Summary of Toxicology Studies with Butachlor
Alan G.E. WILSON and Ayako S. TAKEI
Monsanto Company(Accepted November 20, 1999)

DESCRIPTION OF THE TEST CHEMICAL
Butachlor was developed by Monsanto Company (USA) and introduced in 1968
for the pre-emergence control of undesirable grasses and broadleaf weeds intransplanted, direct seeded rice and barley fields. Butachlor was first introduced inJapan in 1973 and has been used for the control of grass weeds and broadleaf weeds
in transplanted rice paddies.
Butachlor is a member of the chloroacetanilide class of chemistry and is the herbicidal
active ingredient in MACHETER® EC. This report summarizes the results of laboratory
toxicology studies conducted with butachlor. The physical and chemical propertiesof butachlor are given below:
Common name: Butachlor
Chemical name: 2-chloro-2',6'-diethyl-N-(butoxymethyl)acetanilide
Chemical structure:
CH3
O
N
CH2Cl
O

Molecular formula: C17H26NO2Cl
Molecular weight: 311.9
Physical state: clear amber liquid room temperature
Melting point: 4-5˚C
Vapor pressure: 1.8 x 10-6mm Hg 25˚C
Decomposition temperature: 156˚C
Water solubility: 20ppm 25˚C
Butachlor is miscible with alcohol, ether, acetone and other organic solvents.
ACUTE TOXICITY STUDIES
1. Butachlor Technical Material
The results of acute toxicity studies with butachlor are shown in Table 1. In thesetests, no distinctive signs of toxicity were observed following exposure via the oral,
dermal and inhalation routes.

Table 1 Results of acute toxicity tests with butachlor.
Route ofadministration

Species

Sex

Median lethal dose

Oral

Rata)

♂
♀
♂
♀
♂♀
♂♀

2620mg/kg
3050mg/kg
4140mg/kg
5030mg/kg
13,000mg/kg
5.3mg/lMouseb)
DermalInhalation

Rabbitc)
Ratd)a)Institute of Environmental Toxicology, 1980. b) Institute of Environmental
Toxicology, 1976. c) Bio/dynamics Inc., 1979. d) Monsanto Safety EvaluationNewstead, 1998.
Table 2 Results of acute toxicity tests with Machete® EC herbicide.
Route ofadministration

Species

Sex

Median lethal dose

Oral

Rata)
Mouseb)

DermalInhalation

Rabbita)
Ratc)

♂♀
♂
♀
♂♀
♂♀

2800mg/kg
4604mg/kg
5675mg/kg
2000mg/kg
1.63mg/la)

Bio/dynamics Inc., 1979: Study conducted with butachlor 60% EC. b)
Bozo Research Center, Inc., 1993: Study conducted with butachlor 32%
EC. c) Bio/dynamics Inc., 1981: Study conducted with butachlor 60% EC.

2. Machete®EC Herbicide
Machete® EC Herbicide is an organic solvent-based liquid formulation containing
approximately 32% or 60% butachlor. The results of acute toxicity studies with theformulated product are shown in Table 2. In these tests, no distinctive signs of
toxicity were observed following exposure via the oral, dermal and inhalation routes.
IRRITATION STUDIES
1. Primary Dermal Irritation
Butachlor was applied topically to the shaven backs of 6 New Zealand whiterabbits (2 sites per rabbit). Continuous 24hrs exposure resulted in slight erythemaand edema. The primary irritation index was calculated to be 3.5 on a scale of 8.0.

Butachlor was moderately irritating to the rabbit skin.

(Bio/dynamics Inc., 1979)

2. Primary Ocular Irritation
Butachlor was placed into the conjunctival sac of the right eye of 6 New Zealandwhite rabbits. The contralateral eye served as the control. Only a slight degree of
irritation of the conjunctivae was seen in 2 of 6 rabbits. No effect on the cornea oriris was observed. Eyes regained their normal appearance by 3 days after exposure.
Butachlor was slightly irritating to the eyes of rabbits.
(Bio/dynamics Inc., 1979)
DERMAL SENSITIZATION STUDIESThe potential of butachlor to induce dermal hypersensitivity was evaluated in a
modified Buehler assay.1,2) Induction involved topical exposure of 5 male and female
Hartley albino guinea-pigs each, to undiluted butachlor for 6 hours, 3 days per weekfor 3 weeks. Treated and naive guinea-pigs (3 per sex) were challenged with 50 %butachlor in acetone 2 weeks after the last induction dose. Butachlor produced slightirritation following initial application; moderate irritation was observed after thesecond application; and moderate to severe erythema with edema was observed in all
animals after the third application. On challenge, 5 out of 10 animals exhibited apositive skin response. After the second challenge which was conducted to confirm
the response, all animals exhibited a positive skin response. Butachlor is considered
to be a dermal sensitizer in the guinea-pig.
(Bio/dynamics Inc., 1983)
SUBCHRONIC TOXICITY STUDIES
1. Ninety-Day Feeding Study in Rats
Butachlor was administered in the diet to groups of 12 male and female Fischer-344(F-344) rats each, at concentrations of 0, 300, 1000, 3000 and 5000ppm for 90 days.
The animals were housed individually in stainless steel cages. Decreased bodyweight gain was observed in both sexes at the 1000ppm dietary level and above.
Mild anemia was observed in both sexes at 5000ppm and in females at 3000ppm.
Evidence of liver toxicity (e.g. increased γ-glutamyl transpeptidase, alanine
aminotransferase, cholesterol and liver weights as well as diffuse hepatocellularswelling) was observed in males only at the 1000ppm dietary level and above, and infemales only at the 3000ppm dietary level and above.
Early signs ofglomerulonephropathy were apparent in males at the 1000ppm dietary level andabove. Epithelial hyperplasia of the urinary bladder was apparent in males at the
5000ppm and in females at the 1000ppm dietary level and above. The No Observed
Effect Level (NOEL) was considered to be 300ppm (17.5mg/kg/day for males and19.0mg/kg/day for females).
(Institute of Environmental Toxicology, 1987)
2. Ninety-Day Feeding Study in Wistar Rats

Butachlor was administered in the diet to groups of 15 male and female Wistar
Imamichi rats each, at concentrations of 0, 500, 1000, 2500, 5000, 10,000, 20,000
and 40,000ppm for 90 days. Reduced body weight gains were observed in males andfemales at the 5000ppm dietary level and above. Statistically significant decreases in
feed consumption were found in male and female rats at 10,000ppm. There wasmortality at the 10,000ppm dietary level or above but not at the 5000ppm or lower.
At the dietary levels of 2500ppm and higher, hemoglobin concentration, hematocrit
value and RBC were decreased, suggesting possible anemia.
Decreased RBC CHE values in males at the 2500ppm dietary level and above, andfemales at 10,000ppm were noted. Plasma CHE was decreased in males at 10,000ppm
but in females no decrease was found for plasma or brain CHE values.
Statistically significant differences were found in brain, pituitary, ovary, uterus andheart weights, but there were no histopathological changes, and therefore, these organ
weight changes were not considered biologically significant. Decreased kidney weights
were observed in females at the 5000ppm dietary level and in males at the 10,000ppm
dietary level. Spleen weight was reduced in both males and females at dietary levelsof 2500ppm and higher. Histopathologically, dose-related changes were found in the
spleen, kidney and liver of animals at dietary levels of 2500ppm and higher. Atrophy
was observed in the ovaries and testes at 40,000ppm; these findings may have been
related to malnutrition at this very high dose level. The NOEL for this study wasconsidered to be 1000ppm (68.0mg/kg/day for males and 72.2mg/kg/day for females).
(Nihon University of Veterinary Medicine and Zootechnology, 1971)
3. Ninety-Day Feeding Study in Sprague-Dawley Rats
Butachlor was administered in the diet to groups of 20 male and female SpragueDawley (S-D) rats each, at concentrations of 0, 1000, 5000, 7500 and 15,000ppm.
Animals were housed individually in stainless steel cages. Physical signs of toxicity(hair loss and emaciation) and mortality (one female) were noted only at the 15,000ppm
dietary level. These animals also exhibited markedly decreased body weights, decreasedfood consumption, changes in hematology, serum biochemistry and urinalysis
parameters, liver and thyroid organ weight changes, histopathological evidence ofliver damage, and gross and histopathological evidence of kidney damage. Most ofthese effects were also noted at the 5000 and 7500ppm dietary levels. The animals at
the 1000ppm dietary level exhibited increased liver and thyroid weights. Because of
the lack of corresponding histopathological effects, these organ weight changes (liver
and thyroid) were considered equivocal evidence of toxicity and the NOEL of thisstudy was therefore determined as 1000ppm (67.9mg/kg/day for males and97.7mg/kg/day for females).
(Bio/dynamics, Inc./Institute of Environmental Toxicology, 1980)

4. Ninety-Day Feeding Study in DDY Mice
Butachlor was administered in the diet to groups of 7 8 male and female DDY
mice each, at concentrations of 0, 500 (females only), 1000, 2500, 5000, 10,000 and
20,000ppm. No mortality was found in mice at the 5000ppm dietary level or lower.
At the 10,000ppm dietary level, 60% of the mice died within 3 weeks on study; at the
20,000ppm dietary level all mice died within 1 week on study. All of these deaths
were considered to be treatment related. Slightly decreased feed consumption wasnoted in both sexes at the 2500ppm and 5000ppm dietary levels. At the 10,000ppm
dietary level, decreased feed consumption was noted in females. Decreased mean
body weights and mean body weight gains were noted in females at the 2500ppm
dietary level, and in both sexes at the 5000ppm and 10,000ppm dietary levels. A
slight but statistically significant increase in serum glutamic-pyruvic transaminase
values was observed in males at the 5000ppm and 10,000ppm dietary levels; because
there were no histopathological findings observed in the liver corresponding to theseclinical changes it was concluded that these results were not treatment related. RBC
CHE activity was reduced in females from the 10,000ppm group, but not in males atthe same dose.
Slight to moderate decreases in spleen weight were observed in females at the
1000, 5000 and 10,000ppm dietary levels. There were also sporadic changes in otherorgan weights (brain, pituitary, gonads, uterus, liver, adrenals, kidneys, heart). Since
none of these findings exhibited a dose-related response and were not accompaniedby corresponding histopathology changes, these findings were not considered to betreatment-related. Except for an increase in brown pigmentation of the splenic
medulla, there were no treatment related histopathological changes observed. The
NOEL for this study was considered to be 1000ppm (123.3mg/kg/day) for males and2500ppm (278.1mg/kg/day) for females.
(Nihon University of Veterinary Medicine and Zootechnology, 1971)
5. Ninety-Day Feeding Study in CD-1 Mice
Butachlor was administered in the diet to groups of 30 male and female CD-1 mice
each, at concentrations of 0, 1000, 3000, and 6000ppm for at least 91 days. The
animals were housed individually in suspended stainless steel cages. The animals at
the 6000ppm dietary level exhibited decreased body weights, hematological changes,
decreased blood urea nitrogen levels, decreased brain cholinesterase activity, organ
weight changes (brain, liver, and ovaries) and histopathological evidence of liver andkidney damage. Decreased body weights, and increased blood urea nitrogen, plateletlevels, and liver weights were noted in the animals at 3000ppm dietary level. The
animals from the 1000ppm group exhibited increased liver weights, but nocorresponding histopathological changes and is therefore, considered to be equivocal
evidence of toxicity. The NOAEL for this study was considered to be 1000ppm

(213.9mg/kg/day for males and 247.9mg/kg/day for females).
(Bio/dynamics, Inc., 1980)
6. Twenty-one Day Dermal Toxicity Study in Rabbits
Butachlor was administered dermally to groups of 10 male and female New ZealandWhite rabbits each, 5 days/week for 3 consecutive weeks at dose levels of 0, 100, 500
and 2500mg/kg/day. The animals were housed individually in suspended stainlesssteel cages. The only significant finding was an increase in glucose values in thefemales at the 2500mg/kg dose level. These changes in glucose were not consideredbiologically relevant or an indication of toxicity. Butachlor produced no other systemic,
clinical chemistry or pathological changes. Local skin changes consisting of mild
dermatitis were observed at lower dose levels while severe effects were seen at
2500mg/kg/day, the highest level tested. The NOEL for systemic toxicity fromrepeated dermal application of butachlor was considered to be 2500mg/kg/day.
(International Research and Development Corporation, 1982)
CHRONIC TOXICITY AND ONCOGENICITY STUDIES
1. Twelve-Month Oral Study in Dogs
Butachlor was administered orally via gelatin capsules to groups of 6 male andfemale beagle dogs each, at dose levels of 0, 1, 5, and 25mg/kg/day for 12 months.
The animals were housed individually in stainless steel cages. No treatment-related
effects of butachlor were observed on survival, body weights, hematology, urinalysis
or gross pathology. Target organ effects (alkaline phosphatase, organ weight increases
and histopathology findings) were observed in the liver and pancreas in dogs
administered 25mg/kg/day butachlor. The NOEL for this study was considered to be
5mg/kg/day.
(Institute of Environmental Toxicology, 1987)
2. Two-Year Chronic Toxicity/Oncogenicity Study in Fischer-344 Rats
Butachlor was administered in the diet to groups of 90 male and female Fischer-344(F-344) rats each, at concentrations of 10, 100 and 1000ppm for 24 months. Treatmentrelated signs of toxicity at the 1000ppm dietary level included an approx. 9% reduction
in body weight gain, slight increases in liver, kidney, adrenal and thyroid weights,
plasma chemistry and histopathological evidence of liver, kidney, bladder, and ocular
toxicity. A maximum-tolerated dose (MTD) was achieved at the 1000ppm dietarylevel. Butachlor was not oncogenic in the F-344 rat. The chronic NOEL in F-344
rats was considered to be 100ppm (3.6mg/kg/day for males and 4.33mg/kg/day forfemales).
(Institute of Environmental Toxicology, 1990)
3. Two-Year Chronic Toxicity/Oncogenicity Studies in Sprague-Dawley Rats
(1) Butachlor was administered in the diet to groups of 80 male and female

Sprague-Dawley (S-D) rats each, at concentrations of 0, 100, 1000 and 3000ppm for
26 months. The animals were housed individually in stainless steel cages. Increased
deaths were observed in both sexes at the 1000ppm and 3000ppm dietary level during
the last few months of the study. Reduced body weight was observed in males fromthe 1000ppm and 3000ppm dietary levels and in females from the 3000ppm dietarylevel. Food consumption was increased at these same dietary levels. Cholesterollevels were increased in males treated at the 3000ppm dietary level throughout the
study and in males at the 1000ppm dietary level during the latter portion of the study.
No other treatment-related biochemical alterations were observed. Increased urinary
protein levels were seen in male and female rats at the 3000ppm dietary level andwere considered to be treatment-related.
At sacrifice (interim and final) increases in liver, kidney, and thyroid weights anddecreases in adrenal and spleen weights were observed only at the 3000ppm dietarylevel, however most of these changes were not statistically significant. Neoplasticchanges were observed in the glandular stomach, nasal mucosa and thyroid gland, but
only at very high toxic dose levels (i.e. >1000ppm). Other chronic, non-neoplasticchanges were hepatocellular swelling and chronic nephropathy observed in animals
from all treated levels. A NOEL for neoplasms was established at 100ppm. A
definitive NOEL for non-oncogenic effects could not be established for this study.
(Bio/dynamics, Inc./Institute of Environmental Toxicology, 1983)(2) A second chronic study was conducted with S-D rats to follow up on effects
observed in the previous study and to establish a definitive NOEL for non-oncogenic
effects. Butachlor was administered to groups of 80 male and female rats each, inthe diet at concentrations of 0, 5, 20, and 100ppm for 24 months. The animals werehoused individually in suspended stainless steel cages. The outcome of this second
chronic rat study resulted in no observable adverse chronic effects in rats treated withbutachlor at levels of 100ppm for 24 months. Specifically, there was no increase in
the incidence, time of onset, or severity of kidney or liver pathology observed between
untreated control rats and rats given 100ppm butachlor. No evidence of other chronic
toxicity or oncogenicity related to butachlor treatment was observed. It was concluded
that 100ppm produced no butachlor-related toxicity and was a definitive NOEL in
this study (4.9mg/kg/day for males and 6.1mg/kg/day for females).
(Bio/dynamics, Inc./Institute of Environmental Toxicology, 1988)(3) To determine the absolute NOEL for chronic toxicity in S-D rats, the effects
observed in liver and kidney in two chronic studies were reviewed. The hepatocellularswelling observed in the first study at 100ppm was not considered to have been
related to treatment since it was not repeated in the second study and the increased
incidence in treated groups from the first study did not vary in response to dose. In
fact, the lowest dose group in males from the first study had the highest incidence of
this lesion, and the highest dose in females had the lowest incidence among thetreated groups. Therefore these changes were interpreted to have been spontaneousand not related to treatment.
An apparent increase in chronic nephropathy was observed in all treatment groups
in the first study, however there was no increase in severity with respect to controls.
In a repeat study there was no evidence of increased chronic nephropathy in any
treatment group. The NOEL for butachlor chronic toxicity in S-D rats was considered
to be 20ppm (1.0mg/kg/day for males and 1.2mg/kg/day for females).
4. Twenty-Four-Month Oncogenicity Study in CD-1 Mice
Butachlor was administered to groups of 100 male and female CD-1 mice each, inthe diet at concentrations of 0, 50, 500, and 2000ppm for 24 months. The animals
were housed individually in suspended stainless steel cages. No effects on survival
were observed. Mean body weight was decreased in both sexes at the 2000ppm
dietary level and males at the 500ppm dietary level. Mean food consumption wasincreased at the 2000ppm dietary level relative to control. Serum alkaline phosphatase,glutamic oxaloacetic transaminase and glutamic-pyruvic transaminase values observedin females at the 2000ppm dietary level were statistically greater than control values.
After 54 weeks of treatment, elevated total cholesterol values were observed in both
sexes at the 2000ppm dietary level. Organ weight changes (decreased brain, increased
liver and kidney) were observed in animals at the 500 and 2000ppm dietary levels.
Evidence of microcytic anemia was evident in both sexes at the 2000ppm dietarylevel. While decreased hematologic parameters were noted clinically, no corresponding
effect on tissues capable of hemopoietic element production was observed
macroscopically. At week 53, an increased incidence of retinochoroidal degeneration
was noted in males from the treated groups, but this effect did not persist at subsequentevaluation intervals. At week 104, the incidence of cataracts was increased in animals
from the 500 and 2000ppm dietary levels. The cataracts appeared to be similar
pathologically to spontaneously occurring lesions and as is often seen at similar oreven higher incidences in aged mice than those reported in this study.
Necropsy findings in animals were limited to the kidneys and lungs and correlatedwith the histopathologic findings reported below. Microscopic examination revealed
effects in the kidneys (nephrosis), eyes (cataracts), gallbladder (hyperplasia in highdose females only), and lung (alveolar/bronchiolar epithelial hyperplasia; increased
macrophages) of both sexes at the 500ppm and 2000ppm dietary levels. No effects
were observed in the gastrointestinal tract or the nasal region. There was no statisticallysignificant increase in either pulmonary adenomas or carcinomas in mice at theterminal sacrifice at any dose level. The small increase in the number of multiple
adenomas/carcinomas observed in males from all 3 treatment groups can be attributed
to an unexpectedly lower incidence found in the control male group, when comparedto historical control values with this strain of mouse at the testing laboratory. The

NOEL for this study is considered to be 50ppm (7.13mg/kg/day for males and8.56mg/kg/day for females).
(Hazleton Laboratories/Institute of Environmental Toxicology, 1985)
EVALUATION OF THE ONCOGENIC POTENTIAL OF BUTACHLOR
As noted above, neoplastic responses attributable to butachlor administration wereobserved in the stomach, thyroid, and nasal tissue of rats. A significant increase instomach tumors was restricted to the female rat at the highest dose tested (i.e. 3000ppm).
The incidence of thyroid and nasal tumors increased significantly in the females fromthe 3000ppm and 1000ppm groups but in males at 3000ppm only. The highest dose,
3000ppm exceeded the MTD as evidenced by excessive body weight loss ( 30%),
hepatocellular necrosis, and decreased survival. The MTD was shown to be 1000ppm.
The production of tumors only under these extreme conditions of exposure in rats isof doubtful relevance for humans. Also, a weight-of-evidence evaluation of dataobtained from extensive mutagenicity testing reveals that butachlor has no significant
genotoxic activity in mammalian systems. These data indicate that the tumors observedin the rat arise through non-genotoxic, threshold-sensitive mechanisms. To better
understand the mechanisms involved in the production of these rodent tumors andtheir relevance to humans, a series of additional mechanistic investigations were
undertaken with butachlor, as well as with alachlor, a close structural analog ofbutachlor which produces the same neoplastic response. These additional studies
have been communicated elsewhere,3) and the results are summarized below.
1. Rat Nasal Tumors
Previous studies have demonstrated significant species differences in the metabolism
and pharmacokinetics of alachlor and have provided a mechanistic basis for the
rat-specific production of nasal tumors. Butachlor is metabolized in a similar mannerto alachlor. Additional studies conducted with butachlor have further demonstrated
that a common mechanism of action is operative for the formation of rat nasal tumors
with butachlor and alachlor. Alachlor and butachlor are metabolized by common
metabolic processes leading to the formation in rat nasal tissue of the quinoneimine
metabolite of diethylaniline (DEIQ). This metabolite has been shown to localize andaccumulate selectively in rat nasal tissue but not in the nasal tissues of other species.4)
This metabolite forms protein adducts,5) which leads to cytotoxicity, prolonged cellproliferation and the eventual development of the predominantly benign, microscopic
nasal tumors observed. Increased cell proliferation is considered to be a prerequisite
for nasal tumor development, and its induction is threshold-sensitive, i.e., there is adose below which cell proliferation and the ensuing tumor formation do not occur.
Critical differences in enterohepatic circulation and target tissue metabolism resultin greater formation of DEIQ in the rat nasal mucosa as compared to other species.

Rat nasal tissue contains high levels of enzymes which metabolize alachlor or butachlorto 4-amino-3,5-diethylphenol (DEA-phenol), the precursor to DEIQ. For example,
the ability of rat nasal tissue to convert the secondary sulfide metabolite of alachlor to
2,6-DEA-phenol, the proximate metabolite of DEIQ, is more than 30 times greaterthan that of monkeys6) and 751 times greater than that of human nasal tissue.7)
Species differences are even larger when the relative enzymatic rate of the initial
metabolic step (alachlor conjugation to glutathione) is included in the overall rate of
DEA-phenol formation from alachlor. When this is done, the overall ability of rats toconvert alachlor to DEA-phenol is 3000- and 22,000-fold higher than that of humans
when the initial glutathione conjugation occurs in the liver or nose, respectively.7)
These data indicate that the potential for the formation of the reactive DEIQ metabolite
in human nasal tissue is negligible and demonstrate that with respect to alachlor aswell as to butachlor, the rat is not an appropriate model for the assessment of nasal
oncogenic hazard to humans.
2. Rat Stomach Tumors
An extensive research program was conducted to elucidate the mechanisms throughwhich butachlor induced stomach tumors in rats. A stomach tumor initiation-promotion
study was conducted with butachlor as part of this research program.8) Butachlor hadno activity as an initiator, but did promote tumor formation following treatment witha known initiator. Thus, the results from this study produced direct experimental
evidence indicating a non-genotoxic mode of action for stomach tumorigenicity.
Mechanistic studies with butachlor have shown that high dose exposure leads tomucosal atrophy with an accompanying profound loss of parietal cells as the initiallesion.9,10) The resulting gastric hypochlorhydria and increased pH induce acompensatory production of gastrin. Together, the progressive mucosal atrophy andexcessive gastrin secretion drive a proliferative response which culminates in tumor
formation. Butachlor did not produce mucosal atrophy at a lower, non-oncogenic
dose level. The results of these mechanistic investigations provide strong evidence
that butachlor produces stomach tumors in rats through a non genotoxic, thresholdbased mechanism.
This indirect mechanism of chloroacetanilide induced stomach tumors does notpresent a human risk, since the conditions driving the requisite threshold sensitive
events are operative only in rats chronically exposed to very high dose levels. The
prolonged exposure to excessively toxic doses required for tumorigenesis in rats
could not occur in humans. Even if such exposure did occur, a study with rhesus
monkeys showed that the key preneoplastic events (mucosal atrophy) do not occur inprimates even at a dose that is twice that required to produce stomach tumors inrodents. Thus, there is compelling data supporting the conclusion that the stomach
tumors are of no relevance to humans.

3. Rat Thyroid Tumors
Results from mechanistic work have shown that high dose butachlor exposure
results in increased thyroid hormone clearance via induction of hepatic UDPGT
activity, thereby causing a long-term, compensatory increase in circulating TSH
levels.11) It is widely recognized that such prolonged stimulation of the thyroid gland
via elevated TSH secretion leads to follicular cell hyperplasia and neoplasia inexperimental animals.12-14) This hormone-mediated mechanism of carcinogenesis isconsidered to be a threshold phenomenon to which rats are particularly susceptible.
The results with butachlor provide strong evidence that this non-genotoxic mechanismis operative in the production of butachlor-induced thyroid tumors in rats.
GENOTOXICITY STUDIESThe genotoxic potential of butachlor has been evaluated in numerous assay systems
using a variety of species, metabolic activation conditions and endpoints. These
assays tested Monsanto-produced butachlor in well-validated systems using establishedtesting guidelines according to Good Laboratory Practice standards. Butachlorproduced no evidence of a mutagenic response in the E. coli wp2 reverse mutation
assay, but gave weak and inconsistent positive responses in the Ames/Salmonella
reverse mutation assay. Increased revertant colonies caused by high doses of butachlor(0.5 to 10mg/plate) were less than 3-fold above control values and occurred only in
the TA-100 strain of Salmonella. In contrast, when tested in cultured Chinese
hamster ovary (CHO) cells, no evidence of mutagenic activity was detected in the
hypoxanthine-guanine phosphoribosyl transferase (HGPRT) forward gene mutation
assay. The result of a CHO in vitro cytogenetics assay was also negative for clastogenic
effects. Four in vivo mammalian tests have been conducted with butachlor. A bonemarrow cytogenetics study with S-D rats at intraperitoneal (i.p.) dose levels of 75,
250 and 750mg/kg, and a micronucleus test with Swiss-Webster mice at i.p. doselevels of 250, 500 and 1000mg/kg/day both revealed signs of general toxicity, but noevidence of adverse chromosomal effects. Exposure of CD-1 male mice to butachlorfor 7 weeks at dietary concentrations of 100, 1000 and 5000ppm revealed significant
body weight depression, but no evidence of dominant lethal effects in male germ
cells. An in vivo/in vitro DNA repair assay with F-344 rats at oral dose levels of 50,
200 and 1000mg/kg showed no increase in hepatocyte unscheduled DNA synthesis.
The weight of evidence supports the conclusion that butachlor does not havesignificant genotoxic potential in mammals. This conclusion is supported by ananalysis of the butachlor genotoxicity data using a scoring system developed by theInternational Commission for Protection against Environmental Carcinogens andMutagens.15,16) This system generates an overall score using data from multiple test
systems incorporating various weight of evidence considerations (e.g. doses used,species tested, in vitro vs. in vivo). The score of -12 for butachlor clearly positions it
well in the non-genotoxic range of the evaluation system and provides objective
confirmation of the conclusions from careful expert evaluation that butachlor does
not have general genotoxic potential. Furthermore, the butachlor genotoxicity results
support the conclusion that the rat-specific oncogenic responses do not result fromgenotoxicity but rather from butachlor's effects on non-genotoxic processes.
REPRODUCTION AND DEVELOPMENTAL TOXICITY STUDIES
1. Two-Generation Reproduction Study in Rats
Groups of 25 male and female Sprague-Dawley rats each, were fed butachlor in
the diet at concentrations of 0, 100, 1000, and 3000ppm. Treatment began 10 weeks
prior to the first mating to insure that a complete spermatogenic maturation cycle had
occurred during butachlor treatment and continued throughout mating, pregnancy,and lactation periods for 2 successive generations. Each parental generation wasmated to produce two litters per generation. Offspring from the second litters wereselected to be parents for the subsequent generation. All animals were given a gross
necropsy. Mating and fertility indices, as well as offspring weight/survival at birthand throughout lactation was evaluated for all litters. Histopathological evaluations
were conducted on both sexes in the control and 3000ppm dietary level of parents
from both generations as well as 6 male and female pups from the second litter of thesecond generation (F2b). No adverse effects were observed on any reproductive
parameter in any generation. Reductions in pup weights occurred at the 1000ppm
and 3000ppm dietary levels during lactation but maternal and/or parental toxicity was
also observed at these dose levels. The NOEL for reproductive toxicity in rats wasconsidered to be 3000ppm (198.10 283.33mg/kg/day for males and 245.65
319.73mg/kg/day for females). The NOEL for general toxicity in parents and pups
was considered to be 100ppm (6.74 8.13mg/kg/day for males and 8.40 9.58mg/kg/day
for female).
(Bio/dynamics, Inc., 1984)
2. Developmental Toxicity Study in Rats
Butachlor was administered by gavage to 3 groups of 25 mated C-D female rats at
dosages of 49, 147, and 490mg/kg/day on days 6 through 19 of gestation. An
additional 25 mated females received distilled water and served as controls. On
gestation day 20, fetuses were examined externally, weighed, and processed for skeletaland soft-tissue examinations. Maternal toxicity occurred in the high dose group as
evidenced by a decrease in maternal body weight. No butachlor-related effects wereobserved on total implantations, post-implantation loss, viable fetuses or fetal sex
distribution at any dosage level. No effects considered related to butachlor exposure
were seen following external, soft-tissue or skeletal examination of fetuses from all 3
dose levels tested. The NOEL for maternal toxicity in the rat was considered to be147mg/kg/day. Butachlor did not induce a teratogenic effect in rats.
(International Research and Development Corp., 1980)
3. Developmental Toxicity Study in Rabbits
Three groups of 16 mated female Dutch belted rabbits were administered butachlorby oral gavage at dosages of 49, 147, and 245mg/kg/day and an additional 16 matedfemales received distilled water and served as controls. All animals were dosed daily
from gestation day 7 through 27. All surviving females were sacrificed on gestation
day 28 and routine uterine examinations were performed. All fetuses were weighed
and examined for external malformations. Each fetus was then examined internallyby dissection and then stained for skeletal evaluation.
The administration of butachlor resulted in significant maternal toxicity at the
147mg/kg/day and 245mg/kg/day dose levels. In addition to treatment-related deaths
and abortions, mean maternal body weight losses, increases in post-implantation loss
and decreases in fetal body weights were observed. The NOEL for maternal toxicity
in the rabbit was considered to be 49mg/kg/day. Butachlor did not induce a teratogenic
effect in rabbits.
(International Research and Development Corporation, 1980)
PHARMACOLOGY STUDIES
Butachlor was administered intraperitoneally to groups of 5 male and female ICR
mice each, at single doses of 0, 125, 215, 350, 600, and 1000mg/kg. All animals
given the highest dose level died within 4 to 24 hrs after administration. Animal
awareness, motor activity and autonomic nervous system responses were suppressedat levels of 600mg/kg or above. Male and female mice given butachlor at a dose of
1000mg/kg exhibited abnormal signs of toxicity characterized by excitation of thecentral nervous system, writhing, exophthalmos, hypothermia, lacrimation and softstool. Most of the signs observed in the 600mg/kg group were diminished within 24
hrs after administration. No signs of toxicity were observed in animals given butachlorat dose levels of 350mg/kg or less.
After acute oral administration of butachlor to 3 male and 3 female albino rabbits
at dose levels of 0, 1000, 2300, and 5000mg/kg, no abnormalities were observed at
any of the levels tested. Butachlor was administered intravenously to groups of 3
male rabbits at dose levels of 50 and 150mg/kg. In the 150mg/kg group, respiration
was increased, and transient decreases in blood pressure, heart rate and blood flow
were observed after administration. No abnormality was observed in the 50mg/kg
group.
In further experiments to explore the effects on the autonomic nervous system,
gastrointestinal tract, skeletal muscle, or blood, butachlor produced no significant
effect. In conclusion, butachlor has little or no effect on standard physiologicalparameters and pharmacological end-points. The effects observed occurred primarily

at acutely lethal dose levels. The high dose levels required to produce severe effects
confirm that the acute toxicity of butachlor is relatively low.
(Nippon Experimental Medical Research Institute Co., Ltd., 1993)
EPIDEMIOLOGY STUDIES
Three epidemiology studies have been conducted on the manufacturing workers at
the Monsanto Muscatine plant in Iowa motivated primarily by the findings from theavailable toxicology studies with alachlor in which ocular and oncogenic effects wereobserved in the Long-Evans rat. The same types of tumors were also observed in the
toxicology studies with butachlor in the Sprague-Dawley rat. The outcome of extensive
research investigations has proven that the same mechanisms are operative for theformation of stomach, nasal and thyroid tumors in rats with these compounds. The
history of the Muscatine plant indicates that there is a high correlation of alachlor andbutachlor exposure among manufacturing workers, and therefore, the results from thealachlor epidemiology studies are also relevant for butachlor.
A group of the highest-exposed workers was examined for the presence of a
specific eye abnormality, analogous to the initiating lesion in Long-Evans rats, called
pigmentary dispersion syndrome (PDS).17) None of the exposed workers had PDS,
and prevalence rates for other eye abnormalities were similar in exposed and unexposed
individuals. In another study, there was no indication of increased mortality rates
from cancer or any other causes among workers with up to 25 years of follow-up.18)
Likewise, cancer incidence rates over a 24-year period were not elevated in thehighest-exposed alachlor/butachlor workers, nor were there any cases of tumors in
the nose, stomach, or thyroid, the organs in which oncogenic effects were produced
in rats.19) These manufacturing workers had year-round exposure greatly exceeding
agricultural exposure, which is limited to a two- or three week period of application.
In fact, manufacturing exposure was estimated to exceed yearly agricultural exposure
by a factor of 10,000 or more.19) Potential dietary exposures are even lower thanagricultural exposure. Therefore, the absence of eye effects or increased mortality
and cancer rates in manufacturing workers serve as an important indicator of the low
potential for adverse effects among the general population, who are exposed to extremely
low levels of alachlor/butachlor if at all.
CONCLUSIONS
The results of tests in laboratory animals indicate that butachlor has low mammalian
toxicity following acute oral, dermal, and inhalation exposure. Butachlor has somepotential to produce allergic skin reactions following repeated or prolonged exposure.
Subchronic and chronic exposure produces primarily liver and kidney toxicity forwhich large margins of safety exist. Butachlor is not genotoxic and does not interferewith normal reproductive and developmental processes. Butachlor is not oncogenic

in the Fischer-344 rat and CD-1 mouse. Neoplastic lesions were observed in theSprague-Dawley rat in the nasal turbinate mucosa, stomach glandular mucosa, andthyroid follicular epithelium. Significant increases in the stomach, thyroid and nasal
tumors occurred only at or above the MTD dose which produced excessive toxicity.
A large body of data supports the conclusion that butachlor produces these tumorsin rats through non-genotoxic mechanisms for which thresholds exist. The conditions
driving the requisite threshold-sensitive preneoplastic events occur only in experimental
studies employing doses at or above the MTD. Observed species differences inphysiology and metabolism indicate that the rat is particularly susceptible to the
induction of nasal, thyroid and stomach tumors following chronic exposure to thisagent. Consequently, humans would be at significantly lower risk for developmentof these tumors. In fact, substantial margins of safety exist, as human exposure isseveral orders of magnitude below the doses producing tumors in rats. Therefore, it
is concluded that the butachlor-induced tumors in rats are not relevant to humans
under actual conditions of exposure. This conclusion is supported by the lack ofmortality and tumors in manufacturing workers who have the highest exposure tobutachlor.
In summary, an extensive health and safety database has been developed to support
the registration of butachlor and MACHETE® products worldwide. A thoroughevaluation of these data indicates that butachlor products are of generally low toxicity
and present minimal opportunity for human exposure. When used in accordancewith label directions, butachlor will not adversely affect human health.
Based on a review of the studies summarized above, the Japan Ministry of Healthand Welfare (MHW) established an Acceptable Daily Intake (ADI) for butachlor of
0.01mg/kg/day employing the NOEL from the chronic rat study of 1mg/kg/day and asafety factor of 100. This ADI supports the current withholding limits establishedby the Japan Environmental Agency (EA) of 0.1ppm in rice.

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REFERENCES
E. Buehler: Arch. Dermatol. 91, 171 (1965)
H. Ritz & E. Buehler: Current Concepts in Cutaneous Toxicity," ed. by V.
Drill & P. Lazar, Academic Press, New York, pp. 25 40, 1980
W. Heydens, A. Wilson, L. Kier, H. Lau, D. Thake & M. Martens: Hum. Exp.
Toxicol. 18, 363 391 (1999)
P. Feng, A. Wilson, R. McClanahan, J. Patanella & S. Wratten: Drug Metab.
Dispos. 18 373 (1990)
A. Wilson, H. Lau, K. Asbury, D. Thake & W. Heydens: Presented at International
Congress of Toxicology-VII, Seattle, 1995
A. Li, K. Asbury, W. Hopkins, P. Feng & A. Wilson: Drug Metab. Dispos. 20,

616 (1992)
7) A. Wilson, H. Lau, K. Asbury & W. Heydens: Toxicologist 15, Abstract 1398
(1995)
8) A. Wilson, D. Branch, M. Shibata, D. Thake, T. Shirai & A. Hagiwara: Presented
at Annual Conference of International Federation of Societies of Toxicologic
Pathologists, Tours, 1995
9) G. Hard, M. Iatropoulos, D. Thake, D. Wheeler, M. Tatematsu, A. Hagiwara, G.
Williams & A. Wilson: Exp. Toxic. Pathol. 47, 95 (1995)
10) D. Thake, M. Iatropoulos, G. Hard, K. Hotz, C. Wang, G. Williams & A.
Wilson: Exp. Toxic. Pathol. 47, 107 (1995)
11) D. Thake, K. Hotz, C. Reisch & A. Wilson, A Study of the Mechanism of
Butachlor Induced Carcinogenicity in Female Sprague-Dawley Rats," UnpublishedMonsanto Report, MSL-14017, 1995
12) R. Hill, L. Erdreich, O. Paynter, P. Roberts, S. Rosentahl & C. Wilkinson:
Fund. Appl. Toxicol. 12, 629 (1989)
13) G. Thomas & E. Williams: Mutat. Res. 248, 357 (1991)
14) G. Zbinden: Arch. Toxicol. Suppl 12. 98 (1988)
15) D. Brusick, J. Ashby, F. de Serres, P. Lohman, T. Matsushima, B. Matter, M.
Mendelsohn, D. Moore II, S. Nesnow & M. Waters: Mutat. Res. 266, 1 (1992)
16) P. Lohman, M. Mendelsohn, D. Moore II, M. Waters, D. Brusick, J. Ashby &W. Lohman: Mutat. Res. 266, 7 (1992)
17) B. Ireland, J. Acquavella, T. Farrell, M. Anne & T. Fuhremann: J. Occup. Med.
36, 738 (1994)
18) J. Acquavella, S. Riordan, M. Anne, J. Collins, B. Ireland & W. Heydens:
Environ. Health Perspect. 104, 2 (1996)
19) J. Acquavella, B. Ireland, T. Leet, M. Anne, T. Farrell & M. Martens: In
Proceedings of the XII Joint CIGR, IAAMRH, IUFRO International Symposium:
Health , Safety and Ergonomic Aspects in Use of Chemicals in Agriculture andForestry, June, 1993, Kiev, Ukraine: Institute for Occupational Health, p.184,
1994

米国モンサント・カンパニー
ブタクロールは，米国モンサント・カンパニーが開発した一年生イネ科雑草および一部の広葉雑草を対象とする稲作用除草剤である．動物実験の結果，本剤の哺乳動物に対する急性経口，経皮，吸入毒性はいずれも軽微で，眼および皮膚一次刺激性は低い．モルモッ
トを用いた皮膚感作性試験において陽性の反応が認められた．亜急性および慢性毒性試験の結果，主として肝臓および腎臓に検体投与による影響が認められたが，これらの影響には閾値が存在した．ラットを用いた慢性毒性/発がん性併合試験において腺胃，鼻部および甲状腺の腫瘍が認められたが，腫瘍発生のメカニズムに関する試験研究の結果，これらの腫瘍は閾値の存在する非遺伝子傷害性の作用によって引き起こされていることが解明されている．これらの腫瘍の発生に結びつく前段階の症状には閾値があり，最大耐量以上の用量でブタクロールを投与した動物実験においてのみ認められた．また，鼻部の腫瘍の発生には，ラットにおいてのみ認められる種特異的代謝が関与していた．このようにラットにおいて観察された腫瘍をヒトに外挿することは妥当ではない．この結論は，ブタクロールへの暴露量が最も高いと考えられるブタクロールおよびアラクロール製造工場の労働者を対象とした疫学調査の結果，死亡率および腫瘍発症率の増加が認められなかったことか
らも支持されている．本剤には，哺乳動物生体内における遺伝毒性は認められず，正常な繁殖や発生過程を阻害することもなかった．
ここに要約した毒性試験成績の評価に基づき，ラットにおける慢性毒性/発がん性併合試験における無毒性量1mg/kg/日および安全係数100を用い，0.01mg/kg/日のADIが設定されている．
ブタクロールは，マーシェット®粒剤として昭和48年3月登録を取得して以来，水田用の雑草発生前土壤処理剤として広く使用されている．登録保留基準は，米に0.1ppmと設定されている．

Contacts
Monsanto Company, 800 N. Lindbergh Blvd., St. Louis, MO 63167, USA
Regulatory Affairs Team, Agro-Science Division, Monsanto Japan Limited, Mita 43
Mori Bldg., 3-13-16, Mita, Minato-ku, Tokyo 108-0073, Japan

日本モンサント株式会社アグロサイエンス事業部テクノロジー本部規制・環境部〒108-0073 東京都港区三田3-13-16 三田43森ビル