent is divided into five sections. Section I is this
introduction. Section II discusses general information on the product's
chemistry, uses, and tolerances. Section HI addresses the primary purpose of
the review; it compares data on potential adverse effects of IB with the
Agency's criteria for a Rebuttable Presumption Against Registration. Section IV
surraarizss the conclusions of this review of piperonyl butoxide and recaimends
actions to be taken as a result of these conclusions. Sections V and VI
contain tables relating information on the cocacinogenicity of IB and Fteons
and the statistical significance of the Epstein et al. (1079a). Section VII is
a bibliographical listing of the works cited.
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II. Chemical Profile
A. Chemical Identity
The chemical name for piperonyl butoxide (PB) is (butyl carbitol)
(6-propylpiperonyl ether). The Chemical Abstracts Service has assibned to
PB the registry nunber 51-03-6. Hie structural formula of FB is showi
belcw:
FB is a derivative of methylene-dioxyphenol (MDP). Qanpounds containing
the MEP chenical group are widely distributed in essential oils,
alkaloids, and other physiologically active compounds of natural and
synthetic origin. Manbers of this group, H3 among them, are used
as synergists for pyre thrum and certain synthetic pyrethroid
insecticides.
The manufacture of piperonyl butoxide ocmprises three chemical reactions
(Browi et al., 1970):
-The hydrogenation of safrole to dihy3rosafrole (DHS)
-The reaction of CBS with formaldehyde and hydrochloric
acid to furnish chlorcntethyl CHS.,
-The reaction of chlorcmethyl EHS with butyl carbitol.
B. Registered Products, (fees, and Tblerances
Piperonyl butoxide is employed as a pesticide synergist in about 4,200
federally registered formulated products manufactured by approximately 900
registrants. In the United States, the principal manufacturers of
technical IB are airfield American (formerly EMC), Alpha laboratories,
Inc., Rrentiss Drug and Chemical Cfcrrpany, Inc., and Mclaughlin, (brmley,
King Gbntpany. Ihe Agency has developed a yearly aggregated produiduction
estimate of 600 to 1,200 thousand pounds active ingredient (EPA, 1980).
Iblerances for residues of FB have been established cn raw agricultural
cannodities as follows (40 CFR 180.127):
20 ppn frcra post harvest application in or on barley, birdseed
mixtures, buckwheat, corn (including popcorn), rice, rye and wheat,
O
CHjOCHjCBjCCf^CHja^Hg
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8 ppn from post harvest application in or on certain fruit, nut, seed
and grain crops (see 40 CFR 180.127 a complete crop list),
3 ppn in meat, fat, and meat byproducts of poultry,
1 ppra in eggs,
0.25 ppm from postharvest application in or cn potatoes,
0.25 ppn (reflecting negligible residues in milk) in milk fat,
0.1 ppn (negligible residue) in the meat, fat and meat byproducts of
cattle, goats, hogs, horses and sheep,
An exemption from the requirement for a tolerance has been granted IB when
application is made "...to growing crops in accordance with good
agricultural practice." [40 CFR 180.1001 (b)(4)] The apparent
inconsistency created by permitting an exemption frcm the requiranent
for a tolerance for field application, but establishing tolerances for
post harvest applications will be addressed at time of product
re-registration.
PB may additionally be applied in food processing and food storage areas
"provided, that the food is removed or covered prior to such use"
(21 CFR 193.370(a)(5).
C. Exposure
Although in recent years there has been a trend toward the use of
unsynergised synthetic pyre thro id formulations, IB, in combination with
pyrethrun, remains carmen in the marketplace. IB is formulated into total
release pressurized products, intermittent aerosols, fogging concentrates,
emulsions, dusts, wettable powders and other mare specialized products.
In considering potential human exposure, the Agency believes that direct
inhalation is the most significant route. There is, additionally, seme
potential far dietary exposure through the use of aerosol products in food
handling and processing areas of conntercial establishments, heme garden
use and pofet harvest treatment of certain fruits, vegetables and grain.
The Agency has not located any test data relative to either inhalation car
dietary exposure. Theoretical inhalation values, however, have been
calculated (Brow, N.C., 1970). The specific formulation types and
application rates utilized in developing the theoretical values are
consistent with certain products still in use within the thited States.
Although not fully adequate far the development of an exposure analysis,
the Agency will rely upon these data until such time as additional data
might be necessitated by the acquisition of positive chronic toxicity
data.
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III. Pipercnyl Butoxide as a Rjtential REAR Candidate
A. Introduction
As previously noted, piperonyl butoxide was suspected by the Agency of
meeting the risk criteria established under 40 CFR 162.11. Although the
potential oncogenicity of FB initiated Agency review, additional concerns
evolved in response to the Agency's comprehensive review of available
toxicological data. The added areas of concern were co-cncogenicity (co-
carcinogenicity), mutagenicity, reproductive effects, teratogenicity, and
metabolism. Ihe individual areas of concern and the relevant data are
discussed below.
B. Oncogenicity
40 CFR section 162.11 (a)(3)(ii)(A) provides that a "rebuttable
presunpticn shall arise if a pesticide's ingredients).. .(i) induces
oncogenic effects in experimental roanmalian species or in man as a result
of oral, inhalation, or dermal exposure..." Section 162.3 (bb) defines
the term oncogenic as "the property of a substance or a mixture of
substances to produce cr induce benign car malignant tunor formation in
living animals."
1. Sources Originally Suggesting FB as an Choogen
Ihnes et al. (1969) administered FB or Butacide® (the trade name for
PB) to B6C3F1 and B6AKF1 hybrid mice by both oral and
subcutaneous routes. IB vas administered orally by gavage to one
group at 100 mg/kg in 0.5 percent gelatin for three weeks, then
switched to 300 ppn dietary for up eighteen months. IB vas
administered subcutarteously to a second group at 100 rag/kg.
Butacide® was administered to the orally dosed group at a rate of
464 mg/kg by gavage for three weeks. Ftcm week three to the
termination of the study at eighteen months, Butacide® vas
administered at 1,112 mg/kg dietary in 0.5 percent gelatin. A
second Butacide® group received subcutaneous injections of 100 mg/kg
in corn oil. Ihe animals were examined for incidences of
hepatomas, lynphcraas, and pulmonary tunors. Ihe investigators
observed an "Elevated incidence in an tncertain range" in the treated
group when compared to controls and classified FB as a compound
requiring additional study as a *tumorigen".
Ihnes et al. based their conclusion on the results reported in a more
detailed report by B&onetics fesearch labs, Sic. (1968). That
report clarifies the term "uncertain range" used by Ihnes et al.
There was an increased incidence of total tumors in male B6C3F1 mice
treated subcutaneously with Butacide*. Ihere was also an increased
incidence of reticulum cell sarcoma in the same sex and strain of
mice treated orally with IB. This vas regarded as evidence by Ihnes
et al. that an additional study was needed. Since the increased
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incidence of reticulum cell sarcomas was not seen in mice given FB
subcutaneously, the classification of FB as an active "tumcrigen" was
uncertain. In the case of Butacide®, the increased incidence of
total tumors was statistically significant when the treated group
was ccropared to pooled controls, but not when a?cpared to the matched
controls. Cither chemicals tested by Sines et al. were found to
increase the incidence of timors above controls by both oral and
subcutaneous routes of administration. These chemicals were
classified as active or "tunorigenic." Thus, Innes et al.
reccranended that IB be investigated further for potential
carcinogenicity.
Agency scientists (Gardner, R., 1979a and 1980) reviewed Lines' study
and found it to be scientifically valid. However, in the case of
Eutacide®, no statistical significance is found vhen the treated
group is compared with the appropriate matched untreated control
groip or the solvent (corn oil) control grovp. Hie increased
incidence of total tunors is only statistically significant when the
treated group is compared to pooled controls (p=0.05). Thus, the
increased incidence of total tunors cannot be clearly associated with
Butacide0 administration.
The Agency's Carcinogen Assessment Qroup {Byrd, D.M., 1981) stated
that'the Innes stufy showed that FB caused reticulum cell sarccma in
one strain of treated male mice through cne route of administration.
The Agency agreed with the authors that further study was necessary
to more completely evaluate FB's potential carcinogenicity.
3h his review of studies on the toxicology of pesticides, Mrak
(1969) discussed the findings of Innes et al. and supported their
conclusion that further evaluation of FB as an oncogen vbs
indicated. He recormended that the possible interaction of FB with
a variety of toocic si±»stances be investigated and that tests on mixed
function oxidase inhibition be included in the safety evaluation.
2. Other Oncogenic Studies
Hi is section sunmarizes additional oncogenic studies which the Agency
reviewed.
a. The NCI Study
The rational C&ncer Institute (NCI) studied FB in 1978 in its
Carcinogenesis Testing Rrogran. Technical grade IB yes tested.
TV® groups of 50 Fisher 344 Rats of each sex were given FB in
the diet at either 5,000 or 10,000 ppn for 107 weeks. Matched
controls consisted of 20 untreated rats of each sex. One-
hundred BGC3F1 mice of each sex were administered FB at either
2,500 or 5,000 ppn for 30 weeks. Then, because some mice died
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the doses vere reduced to 500 and 2,000 ppn, respectively, for
82 mere veeks. fetched controls consisted of 20 untreated mice
of each sex. Survival was 80 percent of the original; thus
sufficient numbers were at risk for the. possible development of
late-appearing tunars. All the rats and mice were sacrificed
at the end of the period of administration of the test chemical
and were examined.
Mil (1978) found that in the female rats there was a
statistically significant incidence (p=0.020) of lymphomas in
the high-dose groip (15 of 50) when compared to the controls
(1 of 20). The incidence of lynphcmas in historical-control
rats in other studies at the same laboratory, however, has been
19/191 (10%), 7/20 (35%), and 6/20 (30%). Thus, NCI concluded
that the incidence of lymphomas in the control female rats in
this particular assay may have been abnormally lew. The
occurrence of a higher incidence in the dosed groups, therefore,
could not be related to the administration of FB (Byrd, D.M.,
1981). Although the NCI did not perform a
statistical test of significance to compare the incidence of
lymphomas of the treated female rats with that of the
historical controls, the Agency's Cancer Asse^snent Qroup (CftG)
did perform such a test and found p = 7 x 10 (Haberxnan,
B.H., 1981) (significant at 0.001 level).
In the male rats, a significant decrease with increasing dose of
EB was observed in the incidence of neoplastic nodules of the
liver and of adenomas or carcinomas of the pituitary. The
incidence of these tunars in the control group exceeded that of
the dosed groups.
In the female mice, a significant decrease with increasing dose
of IS was observed for the incidence of lymphomas. The
incidence of this tumor in the control group exceeded that of
the dosed groups.
In the male mice, adenomas of the eje or lacrimal gland occurred
at incidences that were dose-related, but in direct
comparisons, the incidences in the individual dosed groups were
not significantly higher than that in the control group
(controls 0/20, low-dose 0/49, high dose 4/50). tCI would
expect, under normal c ire instances, the spontaneous appearance
of a turner in 1 to 3 out of 20 controls; therefore, an incidence
of 4 out of 50 in the high dose group could have occurred merely
by chance (Vbrd, 1980). The JCI report stated that "the
occurrence of this tuner in the male mice was not clearly
related to administration of the test chemical."
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NCI's general assessment of its testing program states that
negative results do not necessarily mean the test chemical is
not carcinogenic, since the experiments are conducted only
under a limited set of circumstances. positive results would
demonstrate that the test chemical is carcinogenic for animals
under the test conditions and would indicate that exposure to
the chemical is a potential risk to man.
NCI cone 1 ix3ed that "under the conditions of this bioassay,
piperonyl butoxide vas not carcinogenic for Fisher 344 rats or
B€C3F1 mice."
Agency scientists (Roger, G., 1979b and 1980), including those
in the CPC (Byrd, D.M., 1981), reviewed and evaluated the NCI
Bioassay and found, however, a statistically significant
increase in the occurrence of lymphomas in treated
female rats when compared to matched controls, or historical
controls fran other NCI studies. The male control rats had an
unusually high incidence (45%) cf lymphomas. Goodman (1979),
for exanple found a 12 percent incidence of lymphomas far pooled
male control rats. (The NCI study does not describe the
incidence in pooled male control rats.) Ihe high variability in
lymphoma incidence in male and female control rats in the NCI
study makes it difficult to interpret the results of this
carcinogenicity bioassay. The utility of the study must,
further, be viewed from the perspective that the potentially
useful findings relate only to one sex and cne species.
Agency scientists (Gardner, R., 1979b and 1980; Byrd, D.M.,
1981) also noted an increased occurrence of adenomas
of the lacrimal gland in treated male mice when compared to
matched controls. However, the number of turners was small and
the findings could have occurred by chance variation, as
Dr. V&rd (1980) of NCI stated.
Agency scientists concluded that further testing is necessary;
the results of the NCI Bioassay are not definitive enough to
judge whether IB is positive or negative for carcinogenicity.
The Agency, at the time of development of protocols for the
required testing, will assist in identifying rat and mouse
strains having historically a lew incidence of spontaneous
lymphomas. This, coupled with increased test group sizes, is
anticipated to provided more definitive results.
b. The amter long-Term feeding Study
Hunter et al. (1976) conducted a long-term feeding study with
PB and pyrethrun, the insecticide most often used vdth IB.
Pyrethrun and PB mixed in a ratio of 1 to 5 were administered
to 45 male and 45 female Sprag ue-Eawley rats. Hi treated
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controls also consisted of 45 ££r ague-Da wley rats of each sex.
After two years of treatment, examination of tissues for tumors
revealed histopathological changes in the treated as well as the
control groups.
Observation revealed, in both treated and control groups,
enlarged pituitaries (sane with tunors), siiscutaneous masses,
areas of fibrosis, liver changes, testicular atrophy, ovarian
cysts, and tunors of the manmary gland. There was no
significant difference in the frequency of turners in the
treated group as canpared to the controls.
Agency scientists Edwards, W.T« (1978) reviewed this study and
found it to be valid; however, because animals were fed a
mixture and rot FB alone, no conclusions can be made about the
carcinogenicity of FB itself. Again, the Agency concluded that
further testing is necessary to determine EB's potential for
causing carcinogenic effects.
3. Ebssible Chcogenic Gbntaminants or Enpurities
The Agency's Guidelines provide that "The composition of each lot of
the test substance shall be determined, including the name and
quantities of knovn contaminants and impurities, as far as is
technically feasible. The determination shall include quantities of
unknown materials, if any, so that 100 percent of the test sample is
accounted for." (43 FR 37352)
Brovn, (1970) indicated that the first step in the manufacture
of IB is the hjdrogenaticn of safrole to dihydrosafrole, both of
vftich are known carcinogens (Innes et al. 1969). Theoretically,
safrole or dihydrosafrole could possibly contaminate the final IB
technical product. Since the Agency has no information cn the
details of the manufacturing process for IB, the registrant s) must
supply this information cn product chemistry in order to rule out the
possibility that technical IB is contaminated with known
carcinogens. This information is necessary so that the Agency can
take the appropriate steps to protect the public in the event that
there is product contamination. In addition, when testing PB for
oncogenic effects, it is imperative that the most pure technical be
used. In this way, any resulting toocac effects can be attributed to
the test chemical alone and not to any product contamination.
C. Gbcarcinogenicity (Gfc>-oncogenicity)
Che stirJy investigated the possibility -that IB was a co-carcinogen.
Ce>-carcinogenicity is a property of two chemicals, not necessarily
carcinogenic by themselves, that induces tunors when the chemicals are
administered together. Ejpstein et al. (1979a) hypothesized that IB mixed
12
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with "Preens" (fluorocarbons used as propeHants with pressurized
aerosols o£ pesticides), might induce carcinogenicity; that is, IB could
be a cocarcinogen. In order to study this hypothesis, neonatal Swiss
mice were subcutaneously injected in the nape of the neck with a 5 percent
solution of IB in redistilled tricaprylin (the solvent) in volumes of
0.1 ml at ages cne and seven days and 0.2 mis at ages 14 and 21 days.
"Efceons" 112 and 113 were injected in combination with IB to these animals
and also separately to tvo other groups. Gbntrol animals were injected
with the solvent alone. Animals were allowed to survive until the
experiments ended in 50 to 52 weeks. It was found that hepatomas occurred
only in males, the highest incidence being in the groups given both IB
and "ft:eons." The results are summarized in Table I.
The hepatoma incidence in male mice far the combination treatment was 17
percent (Rreon 113 & PB) and 31 percent (Rreon 112 & PB) in contrast to a
5 percent (Ffceon 113) and 0 percent (Rreon 112) incidence in the separate
treatment groups. The incidence of obstructive uropathy in the solvent
controls was equal to or higher than the incidence in groups given Rreons
112 and 113 alcne. Epstein et al. suggested that FB may alter the
metabolism of "Rreonperhaps by inhibiting dechlorination. The authors
did not evaluate the statistical significance of increases in tumor
incidence far animals treated with IB alone car in combination with the two
Rrecns.
Agency scientists (Mishra, L.C., 1978 and Gardner, R., 1980) reviewed this
study and applied statistical tests for significance of the data. The
results are fouid in T&ble II. There was an apparently statistically
significant increased incidence of hepatomas in neonatal Swiss mice
treated with IB and Ft eon 112 when compared to that of the solvent control
group (p=0.047). These results do not clearly associate the increased
incidence of hepatomas with IB and Rreon 112 because there are no negative
control data with which to draw a comparison against the solvent control
group. The incidence of tuners in the male solvent controls was 8 percent
(4/48). This incidence was significantly different from that of the
female solvent controls (0/68). Also, seme animals, which were severely
autolysed and cannibalized were not examined histopathologlcally. Had
they survived, the significance of the tumor incidences may have been
different.
The incidence of tuners in animals treated with IB and Rreon 113 was not
significantly different frcm the solvent control group, though the
incidence of hepatomas in the animals treated with IB and Ereon 112 was,
as stated above, significantly different frcm the solvent controls.
This suggests the possibility of specificity with regard to the Rreons
with which PB could be co-carcinogenic, towewer, when the results of the
tvo IB combinations are compared, no significant difference is found
(p=0.222). The number of tumors in the IB plus Rreon 112 group was
significantly different from the number in the IB plus Fir eon 113 group.
13
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Agency scientists t Mishra, L.C., 1978 and &rdner, R., 1980) indicated
that the results of this study do not clearly establish vtoether IB is or
is not a co-carcinogen with the frecns tested. The Frecns 112 and 113
used in this experiment are not the frecns used as prope Hants with
aerosol cans of pesticides containing EB. Those freqns are Freons 11 and
12. The Agency is not avore of any studies viiich tested EB and Ft eons 11
and 12 far co-carcinogenicity.
At the present time, the Agency does not have a policy which addresses the
problon of potential toxicity due to interactions of chemicals. Ihere is
no policy indicating how cx>-carcinogenicity might be assessed and
co-carcinogenicity is not cited in the guidelines as a risk criterion
vhich would initiate the KFAR process. Also, current risk assessment
methods are inadequate far estimation of hunan risk based on this type of
laboratory data. Therefore, the Agency will not at this time require the
registrants to perform further tests to assess H3's potential far causing
co-carcinogenic effects.
D. Mutagenicity
40 Cre 152.11 (a) (3)(ii)
-------
intraperitoneally, an effect on early fetal deaths was demonstrated only
at the lover dose. The authors assigned a "borderline significance at the
5% level" to the effect of higher early fetal death in animals
administered IB.
Agency scientists (feuer, L., 1978b and 1980) evaluated the study and
stated that because of the snail number of animals used, administration of
only cne dose orally, dose reversal in the nunber of early deaths
following intraperitoneal injection, and the borderline significance of 5
percent for an cared dose, the Agency would require additional -testing
before PB could be evaluated for mutagenic potential.
Ashvoqd-Smith et al. (1972) tested the mutagenicity of IB in bacteria. A
10 percent concentration of Butacid^ (containing 80 percent PB and 20
percent "related compounds") vas added to cultures of the auxotrophic
mutant WP2 Try-(tryptophan requiring) of Escherichia coli.
Mutants/revertants not requiring tryptophan were scored after 48 hours of
incubation. IB was reported to be negative in this test for
mutagenicity. Agency scientists (Mauer, I., 1978a and 1980) ccranented
that the test vas performed without a manmalian metabolic activation
system, which might generate mutagenic metabolites. Also, the high
concentration (20 percent) of unidentified con pounds could have interfered
with the potential for direct mutagenicity at the single concentration
used. Therefore, the results of the .study are inconclusive and further
testing is necessary.
Friedman and Senders (1976b) used an _in vivo mouse assay
to determine what effects PB might have on the metabolisn and mutagenicity
of dimethyl nitrosamine (EMN) (which requires metabolic .activation for
mutagenic activity). IB vas injected intranuscularly (ijn.) into male
S*iss-Webster ICR mice at doses of 10, 40, or 640 irg/>g. EMN, at a dose
of 500 mg/kg, was injected i jn. at the same time as an intraperitoneal
injection (45 minutes later) of Salmonella typhimurium G-46 cells. It
vas found that IB inhibited liver demethylase activity resulting in a
decrease in metabolism of EMN and a consequent reduction of DIN
mutagenicity, as shown by a decrease in the nunber of Salmonella
revertents canpared with the nunber when DIN vas administered alone.
Agency scientists (feuer, X., 1978d and 1980) in their review stated that
since IB vas not administered alone, the authors could not male any
conclusions about the mutagenicity of IB itself.
Efciedman and Staub (1976) proposed the inhibition of mouse testicular DNA
synthesis by mutagens as a potential manmalian assay for mutagenesis. in
their study, five male Swiss mice were given 640 mg/hg PB by
intraperitoneal injection. Three hours later each mouse received 10
microcuries of radioactively labeled thymidine. The mice were then
sacrificed and the testes were examined for UNA concentration and
radioactivity. There vas no difference found between the treated group
and controls.
15
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2h their review of the paper, Agency scientists (Mauer, I., 1978c and
1980) pointed out that there may not necessarily be a correlation
between mutagenic activity and inhibition of testicular DNA synthesis. Si
addition, the Agency submits that the expected exposure may not cause
assimilation into the testes. Ihis type of test may, ^therefore, have
little or no relevance for assessing mutagenesis.
The results of these four studies are inadequate to assess whether the
criterion for mutagenicity has been met or exceeded; further testing is
necessary to assess potential mutagenic effects of IB.
E. Chronic toxicity
40 CFR 162.11 (a) (3)(ii)(B) provides that a rebuttable presumption shall
arise if a pesticide's ingredient(s)" produces any other chronic or
delated effect in test animals at any dosage up to a level, as determined
by the Administrator, which is substantially higher than that to which
hunans can reasonably be anticipated to be exposed, taking into account
ample margins of safety."
1. Reproductive and Etetotoxic Effects
Because products containing IB have nunerous heme and garden uses,
it may be anticipated that men and vcmen of reprocutive age will be
exposed to a significant degree. It is due to this exposure
potential that reproductive, fetotcocic and teratogenic effects take
on added significance.
A single reproduction study, Sarles and-Vandegrift (1952), has been
located and reviewed by Agency scientists. The study has been found
deficient. The test groip sizes were snail at the outset of the
study, with subsequent sacrifices and high mortality further
diminishing the population. The two highest dose levels (10,000 and
25,000 ppn) decreased food consumption and compromised nutrition so
that ocmpoirtd related effects could not be evaluated. Ih addition,
only two litters were used far the second and third generations.
The above noted study is not sufficient as an indicator as to
whether or not the criterion far reproductive effects has been met or
exceeded. The Agency, therefore, has determined that an additional
study, to be conducted by the registrant, is required (Gardner, R.,
1981b.)
2. Teratogenic Effects
TWo studies dealing with PB's potential teratogenic effects have been
located by the Agency.
16
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3h a study conducted by 3BT {Industrial Rio-Tfest) (Adler, G. and
S. Snith, 1978) for Hrlaughlin GDrmley King Obmpany (1976), albino
rats were used to assess the effects of PB an mortality, behavior,
reproduction and teratogenicity. Agency scientists oould not ccmment
en this study, because results of studies done ijy IBT are currently
suspect due to questionable laboratory practices. All IBT stud ies
must be audited before they can be validated by the Agency. This
particular study is currently being audited and will then be reviewed
by the Agency. The Agency does -not, however, consider this study to
be critical. The following study by ftiera et al. (1979) provides
information sufficient to satisfy current Agency needs.
Riera et al. (1979) investigated EB's teratogenic potential
utilizing female WLstar rats mated with proven males. E^ch dose
group consisted of 18-20 mated females. Technical grade IB in
distilled vater was administered once daily by esophageal intubation
from days 6-15 of gestation. The doses were 62.5, 125, 250, and 500
mg/hg. The fenales were killed on day 22, and the carcasses were
weighed after the uterine contents were removed. Ffetuses were
weighed and examined fear viability and external malformations.
Resorptions and dead fetuses were recorded. TVjo-thirds of the
fetuses were stained with alizarin red and exanined for skeletal
defects. The remaining one-third vere fixed and freehand sectioned
and examined for visceral anomalies.
Ebses of 500 mg/kg of FB produced no signs of toxicity or
statistically significantly reductions in body weight gain in dans
during gestation.
PB, at the maximum dose tested (500 mg/kg), manifested no prenatal
effects. The incidence of anomalies in treated groups was comparable
to that in the controls.
The Agency has ooncluded that the ttoera et al. study serves as an
adequate indication of FB's nonteratogenicity (Gardner, R., 1981).
Although Ajericy guidelines customarily require teratogenicity
testing in at least tvo marroalian species (43 FR 37383), the negative
findings of the Riera et al. study do not trigger the need for
additional data in relation to the Agency's investigation of PB as a
potential BPAR candidate. Additional teratogenicity testing may,
however, be required in relation to future reregistraticn actions.
3. Metabolic Effects: Nixed Rmction Qcidase Inhibition
Several studies dealt with the inhibitory effect of PB on mixed
function oxidases (info) in microsomes in various tissues of different
animals. Microsomal enzynes, in metabolizing a given chemical other
than EB, cause the chemical to be activated or inactivated. If a
17
-------
metabolite of the chemical is the active form, PB could diminish the
chemical's effect by inhibiting breakdown of the chemical. If, on
the other hand, the chemical itself is the active form, then PB oould
enhance the chemical's effect, by inhibiting its breataiowi.
a. Inhibition Studies in vitro
Generally, in the in vitro inhibition studies, microsomes
containing the info's were prepared frcm the livers of rats,
mice, or hamsters and were incubated with EB and various test
enzynes and chenicals.
Qraham et al. (1970), using the livers of rats, found that IB at
6 x 10 M _in vitro interfered vith the action of the
enzynes aunopyrine demethylase and aniline hydroxylase by
competitive inhibition.
Friedman and Cbueh (1974) confirmed these findings using mice
which were administered 500 mg/kg PB by intraperitoneal
injection (i.p.)-.
Friedman and Epstein (19??) observed that IB administered i.p.
at 2.5 wg/yg or 10 mg/kj first caused an induction of
microsomal aninopyrine demethylase activity and later an
inhibition, 48 hours after treatment with IB. Badgscn and
C^sida (1961), in their review article on microsomal metabolism
found that IB also inhibited the metabolisn of seme N,N-dialkyl
carbamates in rat liver. Using hamsters, Hinson et al. (1975)
found that FB administered to rats at 1500 mg/kj i.p. inhibited
the action of the enzyme p- chloroactanilide (PCAA)
hyfcoxylase, causing a decrease in. the product ion of N-hjdroxy-
PCAA, a metabolite of FCM.
Baker (1974a) in his abstract cn the mfb system determined that
PB (dose not specified) inhibits microsomal enzyoe activity by
acting both as an alternate substrate (ccmpetetive inhibition)
and by binding to Cytochrome P-450, a component of the
microsomal mfb system. The review by Rsdgsan et al. (1973)
supports E&Jker's conclusion.
These _in vitro studies show that IB inhibits microsomal
mfo's. Agency scientists (Marquardt, G.M., 1978d,e,h,i ,j,k,l)
reviewed these studies and carmen ted that the results and
conclusions are valid.
b. Inhibition Studies - in vivo
In vivo studies of mfib inhibition by IB confirm the in
vitro findings. Generally, IB was orally or intraperitoneal!y
18
-------
administered to rats or mice before treatment with test
chemicals, such as hexobarbital, aoxaaDlamine, and dietary
hydrocarbons*
Fine and folloy (1964) foind that IB administered to mice at
50 mg/^g i.p. caused a three-fold increase in sleeping times due
to prolonged action of hexobarbitOl. Flijii et al. (196S)
confirmed this finding and also reported an increase in
paralysis times, due to prolonged action of zoxa diamine. Albro
and Fishbein (1970) found an increase in the concentration of n-
octadecane and n-ncnacosane, dietary hydrocarbons, in the blood
and tissues of rats pre-treated pjo. with 1 g/hg IB.
Cbrmey et al. (1972) administered IB to rats i.p. at 2000
wq/yq, 500 mg/Jqf and 50 mg/kg. It was found that 50 mg/kj, had
no effect an antipyrine metabolism. At the higher doses,
antipyrine metabolism was inhibited. In mice, the no-observed-
effect-level (NCEL) for inhibition of antipyrine metabolism vas
0.5 mg/)g 18; thus, mice are 100-fold more sensitive than rats
to the effects of IB.
Results of all _in vivo studies revieved indicate IB inhibits
the mixed function oxidase systen of metabolizing enzynes, which
would normally break dovn aid inactivate the test chemicals.
The activity-of the test chemicals is, therefore/ increased.
Agency scientists (terquardt, G.M., 1978a,g,m,o) reviewed these
studies and found their results and conclusions to be valid.
c. Inhibition Studies with Riovn Carcinogens car Mj tag ens
E&lk et al. (1965) investigated the influence of IB in rats on
the metabolisn of bens(a)pyrene, a knovn carcinogen. Since IB
inhibited the hepatic metabolizing enzynes, there vas a decrease
in the elimination of benzo(a)pyrene in the bile.
Bi a similar experiment by Cbnney et al. (1972) 2000 mg/Jag IB
administered i.p. to rats caused a decrease in the metabolisn of
benzo(a)pyrene. lt> effect was found when using 50 mg/kj IB.
In the study by Ftiedman and Senders (1976b), already discussed
in the mutagenicity section of this docunent, IB was found to
diminish the mutagenic effects of dim ethyl-nitrosamine.
Nd studies were found indicating that IB enhanced any mutagenic
effects.
19
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d. Inhibition Studies in Humans
Although IB causes inhibition of mib's in laboratory animals,
it is important to know if the sane result could occur in hunans
exposed to IB.
Conney et al. (1972) administered 0.71 mg/kj PB orally to human
volunteers and found that PB did not Inhibit the metaholian of
the chemical antipyrine. They stated that "...since this dose
of IB is 50 times greater than the daily exposure received by
individuals who use sprays extensively in an enclosed area, the
environmental exposure of people to IB is probably insufficient
to inhibit the function of the microsomal enzymes." GDnney et
al. did not indicate their source of daily human exposure data,
but their comparisons are consistent with those data published
in the previously mentioned review article by Brown (1970) (see
page 4).
Agency scientists (Kbrqaardt, G.M., 1978m) commented that the
protoaal was scientifically acceptable, assuming that the
exposure data referred to were valid, the authors' conclusion
that the environmental exposure to IB was probably insufficient
to inhibit the function of the microsomal enzymes is valid under
the conditions of that experiment. However, since this is only
one study, using one dose level to assay the metabolism of a
single chenical (antipyrine), Agency scientists (Brantner, J.,
1979) could not generalize from the authors' conclusions.
Itone of the studies describing the metabolic effect of IB on
mixed function oxidase inhibition met or exceeded the risk
criteria outlined in 40 CFR 162.11. The concentrations of EB
used in the experiments were extremely high; it would be
unlikely that hunans would be exposed to such high levels
(Etovn, 1970). Since mib inhibition by EB has been so
thoroughly studied, no further testing of this metabolic effect
is necessary.
4. Other Chronic Effects
a. Biological Rite of EB and its Metabolites
In order to trace the biological fate of IB, Eishbein et al.
(1969) treated male Spraq ue-Dawley rats intravenously with
radioactive PB (dosages not reported). After eight hours,
samples of lings, liver, kidney, heart, fat, blood, and other
tissues were examined far radioactivity. Although there was a
wide distribution, approximately 12-17 percent of the
20
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administered dose was found in the lungs. Agency scientists
reviewed this study and made the following statements concerning
the metabolisn of IB:
-Metabolites of SB are rapidly excreted into the bile
of treated rats.
-The rates of urinary excretion of IB metabolites were less
than the rates far appearance in the bile.
-Biliary and urinary metabolites of EB were cnly partially
characterized.
-Approximately 40 percent of the administered dose of PB
appeared as COu in the air expired by the rats.
-The lungs contained relatively large anoints of IB (12-17
percent) present primarily in the unchanged form.
The Ajency (ffercpardt, G.M., 1978c) has determined that more
stu3y is needed to substantiate these findings and to suggest
possible modes of action by vhich IB might "preferentially"
accumulate in the lungs. Additionally, as chronic inhalation
testing is extraordinarily difficult, the Agency has elected to
request cnly chronic feeding studies. Metabolism data,
enabling the Agency to draw conclusions from only oral exposure
studies, must be developed.
b. ethology
"In the t*o year feeding study by Hmter et al. (1976), already
discussed in the oncogenicity section of this document, the
rats, which had been treated with pyrethrun and FB administered
together, were also studied fear any clinical abnormalities.
The males had been given cn the average 15.9 mg/hj/day IB. The
doses of insecticide-to-synergist were in a ratio of 1:5,
analogous to some formulated products. Urinalyses indicated a
higher percent of treated females had protein in their urine
than controls did (pCO.Ql). A lover lymphocyte count was found
in treated males, although all groups had values within the
normal range.
Agency scientists (Edvards/W.T. 1978) indicated that although
positive results may have been age-related rather than treatment-
related, it is possible that the clinical effects may have
resulted from the combination of pyre thrum and IB. They
commented further interpretation is limited because only one
dose level was used. At least three dose levels and a control
are needed, with the highest dose clearly showing an effect.
21
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c. natality
Firiedman and fenders (1976a) studied the effect of IB on
mortality in Swiss albino mice due to the known carcinogen
dimethylnitrosamine (EMN). The mice were injected
intraperitoneally with 640 mg/hg IB 45 minutes before injection
with 29. 6 mg/Jsj IMN. values were determined on the
seventh day after treatment. IB treatment did not significantly
alter the mortality of the EMN-treated mice as compared to
controls given IMN alone; there was similar mortality in both
groups.
Agency review (fer guard t, G.M., 1978f) of these data indicate
that the acute mortality prod teed by IMN is probably mediated by
a different mechanism of action than that causing oncogenic car
mutagenic effects. Though JB has been show to inhibit DO!
demethylase (the enzyme catalyzing the formation of the active
EMN metabolite), the EMN- produced lethality is probably not
mediated by this mechanism.
Epstein et al. (1967b) studied the toxicity (nnrtality) of Freon
112, Freon 113, griseofulvin (an antifungal antibiotic), and
bena^fa] pyrene with and without IB. fendcro-bred S*iss infant
mice (ICR/Ha) were injected sibcutaneously with Fireon 112 or
Rreon 113 (0.1 - 0.2 ml/aaimal), griseofulvin (0.125-1.0
ntg/animal) cr benzol a]pyrene (10 ug/an ima 1) with or without IB
(0.1 mg/animal at an initial dose of 2500 mg/Jg). The percent
mortality vbs recorded cn days 1, 7, 14, and 21. It vas found
that mortality due to the test chemicals vas markedly enhanced
by treatment in combination with IB. Agency scientists
(terquardt, G.M., 1978b) aancluted that this
effect vas presunably due to the IB- inhibition of the
microsomal enzymes metabolizing these canpoinds, thus
increasing their toxicity.
There is the theoretical possibility (t"taX, S., 1979) that IB
could enhance the toxic effects of seme drugs or chanicals in
the environment and cause adverse effects in hunans, but it
would be impossible to test IB in combination with every are of .
these chemicals.
F. Effects on Fish and Wildlife
40 CFR Section 162.11(a) (3)(ii) (c) establishes a criterion to protect
against significant population reductions in local, regional, or national
populations of nontarget organisms or fatality to members of endangered
species.
22
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The Agency has determined there is low risk to fish and wildlife based
primarily upon the patterns of use and minimal exposure potential
(Bushcng, C. 1979).
23
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Cbnclusians and Ffeccmmendaticns
A. Summary of Cbnclusions
With respect to piperonyl butoxide as an RH\R candidate, the Agency
concludes that the presently available data on oncogenicity, mutagenicity,
and chronic toxicity including reproductive and teratogenic effects, do
not support a "Ffebuttable Presumption Against Ffegistration" of pesticide
products containing piperonyl butoxide.
1. Oncogenicity
Results of the studies by lanes et al. (1969) in mice and the Nil
BLoassay (1978) in mice and rats are not adequate to determine whether
IB by itself is associated with the production of toners. Further
testing by the registrants will be necessary to properly assess the
potential oncogenic effects of H3.
- Ttesting must be performed as described in 43 FR 37379 with the
strain of rat and mouse to be determined in consultation with the Agency.
- In accordance with 43 PR 37352 information cn any contaninants and
impurities must be submitted.
- Monitoring for oncogenic effects can occur vfcile also assessing other
chronic effects as explained under item 7 of this section.
2. Rroduct Chemistry
Because safrole and dihjdrosafrole, tvo known carcinogens, are involved
in the manufacture of technical S3, the registrant must supply the
Agency with information cn the manufacturing process and the contaminants
or impurities found, as far as is technically feasible, in the finsil IB
product.
- Information cn the product and its manufacturing process must be
s limit ted by the registrants in accordance with 43 PR 29709.
3. Mitagenicity
The results of the four studies by Epstein et al. (1972), Ashwood-Smith
et al. (1972), Rriedman and Sanders (1976a), and Etiedman and Staub 1976)
are inconclusive. Further testing must be done by the registrant to
properly assess EB's potential mutagenic effects.
- For data cn gene mutation, the registrants must conduct an Anes assay
using 5 strains of bacteria and either a point mutaticn in mammalian
cells or a Drosophila test for sex-linted recessive lethals in
accordance with 43 FR 37389.
24
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- For chromosome aberration, the registrants must conduct an _in vivo
cytogenetics test in mice or rats, a daninant lethal test in accordance
with 43 FR 37389, and either an in vitro cytogenetics test in
hutsan cells or a cytogenetic analysis ot blood cells frcm exposed
persons as provided in 43 FR 37401, Aidenduii 3, footnote 2, which
states "...EPAmay require data in addition to those specified in the
proposed guidelines to assess the risks to humans
- R>r ENA damage/repair, the registrant must conduct a sister chromatid
exchange in manmalian cells and either a jeast test for mitotic
recombination or a bacterial ENA repair assay in accordance with 43 FR
37392.
- In addition, the registrant must conduct a micrcnucleus test in mice for
possible non-disjuncticn and a matrialian cell transformation assay as
provided in 43 CFR 37399, Addendum 3, Assessment of Hjman Risk, N>. 8,
which states, "A chemical may cause mutagenic effects by mechanisns such
as disturbed segregation of chranoscmes and suppression of ENA repair
mechanisns. Cbnsideraticns other than those described above will apply
to the evaluation of risk from mutations caused by such mechanisms."
4. ^productive Effects Tfesting
- As piperonjd but oxide possesses a high exposure potential in
relation to hunans of reproductive age, and inadequate data are
available, the Agency has determined that the registrant(s) must
conduct a three-generation reproduction study in accord with 43 FR
37384.
5. Teratogenic Testing
- As a result of the review of Wiera et al. (1979), the Agency finds
no indication that IB possesses any teratogenic potential in rats.
Although Agency guideline requirements have not been fulfilled with
regard to testing in tvo species, 43 FR 37382, no additional data
will be required at this time.
6. ttetabolite testing
- Hie agency believes that the single greatest potential for exposure
to FB rests with the inhalation of spray mist from aerosol and
pressurized spray formulations. As chronic inhalation testing is
extraordinarily difficult, the Agency has elected to request only
chronic feeding studies. Metabolism data, enabling the Agency to
draw conclusions from only oral exposure studies, must, therefore,
be developed. The registrant(s) must submit studies to assess the
uptake and metabolise of EB as described in 43 FR 37394.
25
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7. Chronic Fteeding Study
-Because Hjnter et al. (1976) suggested that administration of FB
plus pyrethruu nay have produced clinical signs of toxicity, it is
necessary for the registrants to conduct a chronic feeding study
using FB alone as described in 43 ET? 37375, Pb specified,
monitoring for effects due to repeated exposure to a pesticide can
be combined with an oncogenic evaluation. Thus, as long as
standards for both types of testing are met, the registrant(s) may
monitor both types of effects during one chronic feeding study.
A Notice will be sent to the registrants pursuant to F1FRA section 3(c)(2)(B)
informing then of the requirement for performing these tests and describing in
more detail the protocols they must fellow and the actions which they must take
to canpLy with the Itotice.
26
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1&ble I: Gbcarcinogenicity of FB and Ffeons (Epstein et al., 1967a)
Treatment group
Solvent control
Freon 112
Fteon 113
Piperonyl butoocide (PB)
Fteon 112+FB
Freon 113-tPB
Number with hepatomas
of total examined
Males
Females
4/48
0/68
0/17
0/19
1/21
0/20
0/20
0/36
4/13
0/24
3/18
0/24
27
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Table II: Statistical Significance of Epstein Stu3y
Comparison _£ value
(Freon
112
+
PB)
with
solvent control
0.-047
(Freon
113
+
PB)
with
solvent control
0.255
(fteon
113
+
PB)
with
(Freon 112 + PB)
0.222
(Freon
113
+
PB)
with
Freon 113
0.208
a/ Significant difference (P less than 0.05)
28
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VII.
Piperonyl Butoxide
Bibliography
Adler, G. and S. Smith (1976) Report to McLaughlin Gormley King Company:
Teratogenic Study With Piperonyl Butoxide Technical in Albino Rats: IBT
No. 8533-0831"7. (Unpublished study received Jun 18, 1976 under ?; prepared
by Industrial BIO-TEST Laboratories, Inc., Northbrook, II; CDL: ? )
Albro, P.W. and L. Fishbein (1970) Short-term effects of piperonyl butoxide on
the deposition of dietary hydrocarbon in rat tissues. Life Sciences 9(11):
"*29-739.
Ashwood-Smith, M.J.; Trevino, J.; Ring, R. (1972) Mutagenicity of dichlorvos.
Nature 240:418-420.
Baker, R. C. (1979) Interactions between the hepatic microsomal mixed-function
oxidase enzyme system and pesticides. Diss. Abstr. Int. 35(10):4792-B.
Bionetics Research Labs, Incorporated (1968) Evaluation of Carcinogenic,
Teratogenic and Mutagenic Activities of Selected Pesticides and Industrial
Chemicals: Volume I: Carcinogenic Study. Prepared for the National
Cancer Institute.
Branter, J. (1979) Memorandum -to Homer Hall thru Christine F. Chaisson dated
Dec 11, 1979. (Concerns the Toxicology Branch review of the Nov 23, 1979
draft of Piperonyl Butoxide.1
Brown, N.C. (1970) A Review of the Toxicology of Piperonyl Butoxide.
Report A 28/52. Prepared by Cooper Technical Bureau, Berchamsted Herts.
The Wellcome Foundation Ltd., Research and Development (V and A).
Bushong, C. (1979) Memorandum sent through Peter McGrath to Homer K. Hall dated
Oct 23, 1979. [Concerns a statement on the lack of risks to wildlife in the
Piperonyl Butoxide Decision Document.]
Byrd, D. M. (1981) Memorandum sent to attendees of the Feb 11, 1980 meeting
dated Mar 4, 1981. {Concerns the Carcinogen Assessment Group's review of
the National Cancer Institute study entitled Bioassay of Piperonyl Butoxide
for Possible Carcinogenicity (1978) and four mutagenicity studies.!
CFR, 21 (1980) Food and Drugs. Subchapter B—Food for Human Consumption.
Part 193—Tolerances for Pesticides in Food Administered by the
Environmental Protection Agency. Subpart A—Food Additives Permitted in
Food for Human Consumption. Part 193.360—Piperonyl butoxide.
CFR, 40 (1980) Protection of Environment. Subchapter E—Pesticide Programs.
Part 162—Regulation for the Enforcement of the Federal Insecticide,
Fungicide, and Rodenticide Act. Subpart A—Registration, Reregistration
and Classification Procedures. Part 162.3—Definitions.
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CFR, 40 (1980) Protection of Environment. Subchapter E—Pesticide Programs.
Part 162—Regulations for the Enforcement of the Federal Insecticide,
Fungicide, and Rodenticide Act. Subpart A—Registration, Reregistration
and Classification Procedures. Part 162.11—Criteria for determinations of
unreasonable adverse effects.
CFR, 40 (1980) Protection of Environment. Subchapter E—Pesticide Programs.
Part 180—Tolerances and Exemptions From Tolerances for Pesticide Chemicals
in or on Raw Agricultural Connodities. Subpart C—Specific Tolerances.
Part 180.127—Piperonyl butoxide; tolerances for residues.
CRF, 40 (1980) Protection of Environment. Subchapter E—Pesticide Programs.
Part 180—Tolerances and Exemptions From Tolerances for Pesticide Chemicals
in or on Raw Agricultural Connodities. Subpart D—Exemptions From
Tolerances. Part 180.1001—Exemptions from the requirement of a tolerance.
Conney, A.H.; Chang, R.Levin, W.M.? Gar but, A.; Munroe-Faure, A.D.; Peck,
A.W.; Bye, A. (1972) Effects of piperonyl butoxide on drug metabolism in
rodents and man. Arch. Environ. Health 24:97-106.
Edwards, W.T. (1978) Data Evaluation Record. (Toxicology Branch review of
Hunter et al., 1976.1
EPA (1980) Preliminary Quantitative Usage Analysis of Piperonyl Butoxide.
Economic Analysis Branch, Benefits and Field Studies Division, Office
of Pesticide Programs, U.S. Environmental Protection Agency.
Epstein, S.S.; Andrea, J.; Clapp, P.; Mackintosh, D.; Mantel, N. (1967a)
Enhancement by piperonyl butoxide of acute toxicity due to freons,
benzofalpyrene, and griseofulvin in infant mice. Toxicology and Applied
Pharmacology 11:442-448.
Epstein, S.S.; Arnold, E.; Andrea, J.; Bass, W.: Bishop, Y. (1972) Detection of
chemical mutagens by the dominant lethal assay in the mouse. Toxicology
and Applied Pharmacology 23:288-325.
Epstein, S.S.; Joshi, S.; Andrea, J.; Clapp, P.; Falk, H.; Mantel, N. (1967b)
Synergistic toxicity and carcinogenicity of 'freons' and piperonyl
butoxide. Nature 214:526-528.
Falk, H.L.; Thcnpscn, S.J.; Kotin, P. (i965) Carcinogenic Potential of
pesticides. Arch. Environ. Health 10:847-858.
Federal Register (1978) Proposed Guidelines for Registering Pesticides in the
United States, July 10, 1978. Federal Register 43 (132):29709.
Federal Register (1978) Proposed Guidelines for Registering Pesticides in the
United States; Hazard Evaluation: Humans and Domestic Animals, August 22,
1978. Federal Register 43 (163):37352, 37375, 37379, 37382, 37383, 37384,
37392, 37389, 37399, 373401.
FIFRA (1978) The Federal Insecticide, Fungicide and Rodenticide Act as Amended.
Public Laws 92-516, 94-140, 95-396. 7 U.S.C. 135 et. seq.
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Fine, B.C.; Molloy, J.O. (1964) Effects of insecticide synergists on duration
of sleep induced in mice by barbituates. Mature 204:789-790.
Fishbein, L.; Falk, H.L.; Fawkes, J.; Jordan, S.; Corbett, B. (1969) The
metabolism of piperonyl butoxide in the rat with C in the
methylenedioxy or -methylene group. Journal of Chromatography 41:61-79.
Friedman, M.A.; Couch, D.B. (1974) Inhibition by piperonyl butoxide of pheno-
barbital mediated induction of mouse liver microsomal enzyme activity.
Research Communications in Chemical Pathology and Pharmacology
8(3):515-526
Friedman, M.A.; Epstein, S.S. ( ? ) 39. Inhibition of microsomal enzyme
induction by piperonyl butoxide. Pages 287-288, In Abstracts:' Ninth Annual
Meeting of the Society of Toxicology; Mar 15-19,~T970.
Friedman, M.A.; Sanders, V. (1976a) Acute toxicity of dime thy lnitros amine in
the presence of inhibitors of DMH demethylase. Experientia 32(4):495-496.
Friedman, F.A.; Sanders, V. (1976b) Effects of piperonyl butoxide on
diroethylnitrosamine metabolism and toxicity in Swiss mice. Journal of
Toxicology and Environmental Health 2:67-75.
Friedman, M.A.; Staub, J. (1976) Inhibition of mouse testicular ENA synthesis
by mutagens and carcinogens as a potential sinple mammalian assay far
mutagenesis. Mutation Research 37:67-76,
Fujii, K.; Jaffe, H.; Epstein, S. (1968) Factors influencing the hexobarbital
sleeping time and zoxa2olamine paralysis time in mice. Toxicology and
Applied Pharmacology 13:431-438.
Gardner, R. (1979a) Data Evaluation Record. [Toxioology Branch review of limes
et al., 1969.1
Gardner, R. (1979b) [Toxicology Branch review of the National Cancer Institute
1979 study, Bioassay of Piperonyl Butoxide for Possible Carcinogencity.
Tables 1 .to 4 are attached.]
Gardner, R. (1980) Memorandum sent to Carol Langley dated Apr 30, 1980.
(Concerns the attached Data Evaluation Record addenda, which clarify issues
raised by comments on the latest draft of the Piperonyl Butoxide Decision
Document.1
Gardner, R. (1981a) Data Evaluation Record. [Toxicology Branch review of Khera
et al., 1979.]
Gardner, R. (1981b) Data Evaluation Record. (Toxicology Branch review of
Sarles, M.P. and W.B. Vandergrift, 1952.]
Goodman, D.G.; Ward, J.M.; "Squire, R.A.? Chu, K.C.; Linnhart, M.S. (1979)
Neoplastic and nonneoplastic lesions in aging F344 rats. Toxicology and
Applied Pharmacology 48:237-248.
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Graham, P.S.? Hellyer, R.O.; Ryan, R.J. (1970) The kinetics of inhibition of
drum metabolism in vitro by sane naturally occurring compounds.
Biochemical Pharmacology 19:769-775.
Gregorio, C. (1978) Data Evaluation Record. [Toxi oology Brarfch review of
Adler, G. and S. Smith, 1976.1
Haberman., B.H. (1981) Memorandum sent to Carol Langley thru Robert McGaughy
dated Jul 31, 1981. (Concerns their coimients on the Piperonyl Butoxide
Decision Document draft dated Jul 21, 1981. A copy of the document with
their comments is attached.)
Hinson, J.A.; Mitchell, J.R.; Jollcw, D.J. (1975) Microsomal N-hydroxylation of
p-chloroacetanilide. Mol. Pharmacol. 11:462-469.
Hodgson, E.and J.E. Casida (1961) Metabolism of N:N-diaDcyl carbamates and
related conpounds by rat liver. Biochemical Pharmacology 8:179-191.
Hodgson, E.; Philpot, R.M.; Baker, R.C.; Mailman, R.B. (1973) Effect of
synergists on drug metabolism. Drug Metabolism and Disposition
1(1):391-401.
Hunter, B.; Bridges, J.L.; Prentice, D.E. (1976) Long term feeding of
pyrethrins and piperonyl butoxide to rats. Prepared by the Huntington
Research Centre; Huntington Cambridgeshire, England for Dr. O. Okeys, The
Pyrethrum Marketing Board, P.B. Box 420, Nakaru, Kenya, East Africa.
(WR3/76254)
Innes, J.R.M.; Ulland, B.M.; Valero, M.G.; Petrucelli, L.; Fishbein, L.; Hart,
E.R.; Palotta, A.J.; Bates, R.R.; Falk, H.L.; Gart, J.J.; Klein, M.;
Mitchell, 1.; Peters, J..' (1969) Bioassay of pesticides and industrial
chemicals for tumorigenicity in mice: A preliminary note. Journal of the
National Cancer Institute 42(6):1101-U14.
Khera, K.S.; VJhalen, C.; Angers, G.; Trivett, G. (1979) Assessment of the
teratogenic potential of piperonyl butoxide, bipheny 1, and phosalone in
the rat. Toxicology and Applied Pharmacology 47:353-358.
Marquardt, G.M. (1978a) Data Evaluation Record. [Toxicology Branch review of
Albro, P.W. and L. Fishbein, 1970.1
Marquardt, G.M. (1978b) Data Evaluation Record. [Toxicology Branch review of
Epstein et al., 1967b.]
Marquardt, G.M. (1978c) Data Evaluation Record. [Toxicology Branch review of
Fishbein et al., 1969.1
Marquardt, G.M. (1978d) Data Evaluation Record. [Toxicology Branch review of
Friedman, M.A. and D.B. Couch, 1974.]
Marquardt, G.M. (1978e) Data Evaluation Record. [Toxicology Branch review of
Friedman, M.A. and S.S. Epstein, ?.I
Marquardt, G.M. (1978f) Data Evaluation Record. [Toxicology Branch review of
Friedman, M.A. and V. Sanders, 1976a.]
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Marquardt, G.M. (1978g) Data Evaluation Record. [Toxicology Branch review of
Fujii et al., 1968.1
Marquardt, G.M. (1978h) Data Evaluation Record. fToxicology Branch review of
Graham et al., 1970.}
Marquardt, G.M. (1978i) Data Evaluation Record. [Toxicology Branch review of
Hinscn et al., 1975.]
Marquardt, G.M. (1978j) Data Evaluation Record. [Toxicology Branch review of
Hodgson, E. and J.E. Casida, 1961.]
Marquardt, G.M. (1978k) Data Evaluation Record. [Toxicology Branch review of
Hodgson et al., 1973.]
Marquardt, G.M. (19781) Evaluation Report. [Toxicology Branch review of
Baker et al., 1979.1
Marquardt, G.M. (1978m) Evaluation Report. [Toxicology Branch review of Carney
et al., 1972.)
Marquardt, G.M. (1978n) Evaluation Report. [Toxicology Branch review of Falk
et al.r 1965.1
Marquardt, G.M. (1978o) Evaluation Report. [Toxicology Branch review of Fine,
B.C. and J.P. Molloy, 1964.1
Mauer, I. (1978a) Data Evaluation Record. Exhibit 34. [Toxicology Branch
review of Ashwood-Smith et al., 1972.1
Mauer, I. (1978b) Data Evaluation Reoord. [Toxicology Branch review of
Epstein et al., 1972.]
Mauer, I. (1978c) Data Evaluation Record. [Toxicology Branch review of
Friedman, M.A. and J. Staub, 1976.1
Mauer, 1. (1978d) Data Evaluation Record: Exhibit 158A. [Toxicology Branch
review of Friedman, M.A. and V. Sanders, 1976b.]
Mauer, I. (1980) Memorandum sent to Carol E. Langley thru William Butler dated
Dec 16, 1980. [Concerns the reassessment of the hazard potential of
piperonyl butoxide for carcinogenicity, mutagenicity, reproduction and
teratogenicity.]
Mishra, L.C. (1978) Data Evaluation. [Toxicology Branch review of Eostein et
al.# 1967a.]
Mrak, S. (1969) Pesticide interactions with synergists. Pages ?, In Report
of the Secretary*s Coranission on Pesticides and Their Relationship to
Environmental Health. Washington, D.C.: U.S. Department of Health,
Education, and Welfare.
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National Cancer Institute, National Institutes of Health, Carcinogenesis
Testing Program, Public Health Service, D.S. Department of Health,
Education and Welfare (1978) Bioassay of Piperorryl Butoxide for Possible
Carcinogenicity. CHEW Publication No. (NIH) 7B-1375.
Sarles, M.P.; Vandergrift, W.B. (1952) Chronic and oral toxicity and related
studies on aninals with the insecticide and pyre thrum synergist, piperorryl
butoxide. Airier. J. Trap. Med. Hyg. 1(5):862-883.
Ward, J.M. (1980) Personnal communication with Carol E. Langley on Mar 27,
1981. (Concerned the National Cancer Institute's study entitled Bioassay of
Piperonyl Butoxide for Passible Carcinogenicity (1978). ]
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