333

TERBUFOS 333–385 JMPR 2003

TERBUFOS

First draft prepared by

K. L. Hamernik

Office of Science Coordination and Policy, United States Environmental Protection

Agency Washington DC, USA

Explanation.................................................................................................... 333

Evaluation for acceptable daily intake.......................................................... 333

Biochemical aspects ................................................................................ 333

Absorption, distribution and excretion .............................................. 333

Biotransformation .............................................................................. 337

Toxicological studies ............................................................................... 337

Acute toxicity..................................................................................... 337

Ocular and dermal irritation......................................................... 339

Dermal sensitization..................................................................... 339

Short-term studies of toxicity ............................................................ 339

Oral administration....................................................................... 339

Exposure by inhalation................................................................. 354

Long-term studies of toxicity and carcinogenicity............................ 355

Genotoxicity ....................................................................................... 360

Reproductive toxicity ......................................................................... 360

Multigeneration study................................................................... 360

Developmental toxicity................................................................. 364

Special studies.................................................................................... 370

Neurotoxicity ................................................................................ 370

Acute neurotoxicity ...................................................................... 372

Neurotoxicity after repeated doses............................................... 374

Studies on metabolites.................................................................. 376

Observations in humans .......................................................................... 379

Comments...................................................................................................... 379

Toxicological evaluation................................................................................ 382

References ..................................................................................................... 384

Explanation

Terbufos is an organophosphorus compound, classified as a systemic insecticide and

nematocide, and was last evaluated by the JMPR in 1989, when an ADI of 0–0.0002mg/kg

bw was established. Terbufos was considered by the present Meeting within the periodic

review programme of the Codex Committee on Pesticide Residues.

Evaluation for acceptable daily intake

1. Biochemical aspects

1.1 Absorption, distribution and excretion

Rats

In an study of metabolism, 16 male Royal Hart Wistar rats were given a single dose

of [14C]terbufos (purity, uncertain; specific activity of 26.4mCi/mg labelled at the carbon

atom of the thiomethyl portion of the parent molecule (or methylene carbon) in ethanol and
water (1:1) at 0.8mg/kg bw by gavage. Animals were housed individually in metabolism

cages. Urine was collected on dry ice at 6, 12, 24, 48, 72, 96, 120, and 168h after dosing,

and faeces were collected and frozen at 12, 48, 72, 96, 120, and 168h after dosing. After

thawing, urine samples were pooled for each time interval. Groups of three animals were

terminated at 6, 12, 24, and 48h after dosing and the final group of four animals was 

terminated at 168h after dosing. Livers, kidneys, gastrointestinal tract, muscle, skin, fat and

blood were taken at these times.

Radioactivity was extracted from the urine, faeces and tissues, and metabolites in

urine, faeces, liver, kidney and muscle were separated and identified. In addition, the amount

of 14CO2 in expired air over a 72h period was determined in one animal that had received

a dose of 0.2mg/kg bw by gavage. There was no indication of overt toxicity. Elimination

was relatively rapid and fairly complete. About 90% of the administered dose was recovered by the end of the study. Over the entire duration of the study, approximately 83% of

the administered dose was found in the urine, which was thus the major route of elimination; about 72% of the administered dose was excreted by 24h and 80% was excreted by

48h. Over the course of the study, about 3.5% of the radiobel was found in the faeces. The

recovery of the administered dose reached a peak of 31.2% in the urine by 24h, and a peak

of about 2% in the faeces by 48h. Tissue concentrations of radiolabel reached a maximum

at between 6 and 12h after dosing. By 168h after dosing, the concentration of radiolabel

in each body tissue examined was <0.1mg/kg. The total percentage of the administered dose

recovered in tissues by 168h was greatest in the liver (0.34%), followed by the gastrointestinal tract (0.087%), blood (0.036%), kidney (0.034%), muscle (0.024%), skin (0.017%)

and fat (0.003%). No 14CO2 was recovered during the designated interval of 72h.

After extraction and thin-layer chromatography, metabolites that did not contain phosphorus were found to account for about 96% of the radiolabel present in urine. The predominant species appearing by 6h after dosing was CL 202474, with lesser amounts of CL

99843, CL 99844 and CL 99875 (see Figure 1 and Table 1). Small amounts of parent compound and other species containing phosphorus accounted for most of the remainder of the

radiolabel (2–3%) in the urine. Two peaks of radioactivity in urine were not definitively

identified.

In the faeces, at 12h after dosing, about 95% of the radiolabel comprised species containing phosphorus (mostly metabolites CL 92320 and CL 94221, with lesser amounts of

CL 94302 and CL 94301). At subsequent time intervals, in addition to CL 92320, nonphosphorus-containing metabolites CL 202474, CL 99843, and CL 99875 predominated.

Very little parent compound was found in the faeces.

The metabolites identified in urine and faeces were also observed in tissue extracts;

the types of metabolite found at detectable levels depended on the tissue and time after

dosing. In liver, kidney and muscle, the approximate ratio of species not containing phosphorus (four metabolites) to those containing phosphorus (e.g. five phosphorus-containing

metabolites and small amounts of parent compound) at 6h after dosing were 2.6, 11 and 

6 in liver, kidney and muscle respectively, and about 9, 23 and 7 for these same tissues at

12h after dosing. Possible sex differences in the metabolic fate of terbufos were not

addressed by this study (North, 1973). No statements of compliance with quality assurance

(QA) or good laboratory practice (GLP) were provided. The study was not performed

according to a specific guideline. Despite the limitations of the study some useful information can be extracted.

334

TERBUFOS 333–385 JMPR 2003
Radiolabelled terbufos (purity, >98%; specific activity, 61.4mCi/g), labelled with 14C

at the methylene carbon position of the parent molecule, was administered in corn oil by

gavage to groups of fasted male and female Crl:CD®(SD)BR rats. Groups of five male and

five female rats were given single oral doses of 0.1mg/kg bw (lowest dose) or 0.4mg/kg

bw (highest dose) and additional groups of one male and one female were similarly treated

and used for collection of volatiles. In the multiple-dose segment of the study, groups of

rats were given non-radiolabelled terbufos (purity, 97.8%) as single oral doses of 0.1mg/kg

bw in corn oil for 14 days, followed by a single dose of radiolabelled material of 0.1mg/kg

bw on day 15. Additional groups of one male and one female were similarly treated with

terbufos at a dose of 0.1 and used for collection of volatiles. Urine and faeces for all groups

were collected 0–6, 6–12, and 12–24h after the administration of radiolabelled terbufos and

daily thereafter until termination at 168h. Cage rinses were collected as necessary. Volatiles

were collected at intervals of up to 7 days after dosing. Radioactivity was extracted from

urine, faeces and tissues (blood, bone, brain, fat, ovaries, testes, heart, liver, kidneys, lungs,

muscle, spleen, uterus and residual carcass). Metabolites were characterized and identified

only in the urine and faeces and only for the 12–24h interval after dosing.

No toxicity was reported. In all the treated groups, total recoveries of the administered radiolabel was about 93–99% in males and 89–96% in females at 168h after dosing.

Relatively rapid elimination, primarily in the urine, indicated fairly fast and appreciable

absorption via the gastrointestinal tract. Urinary excretion of radiolabel was 76% and 79%

in males and females treated with single low doses of terbufos, and 79% and 69% in males

and females treated with single high doses of terbufos, at 168h after dosing. In animals

receiving repeated low doses of terbufos, urinary excretion of radiolabel was 86% and 85%

of the administered dose in males and females. Faecal elimination in groups of males and

females treated with low and high single doses was 13–17% of the administered dose and

in the groups receiving repeated doses was about 5–7% in both sexes. In males and females

in all treated groups, much smaller amounts of radiolabel, as a percentage of the administered dose, were found in the tissues (about 0.09–0.15%), in the carcass (about 0.90–2%),

in expired CO2 (about 2–4%) and in volatiles (0.06–0.5%), and most of the radiolabel was

eliminated in excreta by 24h after dosing.

The highest concentrations of residues in tissues at 168h after dosing were found in

animals receiving single, high doses. For all dosing regimens, the highest tissue residues

were found in the lungs of both sexes (single low doses, 0.003–0.005mg/kg; repeated doses,

0.005–0.007mg/kg; and single high doses, 0.018–0.022mg/kg) and there was no indication

of bioaccumulation.

Metabolites in urine and faeces were characterized only for the period between 12

and 24h after dosing (Figure 1 and Table 1). Since appreciable amounts of radiolabel were

excreted before this interval, the metabolites cannot be reliably quantified relative to percentage of administered dose. In addition, variation in the radiolabel detected in cage rinses

for individual animals (about 6–56% of the administered dose) contributed to uncertainty

in metabolite quantification during the 12–24h interval.

For all dosing regimens and for both sexes, about 70–90% of the urinary residue of

radiolabel was reported to have been characterized for the 12–24h period. Of this, about

67–80% of the residue was described as non-phosphorus-containing metabolites, about

1–4% as phosphorus-containing metabolites and about 2–9% as unknown substances (two

substances). Of the non-phosphorus-containing metabolites, the major metabolite was CL

335

TERBUFOS 333–385 JMPR 2003
202474; lesser amounts of CL 99843, CL 99844 and CL 99875 were found. The only 

phosphorus-containing metabolite detected was CL 94365; no parent compound was found.

During the 12–24h interval, for all dosing regimens and for both sexes, about 49–80% of

the faecal residue of radiolabel was reported to have been characterized. Of this, about

17–34% was described as non-phosphorus-containing metabolites, about 8–44% as 

phosphorus-containing metabolites, and about 7–22% as unknown substances (five 

336

TERBUFOS 333–385 JMPR 2003

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O O

O O

O O

O

(CH3)3C-S-CH2-S-P-(OC2H5)2

S

(CH3)3C-S-CH2-S-CH3

CL 99843

(CH3)3C-S-CH2-S-CH3

CL 99875

Terbufos (CL 92100)

(CH3)3C-S-CH2-S-CH3

CL 202474

(CH3)3C-S-CH2-S-P-(OC2H5)2 (CH3)3C-S-CH2-S-P-(OC2H5)2

S

CL 94221 CL 94301

Desulfuration Sulfoxidation

Sulfoxidation

(CH3)3C-S-CH2-S-P-(OC2H5)2

S

CL 94320

(CH3)3C-S-CH2-S-P-(OC2H5)2

Desulfuration

CL 94302

(CH3)3C-S-CH2-S-CH3

CL 99844

(CH3)3C-S-CH2-S-P-(OC2H5)2

Sulfoxidation

(s

S-methylation

Sulfoxidation

Sulfoxidation

Sulfoxidation (s–p) hydrolysis

S-methylation

Sulfoxidation

Sulfoxidation

Sulfoxidation

CL 94365

–p) hydrolysis

Figure 1. Proposed metabolic pathway of terbufos in rats

Table 1. Terbufos and its metabolites

CL No. Chemical name Common name

92,100 O,O-diethyl-S-t-butylthio-methylphosphorodithioate Terbufos (parent compound)

94,301 Phosphorodithioic acid, S-(t-butylsulfinyl) methyl O,O-diethyl ester Terbufos sulfoxide

94,320 Phosphorodithioic acid, S(t-butylsulfonyl) methyl O,O-diethyl ester Terbufos sulfone

94,221 Phosphorothioic acid, S-(t-butylthio) methyl O,O-diethyl ester Terbufoxon

94,302 Phosphorothioic acid, S-(t-butylsulfonyl) methyl O,O-diethyl ester Terbufoxon sulfone

94,365 Phosphorothioic acid, S-(t-butylsulfinyl) methyl O,O-diethyl ester Terbufoxon sulfoxide

202,474 Methane, (t-butylsulfinyl)(methylsulfinyl)

99,844 Sulfoxide, (t-butylsulfonyl) methyl methyl

99,843 Sulfoxide, t-butyl (methyl-sulfonyl) methyl

99,875 Sulfone, t-butyl (methyl-sulfonyl) methyl

From North (1973) and Cheng (1992)

From Cheng (1992)
substances). The non-phosphorus-containing metabolites were CL 202474, CL 99843

and/or CL 99844, and CL 99875. The major phosphorus-containing entity was the parent

compound (CL 92100) with lesser amounts of CL 94301 and CL 94365. The proposed

metabolic pathway for terbufos in rats, on the basis of the metabolites found in excreta, is

depicted in Figure 1. Sulfoxidation and desulfuration of terbufos is followed by hydrolysis

of the thiolophosphorus bond (S–P), enzymatic S-methylation and then additional 

S-oxidation.

There were no apparent sex differences in the absorption and metabolic fate of

[

14C]terbufos in Sprague-Dawley rats on the basis of the results of this study (Cheng, 1992).

Statements of compliance with QA and GLP were provided. The protocol was generally

consistent with United States Environmental Protection Agency (EPA) Subdivision F Guidelines (November 1982 and revised, 1984).

1.2 Biotransformation

In a study of the biotransformation of terbufos (purity, 91%; apparently purchased

from a pesticide factory in China), livers of male Wistar rats (180–220g) were perfused in

situ with 100ml of terbufos (0.1mol/l, dissolved in methanol and added to the perfusate

reservoir) for 1h at a flow rate of 5ml/liver per min. Metabolic materials collected from

perfusate effluent were separated with a solid-phase extraction cartridge and were characterized and quantified by gas chromatography–infrared spectrometry (GC–IR) and gas 

chromatography–mass spectrometry (GC–MS). The recovery of terbufos and its metabolites was expressed as a percentage of the concentration of the parent compound entering

the liver. Recovery was incomplete, totaling only about 47.13%. Substances in the effluent

separated into five major peaks. These were identified as terbufos (40.8%), terbufos oxon

(2.13%), and three triakylphosphorothioate metabolites: metabolite I: (C2H5O)2POSCH3

(hydrolysate of terbufos oxon, 0.13%); metabolite II: (C2H5O)2PSSCH3 (hydrolysate of terbufos, 2.65%); and metabolite III: (C2H5O)2PSSC2H5 (methylate of the metabolite II,

1.42%). It was suggested that metabolite III may have formed via a detoxification reaction

involving S-adenosyl-L-methionine methyl transferase. The potential for certain trimethyl

and triethyl phosphorothioates, such as metabolites I, II and III, to cause cholinergic toxicity and/or pulmonary toxicity in rats by a non-cholinergic mechanism or mechanisms was

discussed. Sulfoxide and sulfone metabolites were not detected in the effluent (Li et al.,

1999).

2. Toxicological studies

2.1 Acute toxicity

The acute toxicity of terbufos is summarized in Table 2.

Terbufos is of very high acute toxicity when administered by the oral, dermal, or

inhalation routes. LD50 values for acute oral toxicity in rodents and dogs were similar,

ranging from 1.4 to 9.2mg/kg bw. The acute dermal LD50 was about 1mg/kg bw in rabbits,

and the acute inhalation LC50 in rats ranged from 0.0012 to 0.0061mg/l. Clinical signs

observed were those typical of cholinergic toxicity and, depending on the study, route 

and species, included tremors, salivation, exophthalmos, prostration, decreased activity,

chromodacryorrhoea, diuresis, piloerection, ataxia, urogenital staining, nasal discharge,

anorexia, and laboured breathing. Deaths following acute exposures occurred within

minutes to hours or up to a week after administration. With regard to dermal absorption,

terbufos is rapidly penetrating after dermal or ocular application.

337

TERBUFOS 333–385 JMPR 2003
338

TERBUFOS 333–385 JMPR 2003

Table 2. Acute toxicity of terbufos

Species Strain Sex Route Vehicle LD50 (mg/kg Purity (%) Reference

bw) or LC50

(mg/l)

Mouse CF1 albino Female Oral Corn oil 5.0 85.8 American

Cyanamid

Company A72-

95 (1972a)

Mouse CF1 albino Female Oral Corn oil 9.2 96.7 Morici (1972)

Mouse CF-1 albino Male Oral Corn oil 3.5 96.7 Morici (1972)

Rat Wistar (RH albino) Female Oral Corn oil 9.0b 96.7 Morici (1972)

Rat SD Female Oral Corn oil 1.4d 89.7 Bradley (1996)a

(Crl : CD(SD)BR)

Rat SD Male Oral Corn oil 3.2d 89.7 Bradley (1996)a

(Crl:CD(SD)BR)

Rat Wistar Male Oral Corn oil 1.6d 85.8 American

RH albino) Cyanamid

Company A72-

95 (1972a)

Rat Wistar (RH albino) Male Oral Corn oil 4.5b 96.7 Morici (1972)

Rat SD Female Inhalation; 4 h, Administered as a 0.0012 89.6 Hoffman

(CD) whole body vapour (1.2mg/l) (1987)6

Rat SD Male Inhalation; 4 h Administered as a 0.0061 89.6 Hoffman

(CD) whole body vapour (6.1mg/l) (1987)6

Rat Wistar (RH albino) Male Inhalation; 7 h Administered as a Could not be 96.7 Morici (1972)

vapour calculatedg

Rabbit New Zealand white Female Dermal Report stated 0.93b 89.6 Fischer (1985)

applied as receivedc

Rabbit New Zealand Male Dermal Report stated 0.81b 89.6 Fischer (1985)

white applied as receivedc

Rabbit Albino Male Dermal Report stated 1.0 85.8 American

applied as receivedc Cyanamid

Company A72-

95 (1972a)

Rabbit Albino Male Dermal Report stated 1.1 96.7 Morici (1972)

applied as receivedc

Dog Beagle Female Oral Report stated 6.3d 96.7 Morici (1972)

applied as receivede

Dog Beagle Male Oral Report stated 4.5d 96.7 Morici (1972)

applied as received

Although reports for most of these studies (except as footnoted below) were summary in nature and did not contain GLP or QA

statements, protocols appeared to be generally consistent with the intent of EPA Subdivision F Guidelines (1982 or 1984, revised) a

Detailed report contained QA and GLP statements, but stated there was no confirmation of the concentration of test material; the

protocol was consistent with US EPA Subdivision F Guidelines (1982 or 1984, revised) bReport stated that animals were not fasted

c

Test material was a liquid

dReport stated that animals were fasted

e

Test material in a gelatin capsule was administered to fasted animals f

Detailed report contained QA and GLP statements and protocol was consistent with US EPA Subdivision F Guidelines (1982 or

1984, revised)

gTen animals were exposed for 7 h at 25°C to air that was near-saturated with product vapour at a nominal chamber concentration of

1.99 mg/l. There were two deaths, one on day 5 and the other during days 6–14 after dosing. Clinical findings described as transient

irritation and discomfort were present at 0–15 min after dosing and the lung of one survivor was abscessed at necropsy. The findings

of this study are inconsistent with those of Hoffman, 1987
(a) Ocular and dermal irritation

Rabbit

In a study of primary skin irritation, 0.5ml of technical-grade terbufos (purity, 96.7%)

was applied “as received” to shaved rabbit skin for 24h under an impervious patch. The

product was extremely toxic by the dermal route when administered in a single treatment;

all rabbits (number not specified) died within 24h after dosing. All animals exhibited signs

of cholinesterase inhibition before death. The product (a liquid) was said to penetrate rabbit

skin and mucous membranes very easily. No indications of dermal irritation or corrosion

were reported (Morici, 1972).

In a second study of primary skin irritation, a single application of 0.25ml of 

technical-grade terbufos (purity, 85.8%) was administered “as received” to shaved rabbit

skin in a similar protocol to that of Morici (1972), with similar results. All animals died

within 24h after dosing and showed signs of cholinesterase inhibition before death. No skin

irritation was reported (BASF, 1972a).

In a study of primary eye irritation, 0.1ml of technical-grade terbufos (purity, 96.7%)

was applied “as received” to the conjunctival sacs of six rabbits. The product was extremely

toxic by the ocular route when administered in a single treatment; all animals died on the

day of dosing and were observed to exhibit signs of cholinesterase inhibition before death.

The product (a liquid) was said to penetrate rabbit skin and mucous membranes very easily.

No indications of ocular irritation were reported (Morici, 1972).

In a second study of primary eye irritation, a single application of 0.1ml of 

technical-grade terbufos (purity, 85.8%) was introduced “as received” into the conjunctival

sacs of six rabbits in a protocol similar to that of Morici (1972), with similar results. All

animals died 2–3h after dosing and were observed to exhibit signs of cholinesterase inhibition before death. No ocular irritation was reported (BASF, 1972a). Although the reports

for most of these studies were summary in nature and did not contain statements of compliance with GLP or QA, protocols appeared to be generally consistent with the intent of

EPA Subdivision F Guidelines (1982 or 1984, revised).

(b) Dermal sensitization

A study of skin sensitization of technical grade terbufos was not performed owing to

the severe toxicity observed in the studies of primary skin and eye irritation.

2.2 Short-term studies of toxicity

(a) Oral administration

Mice

In a preliminary study, groups of 10 male and 10 female albino CF1 mice were given

diets containing technical-grade terbufos (CL 92100; purity, 96.7%) at a nominal concentration of 0, 1.0, 4.0 or 16mg/kg (nominally equivalent to 0, 0.218, 0.911 and 3.30mg/kg

bw per day in males and 0, 0.286; 0.988 and 3.70mg/kg bw per day in females) for 31 days.

Data on analysis of concentrations of test material in the diet were not provided, so test

material intake and stability in the feed could not be confirmed. Very few parameters were

evaluated. Cholinesterase activity was not assessed. Six females at the highest dose were

found dead between days 5 and 14 and one female at the lowest dose was found dead on

339

TERBUFOS 333–385 JMPR 2003
day 9. Autolysis prevented attempts to determine the cause of death. Mortality was, however,

only notable in females receiving the highest dose (60%), therefore it is possible that the

increase in mortality was related to treatment. Decreases in body weight and food consumption were reported in males and females at the highest dose. Weights of the two organs

examined (liver and kidney) in four to five animals of each sex per group treated with terbufos were not statistically significantly different from those of respective control groups.

For the parameters examined, no effects were observed in other groups. It was reported that

upon gross pathological examination of four to five animals of each sex per group at study

termination, no gross lesions were found that were attributed to treatment (gross pathology

data were not provided). The no-observed-adverse-effect level (NOAEL) in males and

females was nominally 4.0mg/kg (nominally equivalent to 0.911mg/kg bw per day in males

and 0.988mg/kg bw per day in females; intake of test material could not be confirmed) on

the basis of decreases in body weight and food consumption in both sexes and mortality of

60% at the dose above in females only (Morici, 1972). No statements of compliance with

QA or GLP were provided and the study was not performed to address a specific guideline.

Rats

In a short-term feeding study, groups of five male and five female Sprague-Dawley

rats (aged 4 weeks at study initiation) were given diets containing technical-grade terbufos

(CL 92100; purity, 90.1%), prepared in a vehicle of corn oil and methylene chloride (1:1),

at a concentration of active ingredient of 0, 0.125, 0.250, 0.500, 1.00, 3.00 or 6.00mg/kg

per day (equal to 0, 0.020, 0.039, 0.080, 0.16, 0.49, and 0.77mg/kg bw per day in males

and 0.017, 0.033, 0.066, 0.132, 0.409 and 0.750mg/kg bw per day in females, respectively)

for 14 days. The parameters evaluated were limited, including observations for mortality,

morbidity and clinical signs of toxicity (data for individual animals were not provided for

signs), measurement of body weight and food consumption, organ weight determinations

(liver and kidney only) and a gross examination at study termination. Plasma and erythrocyte cholinesterase activity was assessed before treatment and on days, 1, 4, 7, and 14 in

the control groups and in groups of males and females receiving the four lowest doses (i.e.

0.020, 0.039, 0.080 and 0.16mg/kg bw per day). Cholinesterase inhibition was determined

relative to the value for the appropriate concurrent control group. Brain cholinesterase activity was not measured, and clinical chemistry, haematological, urine and histopathological

examinations were not conducted in this study.

There were two deaths; two females in the group receiving the highest dose 

(6.00mg/kg) died or were sacrificed in a moribund condition on days 11 and 13 from treatment-related causes, respectively. Before death, the animals exhibited severe tremors, salivation and prostration. Clinical signs of toxicity in males and females were reported to start

on day 2 and last until termination in the group receiving the highest dose (6.00mg/kg) and

included, initially, ataxia, tremors and miosis. From day 7 on, more severe tremors developed and exophthalmos and piloerection were also observed. At 3.00mg/kg, clinical signs

(slight tremors) were also noted in both sexes from day 4 until the end of the study. Signs

of toxicity were considered to be treatment-related and were not observed at lower doses.

Changes in body weight and/or food consumption seen at the two highest doses were also

considered to be related to treatment. Statistically significant decreases in body weight and

body-weight gain relative to respective control groups were observed in both sexes (being

more severe in females) at 6.00mg/kg during both weeks. Food consumption was also statistically significantly decreased in males and females at the highest dose in weeks 1 and 2.

At 3.00mg/kg, body weight was reduced (statistically significantly in males) during the first

340

TERBUFOS 333–385 JMPR 2003
week only in both sexes, while food consumption did not appear to be affected. Statistically

significant decreases in liver and kidney absolute weights and weights relative to body

weight were observed in both sexes at 6.00mg/kg, but terminal body weights were also

reduced. At gross necropsy, there were no findings that were attributed to treatment with

the test material in any group.

At 1.00mg/kg (the highest dose assessed), plasma cholinesterase activity was statistically significantly inhibited by 21–37% at all time-points during treatment in males, and

by 27–38% on days 4, 7, and 14 in females. At 0.500mg/kg, plasma cholinesterase was 

statistically significantly inhibited on day 4 in males (15%) and on days 4 and 7 in females

(23%), but no significant inhibition was observed on day 14 in either sex. Erythrocyte

cholinesterase was statistically significantly inhibited at 1.00mg/kg (the highest dose

assessed) in males by 51–61% and in females by 40–52%, on days 4, 7 and 14. At 

0.500mg/kg, a statistically significant reduction in erythrocyte cholinesterase activity was

noted on these same days, ranging from 22% to 25%, in males. In females at this dose, a

significant decrease in erythrocyte cholinesterase activity of only 16% was seen on day 4.

This finding in females was not considered to be of toxicological relevance, although the

study authors considered that the decreases in erythrocyte cholinesterase activity in both

sexes at this dose were associated with administration of the test material. If inhibition of

plasma cholinesterase activity is not considered to be an adverse effect, and considering

that brain cholinesterase activity was not measured in this study, the NOAEL was 

0.250mg/kg (equal to 0.0039mg/kg bw per day) in males and 0.5mg/kg (equal to 

0.066mg/kg bw per day) in females on the basis of statistically significant inhibition of erythrocyte cholinesterase activity (Fischer, 1978).

A statement of compliance with QA, but not with GLP, was provided. The study was

preliminary in nature and was not conducted to fulfil a particular guideline.

In a preliminary study performed to aid in selection of doses for a study of subchronic

neurotoxicity, groups of five male and five female albino rats (outbred) (Crl:CD®(SD)IGS

BR VAF/Plus®) (aged 6 weeks at study initiation) were given diets containing technicalgrade terbufos (AC92100; purity, 89.7%) (dissolved in acetone, mixed with a GRITO’Cobs® carrier) at a concentration (adjusted for purity) of 0 (acetone and carrier), 1.0,

5.0 or 6.0mg/kg in males (equal to 0, 0.11, 0.55 and 0.67mg/kg bw per day, respectively)

and of 0 (acetone and carrier), 0.5, 3.0 or 4.0mg/kg in females (equal to 0, 0.06, 0.33, and 

0.43mg/kg bw per day, respectively) for at least 21 days. Diets were made available to

animals until termination on day 22. The number of parameters evaluated was limited and

included observations for general condition, mortality and clinical signs of toxicity, and

measurement of body weight and food consumption. Plasma, erythrocyte and brain (onehalf homogenate) cholinesterase activities were measured from samples obtained and

processed on day 22 at study termination and stored frozen at -70C until analysis.

Cholinesterase inhibition was determined relative to the value for the appropriate concurrent control group. Clinical chemistry, haematological, urine, organ weight, and gross and

histopathological evaluations were not conducted in this study.

No animals died and no clinical signs of toxicity were observed. Statistically significant decreases in body-weight gain relative to respective control groups, considered to be

treatment-related, were noted during each of the 4 weeks in males at the highest dose 

(6.0mg/kg) and during weeks 1 and 2 in females at the highest dose (4.0mg/kg). Food consumption was statistically significantly decreased only in males at the highest dose during

341

TERBUFOS 333–385 JMPR 2003
the first week of the study. Treatment-related decreases in blood and brain cholinesterase

activities were observed in males and females at the two higher doses. In males at 5.0mg/kg

and 6.0mg/kg, plasma cholinesterase activity was statistically significantly inhibited by 

73% and 85%, respectively. In females, plasma cholinesterase activity was inhibited at 

3.0mg/kg and 4.0mg/kg by 84% (not statistically significant) and 94% (statistically significant), respectively. Erythrocyte cholinesterase activity was statistically significantly inhibited by 98% and 99% in at 5.0mg/kg and 6.0mg/kg, respectively. A smaller inhibition of

erythrocyte cholinesterase of about 35% was noted in males at 1.0mg/kg. In the study

report, the finding was not considered to be associated with the administration of test material, as it was not statistically significant and there were no reductions in either plasma or

brain cholinesterase activity in males at this dose. It may, however, have been related to

treatment as the values for cholinesterase activity for four of the five animals in this group

fell below those in the control group in replicate assays. In females, at 3.0mg/kg and 

4.0mg/kg, respectively, erythrocyte cholinesterase was inhibited by 99% to 100%. Statistically significant decreases in brain cholinesterase of 64% and 81% were observed at 

5.0mg/kg and 6.0mg/kg in males, respectively, as were statistically significant decreases in

females at 3.0mg/kg and 4.0mg/kg, of 68% and 84%, respectively.

If inhibition of plasma cholinesterase activity is not considered to be an adverse effect

and brain (not erythrocyte) cholinesterase inhibition and clinical signs of toxicity are considered to be relevant effects for terbufos, the NOAEL was 1.0mg/kg in males (equal to

0.11mg/kg bw per day) and 3.0mg/kg in females (equal to 0.06mg/kg bw per day) on the

basis of statistically significant inhibition of brain cholinesterase inhibiton at the next

highest dose (Mandella, 1999). Statements of compliance with QA and GLP were provided.

This study was preliminary in nature and was not conducted to fulfil a particular guideline.

In a preliminary feeding study, groups of 10 male and 10 female albino RH Wistar

rats were given diets containing technical-grade terbufos (CL 92100; purity, 96.7%) at a

nominal concentration of 0, 0.125, 0.5 or 2.0mg/kg (nominally equivalent to 0, 0.012, 0.069

and 0.299mg/kg bw per day in males and 0, 0.012, 0.053, and 0.212mg/kg bw per day in

females) for 31 days. Data on analysis of test material levels in the diet were not provided

so test material intake and stability in the feed could not be confirmed. Study parameters

examined included observations for appearance, mortality, and measurement of body weight

and food consumption. In five animals of each sex per group (when possible), haematological and limited clinical chemistry evaluations (glucose, urea nitrogen and glutamicpyruvic transaminase), liver and kidney weight measurements and a gross examination were

conducted. At the end of the study, blood (from fasted animals) and brain samples (onehalf of the cerebrum) were taken from five animals of each sex per group for determination of cholinesterase activity. Brain samples were stored frozen until analysis, and assays

were conducted on homogenates. Inhibition of cholinesterase activity was determined relative to the value for the appropriate concurrent control group.

There were many deaths in the study; four males at the highest dose died on day 3,

17, 31 or 31, one male at the intermediate dose died on day 12, and one male at the lowest

dose died on day 31. One female at the highest dose was terminated in a moribund condition on day 24, two females at the intermediate dose died on day 9 or 24 and one control

female died on day 31. The deaths of five unspecified animals were thought to be the result

of a respiratory infection, and autolysis prevented attempts to determine the cause of death

in the remainder (again, unspecified). Therefore, it was not possible to ascertain whether

any of the deaths were related to treatment. There was no apparent effect of treatment on

342

TERBUFOS 333–385 JMPR 2003
body weight, food consumption, on measured haematological and clinical chemistry parameters, or on organ weights in any group treated with terbufos. Cholinesterase activity was

statistically significantly inhibited only at the highest dose in both sexes. Statistically significant inhibition of cholinesterase activity in plasma (57%), erythrocytes (36%) and brain

(28%) was noted in males at the highest dose, as was (mostly) a statistically significant inhibition in plasma (68%), erythrocytes (37%; not statistically significant), and brain (53%) in

females at the highest dose. These decreases were attributed to treatment. There was also a

decrease of 29% in erythrocyte cholinesterase activity in males at the intermediate dose;

although not statistically significant, this was considered to be a possible result of treatment

owing to the magnitude of the decrease. It was reported that upon gross pathological examination of four to five animals of each sex per group at study termination, no gross lesions

were found that were attributed to treatment (gross pathology data were not provided). An

overall NOAEL could not be identified in this study because insufficient information was

provided about the mortality that occurred at all doses in males and at the intermediate and

highest dose in females. There were a number of unspecified deaths that were judged likely

to be the result of infection in the animal facility. In addition, intake of test material could

not be confirmed (Morici, 1972). No statements of compliance with QA or GLP were provided. This study was not performed according to a specific guideline.

In a feeding study, groups of 20 male and 20 female Sprague-Dawley rats were given

diets containing technical-grade terbufos (purity, 90.1%; prepared in corn oil and methylene chloride, 1:1) at a concentration (adjusted for purity) of 0 (vehicle), 0.125, 0.250, 0.500

or 1.000mg/kg (equal to mean intakes of test substance of 0, 0.011, 0.021, 0.041, and 

0.082mg/kg bw per day in males and 0, 0.012, 0.023, 0.048 and 0.095mg/kg bw per day

in females) for 3 months. Parameters evaluated included observations for mortality and 

clinical signs of toxicity, assessments of body weight, food intake and food efficiency, ophthalmoscopic, haematological, and clinical chemistry evaluations, urine analysis, organ

weight determinations, a macroscopic examination in all animals and a microscopic evaluation of organs and tissues in animals at the highest dose and in the control group only. The

heart, liver, and kidney, any gross lesions or masses, and any other tissues, as indicated by

findings at the highest dose, were examined microscopically in all animals. Plasma and erythrocyte cholinesterase activities were measured on day 1, and at weeks 1 and 2 and months

1, 2 and 3 in 10 animals of each sex per group. Brain cholinesterase activity was measured

in 10 animals of each sex per group at study termination. Inhibition of cholinesterase activity was determined relative to the value for the appropriate concurrent control group.

One female at 0.5mg/kg was mistakenly sexed as male until week 4, when the animal

was put with other females in the group. All animals survived until the end of the study,

except for one female and one male at 0.5mg/kg, which died of accidental causes on days

8 and 51. No clinical signs of toxicity were observed and there were no obvious effects of

treatment on body weight, food consumption, food efficiency, haematological, clinical

chemistry, or urine analysis parameters at any dose. Slight statistically significant increases

in liver weight to body weight ratios but not absolute weights in females at the two highest

doses were not considered to be of biological relevance. Plasma cholinesterase activity was

statistically significantly decreased only at the highest dose, at which activity was inhibited

throughout the study in both sexes. At study termination, decreases in activity at the highest

dose were 33% in males and 52% in females. Erythrocyte cholinesterase and brain

cholinesterase activities were not affected by treatment. The macroscopic examination was

not remarkable. Upon microscopic examination of tissues and organs, increases were

observed in the incidence of mandibular lymph node hyperplasia at the highest dose in

343

TERBUFOS 333–385 JMPR 2003
males (20%) and females (70%) compared with that in male control animals (6.25%) and

female control animals (32%). Also, the incidence of mesenteric lymph node hyperplasia

was increased in females at the highest dose (50% compared with 25% in the control group),

but not in males at the highest dose (25% compared with 30% in the control group). There

was no clear association with treatment for these or other histopathology findings in the

study. In the study report, the mesenteric lymph node lesions were considered to be related

to nematodiasis. Other groups treated with terbufos were not examined for the incidence of

either mandibular or mesenteric lymph node hyperplasia. Some respiratory tract lesions that

occurred with similar frequency in control groups and in groups treated with terbufos were

ascribed to chronic murine pneumonia (said to be a common finding in rodent colonies). If

inhibition of plasma cholinesterase activity is not considered to be an adverse effect, the

NOAEL was ≥1.00mg/kg (the highest dose tested) in males and females (equal to 

0.082mg/kg bw per day in males and 0.092mg/kg bw per day in females) (Daly & Knezevich, 1979). A statement of compliance with QA, but no GLP statement, was provided. The

protocol was generally consistent with US EPA Subdivision F Guidelines (November 1982

and 1984, revised).

In a study of toxicity, groups of 30 male and 30 female CD® (Sprague-Dawley

derived) COBS® rats were given diets containing technical-grade terbufos (AC 92100;

purity, 89.6%) (prepared in corn oil and methylene chloride, 1:1) at a concentration of 

0 (vehicle only), 0.125, 0.5, or 1.0mg/kg (equal to 0, 0.007, 0.028, and 0.055mg/kg bw 

per day for males and 0, 0.009, 0.036, and 0.071mg/kg bw per day for females) for 1 year.

Parameters assessed included observations for general health and mortality, clinical signs

of toxicity, measurement of food consumption and body-weight changes, haematological

and clinical chemistry determinations, urine analysis, organ weight measurements and 

ophthalmoloscopic and macro- and microscopic pathology examinations. Plasma and 

erythrocyte cholinesterase activities were assessed in 10 animals of each sex per group at

week 6, months 3 and 6, and at study termination. Brain cholinesterase activity was determined at study termination. Inhibition of cholinesterase activity was calculated relative to

values for values for concurrent controls.

There were six deaths in the study; two control animals (one male on day 306 and

one female on day 271) and two animals at the intermediate dose (one male on day 351 and

one female on day 262) were terminated in a moribund condition. One male at the intermediate dose was found dead on day 310 and one female at the highest dose died accidently

on day 97. There was no obvious pattern in the deaths in the animals treated with terbufos

that would suggest a relationship with administration of the test material. There was no clear

relationship with treatment for the slight increases observed mostly during the last half of

the study in the group of females at the highest dose in the incidence of excess lacrimation,

chromodacryorrhoea and alopecia compared with the control and other treated groups. As

similar increases were noted at lower doses in males, the findings in females were considered to be likely to be caused by random variation. There were no clear effects of treatment

on body weight. Variation was noted among groups in food consumption over the course

of the study, but there was no consistent pattern of findings that would clearly indicate an

effect of treatment in groups of males or females treated with terbufos. There was no evidence that the results of the urine analyses or those of the ophthalmoscopic and haematological examinations were related to treatment. Statistically significant decreases, of small

magnitude, in blood urea nitrogen and bilirubin at termination in females at the highest dose

were not considered to be clearly of adverse significance in the absence of other findings.

Clinical chemistry evaluations were otherwise not remarkable. Slight, statistically signifi344

TERBUFOS 333–385 JMPR 2003
cant reductions were observed at the highest dose in the absolute weight and weight relative to brain weight (but not relative to body weight) of male testes, and slight statistically

significant decreases in the absolute weight and weight relative to brain weight (but not relative to body weight) of the kidney were noted in females at the intermediate and highest

doses. These organ-weight changes were not clearly adverse in the absence of other supporting indications of toxicity. No obvious effect of treatment was indicated by the results

of the gross and microscopic examinations. There was no evidence of carcinogenity.

Decreases in cholinesterase activity that could be clearly related to treatment were

observed only at the highest dose in both sexes. Plasma cholinesterase activity was statistically significantly inhibited in males at the highest dose by 25% and 29%, respectively, 

at 6 and 12 months. Statistically significant inhibition of plasma cholinesterase activity

(33–51%) was noted at all time-points in females at the highest dose. Erythrocyte

cholinesterase activity was not statistically significantly inhibited at any dose or time-point

in either sex. Brain cholinesterase activity in males was decreased relative to control values

at the lowest, intermediate and highest dose, respectively by 4% (statistically significant),

3% (not statistically significant) and 8% (statistically significant). On the basis of the magnitude and pattern of the response, inhibition at the lowest dose was not considered to be

related to treatment. In females, brain cholinesterase activity was statistically significantly

inhibited only at the highest dose. Virtually no changes relative to the control group was

observed at the lowest and intermediate doses. Although the decrease observed at the highest

dose in males and females may have been related to treatment, because of the relatively low

magnitude of the response and the absence of clinical signs at this dose, it was not considered to be toxicologically relevant.

If inhibition of plasma cholinesterase activity is not considered to be an adverse effect

and inhibition of brain cholinesterase activity of 8–10% is not considered to be toxicologically relevant, the NOAEL was 1.0mg/kg (equal to 0.055mg/kg bw per day in males and

0.071mg/kg bw per day in females), the highest dose tested (Daly, 1987). This study complied with QA and GLP and was consistent with US EPA Subdivision F Guidelines.

Dogs

In a short-term study to assess cholinesterase activity, groups of four male and four

female beagle dogs (except at the highest dose, where two dogs of each sex were used) were

given technical-grade terbufos (AC 92100; purity, 89.6%) at a dose (adjusted for purity) of

0 (vehicle), 1.25, 2.5, 5.0, or 15.0mg/kg bw per day, administered orally in corn oil in gelatin

capsules, once daily in the morning for 29 days. Originally planned for 28 days, dosing was

extended by 1 day through a protocol amendment. The number of parameters evaluated was

limited and included observations for mortality, morbidity and clinical signs of toxicity, and

measurement of body weight and food consumption. Plasma and erythrocyte cholinesterase

activities were assessed before dosing and after 1, 2, and 4 weeks of treatment from blood

samples collected before dosing on the given day. Brain cholinesterase activity was determined at study termination in samples from the cerebrum and cerebellum obtained 20–

24h after administration of the last dose. In the study report, cholinesterase inhibition in

the plasma and erythrocytes was determined and statistically analysed relative to values

obtained before the start of dosing. Inhibition of cholinesterase activity in the brain was

determined relative to the value for the appropriate concurrent control group. Clinical chemistry, haematological, urine, organ weight and gross and microscopic histopathological 

evaluations were not performed in this study.

345

TERBUFOS 333–385 JMPR 2003
There were no deaths in the study and no clinical findings were observed that could

be ascribed to treatment in any group. One instance of vomiting was observed in each of

two dogs at the lowest dose only. There were no appreciable differences in body weights or

food consumption among groups of either sex during the study. Statistically significant

decreases in plasma cholinesterase activity ranging from 33% to 37% were noted at the

highest dose in both sexes at all time-points and were ascribed to treatment. Inhibition of

plasma cholinesterase activity of 20–21% was observed at 5.0mg/kg bw per day in males

after 2 and 4 weeks of treatment and in females at all time-points; these values were not

statistically significantly from those for the respective groups of pre-treatment controls and

were considered to be of marginal biological relevance in the study report. If inhibition of

plasma cholinesterase activity was determined relative to values for the appropriate concurrent controls instead of pre-treatment values (as in the study report), there was not much

difference in the magnitude of the decreases calculated by either procedure for either sex

at any week or dose, except in females at 2.5 and 5.0mg/kg bw per day. In these groups,

slightly greater decreases in plasma cholinesterase activity were measured relative to values

for concurrent controls at all time-points (28–30% at 5.0mg/kg bw per day, and 18–23% at

2.5mg/kg bw per day) than relative to pre-treatment values (20–21% at at 5.0mg/kg bw per

day, and 10–18% at 2.5mg/kg bw per day). Under the conditions of the study, there was no

effect of treatment in either sex or at any dose on erythrocyte cholinesterase activity (relative to pre-treatment values or to values for the appropriate concurrent controls) or on brain

cholinesterase activity relative to the values for the appropriate concurrent controls. If inhibition of plasma cholinesterase activity is not considered to be an adverse effect, the NOAEL

was 15mg/kg bw per day (the highest dose tested) in males and females (Shellenberger,

1987). Statements of compliance with QA and GLP were provided. This was a special study

that was not conducted to comply with a particular guideline.

In a preliminary feeding study, groups of two male and two female beagle dogs (aged

8–12 months) were given diets containing technical-grade terbufos (CL 92100; purity,

96.7%) (prepared in a vehicle of corn oil) at a nominal dose of 0, 0.01, 0.05, or 0.25mg/kg

bw per day administered daily for 30 consecutive days. Food consumption was estimated

by visual inspection and many animals did not always consume all the food offered. In addition, data on the analysis of concentrations of test material in the diet were not provided,

so intake of test material could not be determined with any degree of confidence, and stability of the test material in the feed could not be confirmed. Parameters assessed included

observations for appearance, mortality and measurement of body weight and food consumption, haematological and limited clinical chemistry evaluations (plasma glucose, 

glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase, and urea nitrogen),

liver and kidney organ weight measurements and gross examination of all animals. Plasma

and erythrocyte cholinesterase activities were assessed before the start of dosing, at week

2 and at study termination in fasted animals. Brain cholinesterase activity was measured at

study termination in homogenates of samples taken from the cerebrum. Cerebrum tissue

was stored frozen until analysis. Inhibition of cholinesterase activity was determined relative to the values for the appropriate control group.

There were no deaths in the study and no clinical signs of toxicity were observed. On

the basis of the estimated food refusal (%) during the study, received doses were as much

as 20–35% lower than nominal doses in most animals at the lowest and intermediate doses,

and by as much as 35–50% at the highest dose. Over the course of the study, males at the

highest dose failed to gain weight and females at the highest dose and one female at the

intermediate dose lost weight; these findings were statistically significant for the groups

346

TERBUFOS 333–385 JMPR 2003
receiving the highest dose. There was no apparent effect of treatment on organ weights, or

on the haematological and clinical chemistry parameters measured. No treatment-related

findings were observed during the gross examination. As values for cholinesterase activity

were similar in males and females at the same dose, data were pooled for statistical 

analysis. By week 4, statistically significant decreases in plasma cholinesterase activity of

68% and 84%, and in erythrocyte cholinesterase activity of 35% and 80% were observed

at the lowest and intermediate doses, respectively. Brain (cerebrum) cholinesterase activity

was statistically significantly inhibited only at the highest dose (by 66%). An overall NOAEL

could not be identified because intake of test material could not be reliably estimated

(Morici, 1972). No statements of compliance with QA or GLP were provided. This study

was not performed to comply with a specific guideline.

In a feeding study, groups of four male and four female beagle dogs (aged 10–14

months, i.e. somewhat older than the age recommended in the guidelines) were given 

terbufos (AC 92,100; purity not specified) at a nominal concentration of 0 (vehicle only),

2.5, 10.0, or 40.0mg/l (nominally equivalent to 0, 2.5, 10, and 40mg/kg bw per day) in corn

oil, administered daily for 6 days per week, for 6 months. One ml of the test material in the

vehicle was injected via a syringe on top of each dog’s daily food ration (kibbled dog chow).

Doses were adjusted weekly for each individual animal’s body weight. A set amount of

treated food (apparently totalling 3300g per week) was offered for 1h each day and any

food not consumed in that time was removed and weighed in order to measure food consumption and estimate intake of test material. Neither dosing solutions nor treated diets

were analysed to confirm content or stability of the test material. Parameters examined

included mortality and clinical signs of toxicity, assessments of body weight and food

intake, ophthalmoscopic, haematological, and limited clinical chemistry (alkaline phosphatase, blood urea nitrogen, fasting blood sugar, and serum glutamic-pyruvic transaminase) evaluations, urine analysis, weights of selected organs (adrenals, gonads, kidneys,

heart and liver), and macroscopic and microscopic examinations. Plasma and erythrocyte

cholinesterase activities were measured before the start of dosing and at weeks 0, 4, 12 and

26. Brain cholinesterase activity was assessed at week 26. Although data on cholinesterase

activity were provided for individual animals, in the study report data from both sexes were

combined for statistical analysis (t-test) for each dose and type of cholinesterase activity,

as differences between the sexes were not considered to be remarkable. Data on

cholinesterase activity from groups treated with terbufos were compared with data for the

combined concurrent control group.

Test material intake could not be reliably determined. In addition to a lack of analytical information on the purity, dietary content and stability of the test substance, some

animals did not always consume all their food. Since the solution containing the test material was placed on top of the daily food ration, it is not clear how much of the test material was ingested by animals that did not consume the daily food allotment. Female dogs had

a greater tendency not to consume the entire meal. The approximate percentage of unfinished meals over the course of the study for all females at each dose was 36% in the control

group, 38% at the lowest dose, 40% at the intermediate dose and 23% at the highest dose.

Over the duration of the study, some females received only about 80–90% of the intended

dose of terbufos. The situation was less severe in males; the approximate percentage of all

unfinished meals over the duration of the study for all males at each dose was 31% for the

control group, 5% at the lowest dose, <1% at the intermediate dose and 2% at the highest

dose. Also, initial body weights varied more than might be desirable (20% difference

between some animals of the same sex).

347

TERBUFOS 333–385 JMPR 2003
One male in the control group was terminated in a moribund condition and was found

to have a colon obstruction and peritonitis. Clinical signs of toxicity were not observed in

any animals (individual animal or summary data was not included in the report).

With regard to general condition, one male at the lowest dose, one male at the highest

dose, three females in the control group, one female at the highest dose, and possibly one

female at the lowest dose and and one female at the intermediate dose, had histopathological indications of bronchopneumonia. Body-weight loss (measured over the duration of the

study) was noted at termination at week 26 in individual animals in all groups except

females at the highest dose. The incidence of weight loss (and average body-weight change)

during the study was: in males, 2 (-0.55kg), 3 (-0.45kg), 3 (-1.8kg), and 3 (-0.4kg),

respectively; and in females, 1 (0.35kg), 1 (0.075kg), 1 (0.15kg) and 0 (0.425kg), in the

control group and at the lowest, intermediate and highest dose, respectively. There was no

obvious dose-related pattern in these findings. The greater weight loss in the group of males

at the intermediate dose was due to one animal that was the heaviest animal in the group

at initiation of treatment and that lost 5.7kg in weight during the study, apparently exhibiting no clinical signs or indications of toxicity, and no unusual findings with regard to food

consumption or other parameters examined (except for a slight elevation in erythrocyte 

sedimentation rate at week 26). The only gross or histopathopathological finding in this

animal was diffuse mild leukocytosis in the liver, which the study report stated could, along

with the elevated erythrocyte sedimentation rate, have been related to an infection in this

dog near the end of the study. In other animals, there was no clear effect of treatment on

the haematological, biochemical, urinary parameters or organ weights assessed or in the

gross or histopathological examinations.

Inconsistencies and variability were noted in the data on cholinesterase activity. In the

study report, differences between sexes for a given type of cholinesterase activity and at a

particular dose were considered to be minimal and data were combined for both sexes for

statistical analysis. Under these conditions, there were no statistically significant differences

in brain or erythrocyte cholinesterase activity at the end of the study (week 26). Plasma

cholinesterase activity at week 26 was statistically significantly decreased relative to values

for the combined control group, by 26% at the intermediate dose and 31% at the highest

dose; the study report considered these decreases to be minimal but treatment-related. The

value for plasma cholinesterase activity from the control male that was terminated in a moribund condition was not included in the calculation, although it was not an outlying value

compared with those for the rest of the animals in the group. Data for individual animals

were provided. When plasma cholinesterase activity was calculated for each sex separately

at week 26, without statistical evaluation, there was no clear effect on plasma cholinesterase

activity in males relative to that of the control group, either with the inclusion of the terminated control male (no decrease at the lowest dose, decrease of 35% at the intermediate

dose, decrease of 15% at the highest dose) or without it (decrease of 9% at the lowest dose,

decrease of 47% at the intermediate dose, decrease of 31% at the highest dose) owing to

the lack of a clear dose–response relationship at the intermediate and highest doses. In

females at week 26, decreases in plasma cholinesterase activity relative to that of the control

group were noted at the intermediate dose (19%) and highest dose (32%), indicating a possible, small effect of treatment, particularly at the highest dose. Erythrocyte cholinesterase

activity at the highest and intermediate doses was decreased by 26% and 6%, respectively,

when data for males were taken separately (as compared with reduction of 17% and 13%

(neither statistically significant) at the highest and intermediate doses, respectively, when

348

TERBUFOS 333–385 JMPR 2003
data from both sexes were combined), suggesting a possible minimal, but certainly not clear,

effect of treatment at the highest dose.

There were no clear adverse findings associated with treatment in this study, but

because nominal concentrations of test material in the diet could not be confirmed, intake

of test material could not be reliably estimated and underconsumption of diets containing

terbufos was noted, especially in females, a NOAEL could not be identified (Morgareidge,

1973). Statements of compliance with QA and GLP were not provided. The protocol and

study conduct were considered to be inadequate according to current standards.

Subsequent to the 6-month feeding study in dogs (Morgareidge, 1973), a study was

conducted to attempt to address questions about the 6-days-per-week dosing regimen used

in that study, and to determine whether a 7-days-per-week regimen would influence

cholinesterase activity, particularly in erythrocytes. Groups of two male and two female

beagle dogs (aged 9.5–12 months) were given diets containing technical-grade terbufos

(purity, 88%) at a dose of 50mg/kg bw daily, for (1) 7 days per week for 28 days; or (2) 6

days per week (basal food given on day 7) for 28 days, followed in both groups by a 28 day

recovery period without treatment. Five out of the eight dogs in the study had previously

been exposed to dichlorvos, another cholinesterase-inhibiting chemical, 4–7 months before

their arrival in the testing facility. No other details were provided. A concurrent control

group was not included in the study. The test material was prepared as a solution in corn

oil, added to a fixed amount of food via a syringe at the rate of 1ml/kg bw, and given to

each dog individually in the morning. If treated food was not eaten within 1h, it was

removed and weighed. It was stated that any food treated with terbufos that was not consumed in the morning was re-administered to the animal in the afternoon (regular feed of

plain dog chow), such that any remaining test substance was eaten. Erythrocyte and plasma

cholinesterase activities were measured before treatment on days -7 and -6, on days 1, 3, 7,

10, 14, 18, 21 and 28, and after treatment on days 29, 31, 35, 38, 42, 49 and 56. Average

cholinesterase inhibition for each group (both sexes combined) was calculated relative to

the respective average value before treatment (sexes combined) at each time-point, but

apparently these comparisons were not analysed statistically. Statistical analysis was performed for each time-point (both sexes combined) to determine whether there was a significant difference between the 6-day treatment regimen and the 7-day treatment regimen.

Animals were observed for general condition, signs of toxicity and body-weight changes

during treatment. After the first treatment phase of the study was concluded, a 28-day crossover phase was performed in which the group of animals dosed for 7 days per week were

dosed for 6 days per week for 28 days and the group of animals dosed for 6 days per week

were dosed for 7 days per week for 28 days. Erythrocyte and plasma cholinesterase 

activities were assessed before treatment and on days 1, 3, 7, 10, 14, 18, 21, 25, and 28.

There was no recovery period. Brain cholinesterase activity was not assessed in either the

first phase or the cross-over phase.

Dosing solutions were analysed four times during the first phase of the study and

found to contain 87.6–98.8% of the target amounts (average, about 92%). All animals survived both study phases and no clinic signs of toxicity were observed. In the first phase,

initial body weights in some dogs varied considerably; a difference of about 40–50% was

noted between some animals in the 7- or 6-day dosing group. Three out of four dogs in both

the 7- and 6-day feeding groups lost weight during at least part of the first phase of treatment; this may have been related to treatment, but there were no control groups available

349

TERBUFOS 333–385 JMPR 2003
for comparison. Some weight gain was noted in most animals in both groups during the

recovery period. Recorded weekly food intakes for individual animals in the first phase were

fairly variable week-by-week and between individuals in dogs fed the test material for 7

days per week; it is thus not clear whether all animals in that group ate all the treated food

offered. Far less variability was observed in dogs fed test material for 6 days per week, as

apparent weekly maximums of 2400g of food per dog were commonly consumed.

Owing to the small sample size and variability in the data, it was difficult to determine whether there was a sex difference in cholinesterase activity measurements. In the

study report, it was assumed that there were no sex differences and group means from data

from both sexes combined were compared. In the first phase of the study, on day 28,

although data were apparently not analysed statistically, erythrocyte and plasma

cholinesterase activities decreased by 14% and 68%, respectively, in the group fed test material for 7 days per week, and by 4% and 40%, respectively, in the group fed test-material

for 6 days per week relative to values before treatment (there was no concurrent control

group). The difference between the groups treated for 7 days per week and for 6 days per

week was not statistically significant with regard to erythrocyte cholinesterase activity at

any time-point, either during or after treatment. Differences in plasma cholinesterase activity between the groups treated for 7 days per week and for 6 days per week were statistically significant at weeks 7, 21 and 28. Based on the data provided, recovery to pre-treatment

or almost pre-treatment levels was noted during the recovery phase with both dosing regimens, but progress appeared to be a little more rapid on the 6-day regimen and plasma

cholinesterase activity seemed to recover more rapidly than erythrocyte cholinesterase 

activity on either regimen.

In the cross-over phase of the study, after 28 days of treatment, erythrocyte and plasma

cholinesterase activities decreased by 18% and 55%, respectively, in the cross-over group

fed test material for 7 days per week and by 28% and 52%, respectively, in the cross-over

group fed test material 6 days per week, relative to values before treatment. No statistically

significant differences were found between the groups on the 6- and 7-day feeding regimens

at any time-point assessed. Body-weight losses and variable food intakes were noted with

both groups. The study authors concluded that the decrease in cholinesterase activity noted

was reversible after cessation of treatment. They also concluded that there were no cumulative adverse effects in erythrocyte or plasma cholinesterase activity on either regimen. In

the first phase, however, there did appear to be a slightly larger effect on cholinesterase

activity on the 7-day regimen compared with the 6-day regimen, and in the second phase a

stronger effect on erythrocyte activity was observed relative to the first phase, possibly

implying some carry-over influence between treatments. Owing to the small sample size,

the limited protocol (no assessment of brain cholinesterase activity), the lack of a concurrent control group, the use of only one dose, questions about the intake of test material

based on food consumption data, the variability in data for individual animals and other

uncertainties (e.g. previous treatment of test animals with dichlorvos), this study is not suitable for the identification of a NOAEL or for performing regulatory toxicology assessments

for terbufos. No statements of compliance with QA or GLP were provided and the study

was not performed to comply with a particular guideline (Berger, 1977).

In a 1-year study, groups of male and female beagle dogs were fed gelatin capsules

containing technical-grade terbufos (AC 92,100; purity, 89.6%) at an initial dose (not

adjusted for purity) of 0 (vehicle only), 15, 60, 240 or 480mg/kg bw per day in corn oil for

1 year. Owing to toxicity that resulted in mortality, the dose of 480mg/kg bw per day was

350

TERBUFOS 333–385 JMPR 2003
reduced to 120mg/kg bw per day 1 day before the start of week 6, and the dose of 240mg/kg

bw per day was decreased to 90mg/kg bw per day 2 days after the beginning of week 8.

Eight animals of each sex were assigned to the vehicle control group and six animals of

each sex were assigned to each of the groups receiving terbufos. During weeks 3 and 4, an

error occurred and the doses administered were only 5.2% of those intended. Administration of the test material continued until 20–24h before termination, and animals were terminated during the 5 days following the 1-year period of treatment. Parameters assessed

included general condition, mortality, morbidity and clinical signs of toxicity, measurement

of body weight and food consumption, and clinical chemistry, haematology, urine analysis,

ophthalmoscopy, organ weight, and gross and microscopic evaluations in all animals.

Plasma and erythrocyte cholinesterase activity in fasted animals was assessed before treatment, at months 3 and 6 and at study termination. Brain cholinesterase activity in samples

from the cerebrum and cerebellum was measured from tissues taken at study termination

and stored frozen until analysis. Inhibition of cholinesterase activity was determined relative to the value for the appropriate concurrent control group.

There were three deaths attributable to treatment-related causes. One male and one

female at the highest dose died during week 6, while the dose of 480mg/kg bw per day was

being administered. Clinical signs observed in these animals included vomiting, slight

tremors and inactivity in the male and tremors, diarrhoea, weak hind legs and excessive

salivation in the female. Decreased body weight and food consumption were noted in both

dogs. One female at 240/90mg/kg bw died during week 7, also from causes related to treatment, while receiving the dose of 240mg/kg bw per day. Clinical findings in this animal

included tremors that increased in severity, inactive behaviour, red-tinged faeces, excessive

salivation, dehydration, listless behaviour, rough hair coat and decreased body weight and

food consumption. One other female at the highest dose was sacrificed in a moribund condition at the start of week 31, owing to causes unrelated to treatment (prolapsed vagina).

Clinical signs of toxicity noted in surviving males at 480mg/kg bw per day included

tremors (slight to more severe) and inactivity. In males at 240mg/kg bw per day, clinical

signs of toxicity included excessive salivation, dehydration, red-tinged faeces, listlessness

and inactivity. Symptoms in females were generally more severe than in males at these doses

and included tremors (slight to more severe), inactivity, excess salivation, red-tinged faeces,

vomiting and weak hind legs at the highest dose (480mg/kg bw per day), and slight tremors,

inactive behaviour, excessive salivation, weak hind legs, diarrhoea, and red-tinged faeces at

the lowest dose (240mg/kg bw per day). Decreases in body weight and food consumption

were noted in males at both 480mg/kg bw per day and 240mg/kg bw per day and to a greater

extent in females at these doses during the early weeks of the study before and just after

the decrease in dose. During this time, body-weight decreases reached statistical significance in females at the highest dose, and statistically significant decreases in food consumption were noted for males and females at the highest dose and for females at 240mg/kg

bw per day. Shortly after the two higher doses were lowered to 120 and 90mg/kg bw per

day, respectively, and for the remainder of the study, there was no apparent effect of treatment with terbufos on body weights or food consumption at any dose.

After the two higher doses were reduced, the only clinical findings noted during the

remainder of the study, for which an association with treatment could not be dismissed,

were two instances of severe convulsions in one female at 120mg/kg bw per day during

weeks 46 and 47. There were no obvious effects of treatment on the ophthalmoscopic, clinical chemistry or urinary parameters assessed or on organ weights after doses of terbufos

351

TERBUFOS 333–385 JMPR 2003
of £120mg/kg bw per day. In males, at month 3, slight statistically significant decreases

were found in erythrocyte counts at 90 and 120mg/kg bw per day, and in haemoglobin, erythrocyte volume fraction, and mean corpuscular haemoglobin concentration at 120mg/kg

bw per day. Numbers of platelets were also slightly increased (statistically significantly) in

males at 90 and 120mg/kg bw per day. Slight, statistically significant decreases in haemoglobin and erythrocyte volume fraction were noted at month 3 in females at 120mg/kg bw

per day. These perturbations were transient and had resolved by the next assessment at

month 6. They may have been related to treatment at these doses or may have been residual effects from previous dosing at 240 and 480mg/kg bw per day.

Data on cholinesterase activity reported in this study were difficult to interpret owing

to variability in the values for individual animals and because of generally inconsistent or

sometimes shallow dose–response relationships. Data were apparently not analysed statistically. Plasma cholinesterase activity was inhibited by about 40% or more in all groups

receiving terbufos at all time-points in both sexes. At study termination (week 52), plasma

cholinesterase activity was inhibited at the lowest, low intermediate, high intermediate and

highest doses by 44%, 66%, 67% (shallow dose–response relationship) and 68%, respectively, in males, and by 45%, 68%, 67% (shallow dose–response relationship) and 74%,

respectively, in females. Owing to the magnitude of the relative decreases, a relationship to

treatment could not be excluded at any dose. At study termination (week 52), erythrocyte

cholinesterase activity was lower than the control value at the lowest, lower intermediate,

higher intermediate and highest doses in males by 4%, 13%, 18% and 19%, respectively.

Similar patterns were found at earlier time-points. The decreases at week 52 were considered to be marginal and the changes of slightly higher magnitude at the two higher doses

were considered to be of no toxicological significance. In females, inhibition of erythrocyte

cholinesterase activity varied little from control values at the lowest and lower intermediate doses, and ranged from 18% to 28% at the higher intermediate dose and 27% to 35%

at the highest dose, at all time-points. At study termination (week 52), erythrocyte

cholinesterase activity was lower than the control value in the lowest, lower intermediate,

higher intermediate and highest doses in females by 6%, 15%, 20% and 27%, respectively.

The magnitude and consistency over time of the change at the highest dose was such that

a relationship to treatment could not be dismissed. At week 52, cerebellum cholinesterase

activity was not inhibited at any dose (values were 98% to 125% of those of the controls)

in males. Cerebrum cholinesterase activity in males was lower than the control value at the

lowest, low intermediate, higher intermediate and highest dose by 5%, 10%, 22%, and 22%,

respectively. Despite the lack of a dose–response relationship at the two higher doses and

the large variability in the data, the magnitude of the response in the brain at the two higher

doses was such that a relationship to treatment could not be dismissed. For the same doses,

inhibition of cholinesterase activity in the cerebellum in females was 3%, 3%, 12% and

21%, respectively. The magnitude of inhibition in the brain at the two higher doses could

not be ignored as a possible effect of treatment; however, the decrease of 12% was not considered to be toxicologically relevant. At the lowest and highest doses, cholinesterase activity in the cerebrum of females was similar to that in controls (although activity fell below

the mean for controls by 18% and 24% at the two intermediate doses). Owing to the lack

of a dose–response relationship, a treatment-related effect was not readily supported.

Gross lesions seen in the intestinal tracts of animals that died during weeks 6 and 7

had non-neoplastic microscopic correlates. On histopathological examination, the male 

at 480mg/kg bw per day that was found dead at week 6 had diffuse congestion of the 

352

TERBUFOS 333–385 JMPR 2003
duodenum, jejunum, ileum and colon, congestion of the lungs, kidneys and liver and fibrinous thrombi in the pulmonary vessels of the lungs and in the arteries of the pancreatic

mesentery. Microscopic findings in the female at the highest dose found dead at week 6

were marked haemorrhage and congestion in the mucosa and muscularis of the jejunum

and areas of necrosis in the muscularis, mucosa and Peyer patches of the ileum (possibly

secondary to intussusception). The female at 240mg/kg bw per day found dead at week 7

had congestion of the muscularis and mucosa of the jejunum on microscopic examination.

The study authors considered that these findings were likely to be related to treatment. Other

gross and microscopic findings in the study were considered to be incidental and not related

to treatment.

If inhibition of plasma cholinesterase activity is not considered to be an adverse 

effect, and inhibition of brain (not erythrocyte) cholinesterase activity is considered to 

be a relevant effect for terbufos, the NOAEL was 60mg/kg bw per day in males on the 

basis of a decrease (22%) in cerebral cholinesterase activity for which a relationship to treatment could not be excluded, and 90mg/kg bw per day in females on the basis of a decrease

in cerebellar cholinesterase activity of 21% and instances of severe convulsions in one

female for which an association with treatment could not be dismissed (Shellenberger &

Billups, 1986). Statements of compliance with QA and GLP were provided and the protocol was consistent with US EPA Subdivision F Guidelines (November 1982 and 1984,

revised).

Sheep

In a feeding study, groups of three wethers (males) were given diets containing 

technical-grade terbufos (purity, 89.8%; in corn oil, diluted with an equal volume of methylene chloride) at a concentration (not adjusted for purity) of 0 (with vehicle only), 0.01,

0.1 or 1.0mg/kg (equal to 0, 0.0003, 0.0023, and 0.0245mg/kg bw per day) administered

daily in two portions (half in the morning and half in the afternoon) for 42 days. Parameters assessed included mortality and clinical signs of toxicity, measurement of body-weight

changes and food consumption, ophthalmoscopic examination, haematological and clinical

chemistry determinations, urine analysis, and measurement of heart and respiratory rate.

Necropsies were not conducted at study termination. Erythrocyte cholinesterase activity was

determined before the start of treatment and on days 1, 3, 7, 14, 21 and 42. Erythrocytes

were stored frozen for an unspecified period of time before analysis. Erythrocyte

cholinesterase activity in groups treated with terbufos was compared with that for the appropriate concurrent controls at each time-point. Brain cholinesterase activity was not measured. The study report stated that the blood plasma of sheep had little or no cholinesterase

activity and therefore was not assessed.

No deaths occurred during the study and no clinical signs of toxicity were observed.

There was no obvious effect of treatment with terbufos on any parameter examined, including erythrocyte cholinesterase activity. It is not known whether storage conditions before

assay had any effect on the cholinesterase activity of the erythrocytes. Brain cholinesterase

activity was not assessed. The NOAEL in males (the only sex tested) was ≥1.00mg/kg (equal

to 0.0245mg/kg bw per day) (the highest dose tested) (Garces et al., 1977). No statements

of compliance with QA or GLP were provided. The study report stated that the facility in

which the animals were maintained was fully accredited by the American Association for

Accreditation of Laboratory Animal Care.

353

TERBUFOS 333–385 JMPR 2003
(b) Exposure by inhalation

In a short-term study of whole-body inhalation, groups of 10 male and 10 female

Sprague-Dawley rats were given technical-grade terbufos (AC 92,100; purity, 90.1%) at a

target concentration of 0, 0.005, 0.01, 0.05 or 0.10mg/m3 (not adjusted for purity) as a

vapour for 8h per day, for 5 days per week over a period of 3 weeks, for a total of 15 exposures, followed by a 2-week recovery period. A 2-week treatment period had originally been

planned, but during week 1 of exposure, analytical chamber concentrations were only about

2–44% of target concentrations. During exposure weeks 2 and 3, when vaporization flasks

were heated slightly, analytical concentrations were much closer to those targeted, however,

the range of daily means varied widely suggesting chamber concentrations were not well

maintained. Mean daily analytical chamber concentrations and the range of daily means for

exposure weeks 2 and 3 corresponding to the control, 0.005, 0.01, 0.05 and 0.10mg/m3

groups were, respectively, for males 0, 0.0117 (range, 0.0003–0.0380), 0.0243 (range,

0.0066–0.05669), 0.0458 (range, 0.0098–0.0763) and 0.0946 (range, 0.0394–0.1523)

mg/m3

, and for females 0, 0.0112 (range, 0.0003–0.0380), 0.0256 (range, 0.0066–0.0569),

0.0468 (range, 0.0098–0.0763), and 0.1001 (range, 0.0448–0.01523) mg/m3

. Estimated

inhalation doses on a mg of active ingredient/kg bw per day basis for the control group and

at thes lower intermediate, higher intermediate and highest dose over the last 2 weeks of

exposure, assuming a retained dose of inhaled material of 50%, were: males, 0, 0.0030,

0.0065, 0.0122 and 0.0246mg/kg bw per day; and females, 0, 0.0041, 0.0097, 0.0175 and

0.039mg/kg bw per day, according to the study report. Possible contributions to exposure

via the dermal and oral routes (i.e. from grooming) were not discussed. Owing to mortality, exposure of females at the highest dose was stopped early, on day 18. The study was

terminated for half the animals in all groups at the end of the exposure period on day 21

and for the other half at the end of the recovery period on day 37. Parameters evaluated for

both the treatment and recovery phases of the study included observations for mortality,

clinical signs of toxicity, measurement of body weight and food consumption, haematological and clinical chemistry determinations (in five animals of each sex per group), urine

analysis, selected organ weight measurements (adrenals, heart, kidneys, liver, and lungs)

and an examination for gross pathology. Plasma and erythrocyte cholinesterase activities

were measured in five animals of each sex per group before testing, on days 1, 5, 8, 13 for

all groups, on day 18 for surviving females at the highest dose and five control animals,

and on days 21 and 37 for all groups. Brain cholinesterase activity was measured in five

animals of each sex per group on days 21 and day 37. Cholinesterase inhibition was calculated relative to the appropriate concurrent control group.

All animals survived except for one male at 0.0117mg/m3 that died accidently on day

8 and two females at the highest dose that died on days 17 and 19 from treatment-related

causes. The only clinical findings ascribed to treatment in the study were observed during

the treatment period in the group of females at the highest dose and included body tremors,

body coldness, and rapid or laboured breathing. Body weights were statistically significantly

depressed in females at the highest dose, as was food consumption, particularly towards the

end of the period of exposure. Haematological and clinical chemistry findings, which were

generally statistically significantly different from the respective control group and which

may have been related to treatment, were noted primarily during week 3. These included

decreased haemoglobin, erythrocyte volume fraction and erythrocyte counts and an elevated

clotting time in females at the highest dose, and decreased blood glucose concentrations in

males at the highest dose. The results of urine analysis were not remarkable, although there

were some low level increases in ketones, bilirubin and occult blood in males at the highest

354

TERBUFOS 333–385 JMPR 2003
dose at the end of the recovery period. The only remarkable organ weight change was a

slight increase in the adrenal:body weight ratio of males at the highest dose (0.03580) relative to the control group (0.0272) at the end of week 3. No gross necropsy findings were

considered to be related to treatment.

There were some inconsistencies observed in the data on cholinesterase activity, which

sometimes made interpretation difficult, but it appeared that by the end of the exposure

period (day 18 for females at the highest dose and day 21 for all the other groups), the following changes in blood cholinesterase activity were related to treatment: statistically significant inhibition of erythrocyte cholinesterase activity of 28% and 33% in females at the

intermediate and highest doses; reduced erythrocyte cholinesterase activity of 22% in males

at the highest dose with statistically significant reductions of 33% and 24% seen at the two

previous time-points, respectively (days 13 and 8); statistically significant decreases in

plasma cholinesterase activity of 30% and 61% in females at the intermediate and highest

doses, respectively, and of 12% and 21%, respectively, in males at the intermediate and

highest doses. Blood cholinesterase activity values were not statistically significant at the

end of the recovery period. On day 21, brain cholinesterase activity was statistically significantly inhibited in females at the highest dose (based on data from three animals) by

about 45% and in males by about 15% decreases that were considered to be associated with

treatment (although the decrease in males was not considered to be toxicologically 

relevant). At the end of the recovery phase, brain enzyme activity values were not significantly different from those of controls, but statistically significant inhibition of 29% was

observed in males at the highest dose, possibly indicating a lack of recovery. However, this

interpretation of the data was questionable because statistically significant inhibition of 35%

was also noted in brain cholinesterase activity at 0.0243mg/m3

, but not at the next highest

dose of 0.048mg/m3

.

No other remarkable findings in other parameters, except as already noted, were

reported for the recovery period. If plasma cholinesterase inhibition is not considered to be

an adverse effect of treatment and toxicologically significant brain (not erythrocyte)

cholinesterase inhibition and clinical signs of toxicity are considered to be relevant effects

for terbufos, the NOAEL was 0.0458 (range, 0.0098–0.0763) mg/m3 (reflecting mean daily

analytical chamber concentrations over the 3-week period of exposure and the range of daily

means) in males and females on the basis of a statistically significant decrease and increase,

respectively in blood glucose concentration and the adrenal to body weight ratio at the next

highest dose in males, and mortality, clinical signs of toxicity, decreases in body weight and

food consumption, and haematological changes in females at the next highest dose

(Whitney, 1980). A statement of compliance with QA, but not for GLP, was provided. The

protocol was not done to satisfy a particular guideline but was generally satisfactory for the

intended purpose of the study.

2.3 Long-term studies of toxicity and carcinogenicity

Mice

In a combined long-term study of toxicity and carcinogenicity, groups of 65 male and

65 female CD-1 mice were given technical-grade terbufos (AC 92,100; purity, 89.6%; prepared in a 1:1 solution of corn oil and methylene chloride) at a concentration (adjusted for

purity) of 0 (vehicle), 3, 6, or 12mg/kg equivalent to 0 (vehicle only), 0.45, 0.9 and 

1.8mg/kg bw per day for males and females for 18 months. Ten animals of each sex per

group were scheduled for interim termination at 12 months (week 53) and the remaining

355

TERBUFOS 333–385 JMPR 2003
55 animals of each sex per group for termination at 18 months (week 80). The rationale for

dose selection was not provided. Parameters assessed included observations for general

health and mortality, clinical signs of toxicity, measurement of food consumption and bodyweight changes, haematological determinations, organ weight measurements and macroand microscopic pathology examinations. Cholinesterase activities were not assessed in this

study.

Mortality was greater at the highest dose in both sexes relative to at the other doses

at both interim and final termination. At week 53, mortality in the control group, and at the

lowest, intermediate and highest dose was 3.6%, 0%, 1.8% and 12.7% for males, and 7.2%,

1.8%, 7.3% and 12.7% for females, respectively. At week 80, mortality in these same groups

was 12.7%, 9%, 5.5% and 27.2% for males and 27.2%, 14.5%, 21.8% and 34.5% for

females, respectively. A relationship to treatment for the decreased survival at the highest

dose could not be dismissed (although the study report did not come to this conclusion).

There were no clinical findings in the study that were obviously related to treatment. Body

weight was statistically significantly decreased in both sexes relative to respective control

groups at most weekly measurement time-points throughout the study. At week 54 and study

termination, statistically significant reductions of about 8% were noted in males and of 10%

and 11%, respectively, in females. At the intermediate dose, during the first 4–5 weeks of

treatment, but not thereafter, statistically significant decreases in body weight of up to about

5% in males and up to about 8% in females were observed. These changes may have been

related to treatment as they were not clearly associated with decreases in food consumption

that might have indicated a palatability problem. Group mean body-weight gains over the

entire study were reduced at the highest dose by 10% in males and 20% in females relative

to respective control groups (statistical significance was not assessed) while group mean

body-weight gains at the intermediate and lowest dose in both sexes exceeded those

observed in animals in the control group. Statistically significant decreases in food consumption associated with treatment occurred at various times throughout the study at the

highest dose in both sexes, with females somewhat more affected than males. Food consumption was not clearly affected by treatment at in the intermediate and lowest doses.

There were no remarkable findings in the haematological, organ weight, or gross pathology

assessments for any group treated with terbufos. With regard to non-neoplasic findings noted

microscopically, slight increases in total occurrences of fatty metamorphosis in the liver

were observed in test material-treated groups of males and females. For males in the control

group and at the lowest, intermediate and highest doses, respectively, the incidence was

13%, 13%, 25% and 28% and for females in these same groups, the incidence was 30%,

38%, 34% and 48%, respectively. In the absence of other associated findings (e.g. organ

weights or other histopathological lesions), the increased instances were considered to 

be of a spontaneous nature and not related to treatment (there were no historical control

data available for comparison). Other microscopic findings in the study had no obvious 

relationship with treatment. The NOAEL for systemic toxicity was 3mg/kg, equivalent to

0.45mg/kg bw per day in males and females, on the basis of statistically significant

decreases in body weights in both sexes during the first 4–5 weeks of the study that 

were not clearly associated with decreased food consumption at the next highest dose. There

was no evidence of carcinogenicity (Silverman et al., 1986). Statements of compliance 

with QA and GLP were provided. The protocol was consistent with US EPA Subdivision 

F Guidelines (November 1982 and revised, 1984).

356

TERBUFOS 333–385 JMPR 2003
Rats

In a combined long-term study of toxicity and carcinogenicity, groups of male and

female Long Evans rats were given diets containing terbufos (AC 92,100; purity not 

specified) at a nominal concentration of 0.25, 1.00 or initially 2.00mg/kg (nominally to

0.0125, 0.05, and 0.1mg/kg bw per day) for 2 years. For both sexes, the highest dose was

raised to 4mg/kg (nominally 0.2mg/kg bw per day) at the beginning of week 6 (day 35),

raised again to 8mg/kg (nominally 0.4mg/kg bw per day) at the beginning of week 12 (day

77), and lowered again for females only to 4mg/kg (nominally 0.2mg/kg bw per day) during

week 16 (around day 105). Two concurrent control groups of males and females were

included in the study. Diets containing terbufos were prepared by adding a premix nominally containing 100mg/kg of feed (0.01%) of active ingredient to lab chow to achieve the

desired nominal concentrations. Diets were apparently not analysed for test material content,

homogeneity or stability (no supporting data or information were provided in the study

report), and intake of test material was estimated based on nominal dose and food consumption data. Animals in the control group were given lab chow only. The study was initiated with 60 animals of each sex per group. A subset of this group (five animals of each

sex in the control group and 10 animals of each sex in each group treated with terbufos)

was terminated at 3 months. The remaining 55 animals of each sex per group in the control

group and 50 animals of each sex per group in each group treated with terbufos continued

into the long-term portion of the study, which terminated at 24 months. During both parts

of the study, all animals were monitored for mortality, clinical signs of toxicity, body-weight

changes and food consumption. Also, during the 3- and 24-month portions of the study,

urine analysis, and haematological and limited clinical chemistry examinations (serum 

glutamic-pyruvic transaminase, alkaline phosphatase, fasting glucose and blood urea nitrogen concentrations) were performed on three control animals of each sex per group and six

animals of each sex in groups treated with terbufos. At study termination, a gross examination was performed on five control animals of each sex per group and on 10 animals of

each sex in groups treated with terbufos at 3 months, on all survivors at 24 months and on

all animals terminated in a moribund condition or found dead.

At 3 and 24 months, selected organ weights were measured (heart, kidney, liver and

thyroid) in five control animals of each sex per group and on 10 animals of each sex in each

group treated with terbufos and a histopathological examination was conducted on five

control animals of each sex per group and on 10 animals of each sex treated with terbufos

at the highest dose. Lung, liver, kidneys and heart were evaluated microscopically in a

similar number of animals at the lowest and intermediate doses at 3 and 24 months. Tissues

from other animals in the 24-month study were stored. Subsequent to the release of the 

original study report, tissues from all rats that were not previously processed were examined microscopically and the results of the 24 month exposure study were re-evaluated on

this basis. Ophthalmoscopic examinations were performed only at 24-months. In three

animals of each sex per control group and six animals of each sex in each group treated

with terbufos, plasma and erythrocyte cholinesterase activities were determined for both

sexes at 3, 6, 12 and 18 months, and brain cholinesterase activity was determined at 3 and

24 months. Inhibition of cholinesterase activity was assessed relative to values for the appropriate concurrent control groups.

There did not appear to be any deaths in the first 3 months of the study. At the intermediate dose, one animal, which was mis-sexed as a male, was placed with other females

of that group, resulting in 59 males and 61 females. When the animals terminated at 3

357

TERBUFOS 333–385 JMPR 2003
months were not included in the calculations, mortality compared with that in the combined

concurrent control groups was statistical significantly increased at the highest dose over the

first 12 months of the study in males (28% in treated compared with 6.4% in the combined

controls) and in females (28% compared with 0% in combined controls) and was statistically significantly increased over the 24 months at the intermediate and highest doses in

males (57.1% and 62%, respectively, compared with 38.2% in combined controls) and in

females at the highest dose (60% compared with 32.7%, respectively, in combined 

controls). Although a relationship of mortality rate and treatment could not be dismissed,

the pathology report indicated illness existed among the test animals such as endemic bronchopneumonia (associated with bacterial infection) and pulmonary disease (said to be associated with the inhalation of food particles) and suggested that these conditions could have

compromised animal well-being to a certain extent in some animals and/or contributed to

the demise of others.

Neither individual nor summary data were provided in the study report for clinical

findings or clinical signs of toxicity. According to the discussion in the study report, signs

consistent with inhibition of cholinesterase activity (muscle tremors, excessive salivation,

hyperactivity, hyperpnoea and tachycardia) were first noted in females at the highest dose

during administration of the diet containing terbufos at 8mg/kg (weeks 12–15). After the

dose was lowered, the signs reportedly decreased in incidence in females at the highest dose

and were not observed from months 18 until the end of the study. Some females at the intermediate dose reportedly exhibited some of the clinical signs (not specified) during months

5 and 6. Muscle tremors were reported in eight males at the highest dose during study

months 22–24. Starting at around the time when females at the highest dose were placed

on the die containing terbufos at 8mg/kg, exophthalmos was noted in this group. Eventually, the condition manifested itself in all other groups of females including the controls and

was said to persist until about month 15. The etiology of this condition was not clear. 

Ophthalmoscopic examination at the end of the study revealed an increase in corneal scarring and cataracts in males and females, but particularly in females, at the highest dose.

Statistically significant decreases in body weight and food consumption compared with

respective control values were observed in males and females at the highest dose throughout much or most of the 2-year study. At 24 months, there was no obvious effect of treatment on haematological, clinical chemistry or urine parameters examined. The significance,

at 3 months, of small magnitude perturbations in concentrations of glucose and blood urea

nitrogen in females at the highest dose was difficult to judge due to the changing doses.

Small magnitude perturbations at the highest dose in relative or absolute weights at 3 months

(kidney, heart, liver) and at 24 months (liver, kidney, and heart) may have been related to

decreases in terminal body weights noted in this group.

Despite some variability in the magnitude and consistency of response among the

various time-points measured, patterns of cholinesterase inhibition were noted for which an

association with treatment could not be dismissed. Erythrocyte cholinesterase activity was

statistically significantly inhibited at months 3, 6, 12, 18 and 24 at the highest dose in males

(42–70%) and in females (35–80%). At the intermediate dose, statistically significant inhibition of erythrocyte cholinesterase activity was observed in males at months 6, 18 and 24

(32–40%) and in females at months 3 and 24 (43–46%). Brain cholinesterase activity was

inhibited in males only at the highest dose at both 3 and 24 months (62–63%) and in females

at the highest dose at 3 months (25%) and 24 months (58%) and marginally at the intermediate dose, 10% at 3 months (not statistically significant) and statistically significantly

by 12% at 24 months. Inhibition of brain cholinesterase activity in females at the inter358

TERBUFOS 333–385 JMPR 2003
mediate dose was not considered to be toxicologically relevant. Plasma cholinesterase was

statistically significantly inhibited in females at the highest dose at months 3, 6, 18 and 

24 by 54 to 70% and marginally in males (statistically significant inhibition of about 40%

only at months 12 and 18). Plasma cholinesterase data showed a remarkable degree of 

variability with time and dose.

Because an insufficient number of animals had originally been evaluated for pathology, a re-evaluation of the 24-month exposure period was conducted. Apparently, most of

the tissues and masses from animals not previously processed for microscopic examination

were available for examination. The total number of rats whose tissues were re-evaluated

histopathologically out of the original numbers started on test (60) were for (males/females):

control group I, 54/55; control group II, 53/55; at the lowest dose, 50/50; at the intermediate dose, 44/50; and at the highest dose, 47/49. Animals terminated after 3 months were not

included.

A new gross examination could not be re-conducted, but the previous report was said

to have been used, as far as was possible, to make correlations with regard to gross and

microscopic examinations.

With regard to the microscopic examination, the re-evaluation report for the 24-month

treatment period discussed a number of non-neoplastic findings in both males and females.

Inflammatory lesions in the lung were associated with two conditions. One was the endemic

bronchial pneumonia (thought to be related to bacterial infection) found in increased incidence in the group at the highest dose. There was also a higher incidence at the highest

dose of a second type of pneumonia (granulomatous) attributed to the inhalation of food

particles containing plant fibres which were said to act as foreign bodies but for which treatment with test material may have been a contributing or pre-disposing factor. It was suggested that an increased incidence of oesophageal distension in animals at the highest dose

may have been related to an effect of treatment on muscle contractility, but the pathology

report suggested possible relationship of this finding to the bacterial infection and sequelae of the bronchopneumonia. A higher incidence at the highest dose of gastric mucosal

ulceration and/or erosion was also of uncertain etiology, although a relationship to treatment could not be dismissed.

The report concluded that there was no evidence that the test material had an effect

on tumorigenesis.

The study was inadequate to assess chronic toxicity owing mainly to outstanding questions involving the etiology and/or relationship to treatment and/or dose of certain nonneoplastic findings (including ocular, lung and stomach lesions), and also uncertainty associated with the variability in some of the cholinesterase measurements for which a relationship to treatment could not be dismissed, and lack of sufficient documentation of clinical

signs of toxicity. Therefore, a NOAEL for chronic (systemic) toxicity was not identified.

Despite some variability and inconsistencies, there were apparent response patterns

noted in the data on cholinesterase activity for which an association with treatment could

not be dismissed. If inhibition of plasma cholinesterase activity is not considered to be an

adverse effect and toxicologically significant inhibition of brain (but not erythrocyte)

cholinesterase activity is considered to be a relevant effect for terbufos, the NOAEL for

cholinesterase inhibition was 1.00mg/kg in males and females (nominally 0.05mg/kg bw

359

TERBUFOS 333–385 JMPR 2003
per day) on the basis of statistically significantly decreased cholinesterase at the next highest

dose.

With regard to the carcinogenicity phase of the study, information presented in the

pathology re-evaluation report for the 24-month period of exposure was considered to be

adequate to support the conclusion that there was no evidence of a carcinogenic response

in the study, provided that the nominal intake of test material intake could be justified (Rapp,

1974). No statements of compliance with QA or GLP were provided.

2.4 Genotoxicity

The results of assays for genotoxicity with terbufos are summarized in Table 3.

Most of the tests for mutagenicity with terbufos in vitro and in vivo gave negative

results. However, in one acceptably performed study of dominant lethal mutation in vivo,

results were inconclusive. In a paper from the open scientific literature (Gentile et al., 1982),

positive results were reported in an acceptably performed assay for mitotic gene conversion

in yeast cells (ade locus) with technical-grade terbufos in the presence or absence of a metabolic activation system, and also with a commercial grade of terbufos, without metabolic

activation. However, insufficient purity and analytical data were provided in the paper for

the materials tested.

Although the results of an assay for unscheduled DNA repair synthesis in cells in

primary culture were negative, only male Fischer 344 rat hepatocytes were used; an optimal

protocol would also have included assessment of hepatocytes from female rats.

2.5 Reproductive toxicity

(a) Multigeneration study

Rats

In a two-generation study of reproductive toxicity, four groups of 25 male and 25

female Sprague-Dawley (COBS CD) rats, F0 generation or F1 generation parental animals,

were given diets containing technical-grade terbufos (AC 92,100; purity, 89.6%; dissolved

in a 1:1 solution of methylene chloride and corn oil) from the pre-mating period until termination of the adults. Terbufos was administered at a dietary concentration of 0 (vehicle

only), 0.5, 1.0, or 2.5mg/kg (equal to group mean intakes for the F0 generation during the

pre-mating period of 0, 0.035, 0.07, and 0.18mg/kgbw per day in males, and 0, 0.04, 0.085,

and 0.22mg/kgbw per day in females, and for the F1 generation during the pre-mating

period of 0, 0.0372, 0.0742, and 0.1943mg/kgbw per day in males, and 0, 0.04, 0.089, and

0.24mg/kgbw per day in females. An additional group of F0 generation animals was given

terbufos at a dietary concentration of 5.0mg/kg (equal to group mean intakes of 0.42mg/kg

bw per day in males and females); this group was, however, terminated early in week 6 of

the pre-mating period because of toxicity in the females.

According to a standard protocol, the F0 and F1 generations were mated twice to

produce F1a and F1b, and F2a and F2b litters, respectively. Dosing commenced in the F0 generation at 63 days before mating when parental animals were aged about 7 weeks and continued for about 191 days in males and 210 days in females (during the lactation period for

the F1b litters). Initiation of dosing for F1 generation parental animals (from F1b litters) commenced after weaning of the F1b litters and continued for about 205 days in males and 

360

TERBUFOS 333–385 JMPR 2003
361

TERBUFOS 333–385 JMPR 2003

Table 3. Results of studies of genotoxicity with terbufos

End-point Test object Concentration/dose Purity (%) Results Reference

In vitro

Reverse mutation S. typhimurium 50–5000mg/plate, 89.6 Negative Allen (1985)a,b

TA98, TA100, 1000mg/disc in

TA1535, TA1537, DMSO, ±S9h

TA1538; E. coli

WP2 uvrAPoint mutation Chinese hamster 10–100mg/ml & in 87.8 Negatived Allen & Johnson,

ovary cells (CHO- DMSO, ±S9c,i (1983)a,b

K1-BH4), Hprt locus

Chromosomal Chinese hamster 2.5–100 nl/ml in DMSO, 87.8f (1983)a,b Negative Thilager (1983)

aberration ovary cells ±S9e,i

Unscheduled Primary rat (male, 0.33–33.33mg/well in 87.8f Negative Godek (1983)b,n

DNA synthesis Fischer 344) DMSOg,i

hepatocytes

Mitotic gene S. cerevisiae strain Apparently Technical and Technical- Gentile et al.

conversion D4 (ade and trp 0.33–33mg/tube in commercial grade: positive at (1982)

loci) DMSO, ±S9 grades the ade locus (±S9);

(purities not Commercial-grade:

specified; no weakly positive at

analytical the ade locus (-S9)o

information)

In vivo

Dominant lethal Crl :CD(SD)BR rats 0, 0.1, 0.2, or 0.4 mg/kg 89.6 Inconclusivel MacKenzie

mutation (10 (10 male rats per bw per dayh,j,k in corn oil (1986)a,b

mating cycles) group) by gavage daily for 5

days at the start of the

first mating cyclel

Chromosomal Sprague-Dawley rats Single intraperitoneal 89.6 Negative Putnam (1986)a,b

aberration (20 males, 20 doses of 0, 0.2,0.6,

females per group), 1.5 (females only)m or

bone-marrow cells 1.8 mg/kg bw in corn oili

DMSO, dimethyl sulfoxide

a

Consistent with US EPA Subdivision F guidelines (1984,revised)

bStatements of compliance with GLP and QA were provided

c

Cytotoxicity observed at 75 and 100mg/ml in main study and at higher concentrations in the dose-finding study dSlightly higher mutation frequencies (+S9) observed relative to the concurrent solvent control at 25 and 50mg/ml were generally in

the range of values for historical solvent controls and were attributed to unusually low mutation frequencies in concurrent controls . A

repeat assay (+S9) gave negative results at concentrations of up to and including 50mg/ml e

Cytotoxicity was observed at 100 nl/ml in main study and at higher concentrations in dose-finding study f

Purity information obtained from the sponsor

gCytotoxicity observed at higher concentrations (100mg/well and above) hDose calculations corrected for % active ingredient

i

No correction of dose for % active ingredient

j

No clinical signs of toxicity or effects on body weight were reported at the highest dose, but this dose was approximately one-tenth of

the reported LD50

kAfter treatment, each male was paired with two non-treated virgin females, 5 days per week for 10 weeks. Females were evaluated

for number of implants, viable and nonviable fetuses and number of corpora lutea about 15 days after the mating period mid-point.

Fertility index and implantation efficiency were calculated. The positive control used, triethylenemelamine, was administered

intraperitoneally to 10 males for 5 days at a dose of 0.05 mg/5 ml saline/kg bw before the first mating cycle l

At mating 9, the number of viable implants at 0.4 mg/kg bw per day was reduced slightly compared with the concurrent vehicle

control group (statistically significant) and with respect to the positive control group (e.g. 12 compared with 14 and 13, respectively).

At mating 7, implant efficiencies at the lowest, intermediate and highest doses (95%, 93%, and 89%, respectively) were lower (with a

difference that was statistically significant at the lowest and highest doses) than that of the concurrent vehicle control group (98%) and

the positive control group (97%). A slightly lower implantation efficiency (not statistically significant) relative to all other groups

continued at the highest dose through the remaining three mating cycles numbers 8–10; the study report concluded that the NOAEL in

males treated for 5 days was 0.2 mg/kg bw per day

m Evaluations of structural chromosomal aberration were conducted in five animals per group (including replacement animals) at 12,

24, and 36 h after dosing. Clinical signs of toxicity and mortality were observed after an intraperitoneal dose of 1.8 mg/kg bw in males

and females and 1.5 mg/kg bw in females. Excessive mortality was observed in females at both of these doses, indicating an increased

sensitivity of females to the test material. Protocol was consistent with US EPA Subdivision F guidelines (1984, revised), but only

hepatocytes from males were used; hepatocytes from females should also have been assayed oResults given only for the dose said to give highest recombinogenic activity. Assay protocol appeared to be generally acceptable, but

no rationale for dose selection was presented. Commercial-grade terbufos gave negative results in Zea mays in a field plot assay for

plant mutation in situ (technical grade not tested)
220 days in females. F1 and F2 litters were reduced to eight pups (equal numbers of each

sex, if possible) on day 4 of lactation. F1a, F2a and F2b litters were sacrificed at or after

weaning on day 21 of lactation. Pups were examined externally for abnormalities and gross

malformations. One F1b and F2b pup of each sex per litter, as well as any dead or stillborn

pups, were necropsied and any abnormal tissues found were preserved.

Assessments performed for parental animals included observations for general condition, mortality and clinical signs of toxicity, measurement of body weight and food 

consumption, ophthalmoscopic examinations, evaluation of reproductive performance,

including mating and fertility and pregnancy rate, duration of gestation and data on parturition, litter size and survival until day 21 of lactation, and macroscopic examination. Organ

weight measurements were not made. At termination of each generation, histopathological

evaluation was made of the following tissues: testes, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, pituitary and any gross lesions; this was performed only for

animals receiving the highest dietary concentration (2.5mg/kg) and for control F0 and F1b

adults that had been selected for mating. Offspring parameters examined included assessment of general appearance, viability, litter sex ratio, and body weight (on days 1, 4, 7, 14,

and 21 of lactation). Inhibition of plasma, erythrocyte and brain cholinesterase activities

was calculated relative to the values for the appropriate concurrent control group for adult

F0 males and females (week 27) and F1b adults, and for males and females at termination.

Cholinesterase activity was not assessed in adult animals receiving terbufos at a dietary concentration of 5.0mg/kg, nor in pups at any dose.

Six out of 25 females at the highest dietary concentration tested (5.0mg/kg) died from

treatment-related causes after 22–34 days. Clinical signs of toxicity, including tremors, a

generally poor condition, emaciation, and unkempt appearance, were also noted in females

in this group. There were no deaths among males at 5mg/kg and few, if any, clinical signs

were observed in males in this group during the same period (two males had excess salivation at pre-mating week 4). During certain periods before termination of all animals at

5.0mg/kg at pre-mating week 6, statistically significant decreases in body weight were

reported in both sexes, as were decreases in food consumption.

In the study of four groups, with 2.5mg/kg as the highest dose, parental mortality was

very low and of comparable incidence in groups treated with terbufos and respective control

groups in the F0 and F1 generation. With the possible exception of soft stools in F0 females

at the highest dose during week 28 (12 occurrences compared with 2, 3 and 6 occurrences

in the control group, and at the lowest and intermediate doses, respectively), there were no

clinical findings that could definitively be associated with treatment with terbufos in either

the F0 or F1 parents. Animals with symptoms considered in the study report to be consistent with sialodacryoadenitis viral infection (including dry eyes in females at the lowest and

intermediate doses) were noticed during weeks 26 to 28 (towards the end of the F0 generation). The study report indicated that the outcome of the study was considered not to have

been affected by the presence of the virus at this stage.

There was no apparent effect of treatment on male or female body weight of parents

in either the F0 or F1 generations, except in lactating females at 2.5mg/kg. Over the duration of the lactation period (days 0–20) for F1a, F1b, and F2b litters, females at the highest

dose exhibited statistically significant body-weight loss relative to that in the respective

control groups. Food consumption did not appear to be adversely affected in parental

362

TERBUFOS 333–385 JMPR 2003
animals of either generation. The results of ophthalmoscopic examinations were not 

remarkable.

In F0 adults, statistically significant inhibition of plasma cholinesterase activity was

observed in males at the highest dose (46%) and females at the intermediate and highest

doses (61% and 94%, respectively). Statistically significant reductions were noted in erythrocyte cholinesterase activity in males at the highest dose (11%) and in females at the

intermediate and highest doses (7% and 15%, respectively). Brain cholinesterase activity

was statistically significantly inhibited in males at 1.0 and 2.5mg/kg (8% and 29%, respectively) and in females at 0.5, 1.0, and 2.5mg/kg (7%, 22%, and 66%, respectively).

In F1 adults, statistically significant inhibition of plasma cholinesterase activity was

observed in males (by 20% and 53%, respectively), and in females (by 50% and 87%,

respectively) at the intermediate and highest doses. Erythrocyte cholinesterase activity was

statistically significantly reduced only at the highest dose in males (by 15%) and in females

(by 13%). Brain cholinesterase activity was statistically significantly inhibited at the intermediate and highest doses in males (by 8% and 34%, respectively) and in females (by 21%

and 59%, respectively).

Decreases in brain cholinesterase activity of 7–8% in F0 and F1 adults at the lowest

and intermediate doses, although possibly treatment-related, were not considered to be 

toxicologically significant.

At 2.5mg/kg, a treatment-related reduction in male fertility and the number of pregnant females was observed for the production of F1b litters and F2b litters compared with

values for these parameters in the respective control groups. In the control group, and at

the lowest, intermediate and highest dose, pregnancy rates were 92%, 88%, 96% and 80%

in the first generation (F1b) and 86%, 92%, 82% and 63% in the second generation (F2b),

respectively. The corresponding male fertility index (number impregnating per number

mated) for F1b litters was 91%, 87%, 96% and 79% and for F2b litters, 94%, 95%, 94% and

61%, respectively.

At the highest dose, there were some, generally slight, changes in several F1b and F2a

and F2b neonatal parameters (such as in mean numbers of pups, live pups or dead pups per

litter and total numbers of live or dead pups per litter). When these parameters were looked

at overall, they were suggestive of possible subtle treatment related decreases in pup viability, number and/or survival at 2.5mg/kg. However, they lacked statistical significance or

persistence; the only statistically significant change, a decrease in the number of live offspring in F1a litters relative to that in the control group (9.8 compared with 12.8) before

reduction of litter sizes on lactation day 4, was not found at later time-points).

A more concrete case for an effect of treatment with terbufos on offspring concerned

pup body weights. On days 14 and 21 of lactation, decreases (about 15–17%) in the mean

weight of viable pups relative to those of pups in the the respective control group, were

observed in F1a, F1b, F2a (smaller decreases of 7–9%) and F2b litters and were considered to

be related to treatment. Decreases were statistically significant in F1a litters, F1b litters (a

decrease of 11% was also noted on day 7), and F2b litters (a decrease of 16 % was also noted

on day 7).

363

TERBUFOS 333–385 JMPR 2003
Other parental reproductive or neonatal parameters examined were not obviously

affected by treatment with terbufos. There were no obvious treatment-related findings upon

gross examination of pups or adults, or upon histopathological evaluation of the adult tissues

and organs selected for analysis.

The NOAEL for reproductive effects was 1.0mg/kg for males and females (equal to

0.07–0.074mg/kgbw per day in males and 0.0854–0.089mg/kgbw per day in females) on

the basis of decreases in male fertility and pregnancy rate, respectively. The NOAEL for

parental toxicity was 2.5mg/kg in males (equal to 0.18–0.19mg/kgbw per day) on the basis

of statistically significant decreases in body weight and decreases in food consumption at

the next highest dose and 1.0mg/kg in females (equal to 0.085–0.089mg/kgbw per day) on

the basis of statistically significant body-weight loss during lactation and an apparent

increase in soft stools at the next highest dose. The NOAEL for offspring toxicity was 

1.0mg/kg (equal to 0.07–0.074mg/kgbw per day in males and 0.085–0.089mg/kgbw per

day in females) on the basis of statistically significant decreases in the mean body weight

of viable pups on days 14 and 21 of lactation in F0 and F1 litters.

If plasma cholinesterase inhibition is not considered to be an adverse effect and toxicologically significant brain (not erythrocyte) cholinesterase inhibition is considered to be

a relevant effect for terbufos, the NOAEL for cholinesterase inhibition was 1.0mg/kg (equal

to 0.07–0.074mg/kgbw per day) in parental males and 0.5mg/kg (equal to 0.04–

0.044mg/kgbw per day) in parental females on the basis of statistically significant decreases

in brain cholinesterase activity in the F0 and F1 generations at the next highest doses

(Schroeder, 1989). Statements of compliance with QA and GLP were provided. The protocol was consistent with US EPA Subdivision F guidelines (1982 and 1984, revised).

(b) Developmental toxicity

Rats

In a study of developmental toxicity, groups of Charles River albino rats were given

technical-grade terbufos (AC 92,100; purity not specified) at a dose of 0 (vehicle only; 

20 rats), 0.075 (20 rats) or 0.150 (21 rats) mg/kgbw per day daily on days 6–15 of gestation, inclusive, by gavage in corn oil. The study was terminated on day 20 of gestation. The

basis for dose selection was not discussed. Adult animals were observed for mortality,

unusual reactions, clinical signs of toxicity, and body-weight changes. Other parameters

assessed were group mean numbers of corpora lutea, implantation sites, resorption sites,

and viable fetuses, as well as number of fetuses, group mean fetal body weights, sex ratio

and the incidence of females with one or more resorption sites. Fetuses were examined

externally for effects of treatment and, where possible, approximately equal numbers of each

sex were evaluated for skeletal and internal developmental abnormalities using the Hurley

method of staining with alizarin, or the Wilson and Warkany technique, respectively.

Cholinesterase activity was not measured.

There were no deaths or unusual reactions in adult animals. No treatment-related

effects were noted in any parameter. This study had many deficiencies, including the following: the purity of the test material and the concentration of the test material in dosing

solutions could not be confirmed; it was not clear if randomization techniques were used

in assigning animals to treatment groups; no data for individual adult animals or fetuses

were provided in the study report; the number of dead fetuses was not indicated; maternal

or fetal data were generally reported only as group means; it was not clear how fetuses were

364

TERBUFOS 333–385 JMPR 2003
selected for examinations for developmental abnormality; fetal abnormalities were not

assessed on a litter basis; the number of fetuses examined for abnormalities did not match

the number of fetuses reported; complete necropsies of adult animals were not conducted.

Also, animals were shipped to the laboratory on day 1 of gestation and were allowed no

acclimation period. NOAELs for maternal and developmental toxicity could not be identified owing to the many deficiencies in study design and data reporting (Haley, 1972). No

statements of compliance with GLP or QA were provided, the protocol was not consistent

with EPA or OECD guidelines and was unacceptable by current standards; the study was

conducted in 1972 (before the establishment of guidelines for GLP and EPA guidelines).

In a preliminary study of developmental toxicity, groups of five female Charles River

COBS® CD® rats were given technical-grade terbufos (AC 92,100; purity, 87.8%) at a dose

(adjusted for purity) of 0 (vehicle only), 0.4, 0.8, 1.4, 3, or 6mg/kgbw per day once daily

by gavage in corn oil during days 6–15 of gestation, inclusive. These doses were actually

twice the amounts intended owing to an unintentional protocol error such that the volume

administered was 10ml/kg instead of 5ml/kg. Owing to excessive toxicity and mortality in

all groups treated with terbufos, the study was terminated prematurely (before day 20 of

gestation) and two additional groups of five females were subsequently given the test material at a dose of 0.05 or 0.2mg/kgbw per day administered in a volume of 5ml/kg. This

second phase was terminated as planned on day 20 of gestation. For all groups in both

phases, planned evaluations in adult females included observations for general condition

and clinical signs of toxicity, mortality and moribundity, body-weight changes, abortions,

number of corpora lutea, and terminal necropsy. Litter and fetal assessments included

number of viable and dead fetuses and fetal position in the uterus, number of implantation

sites, early and late resorptions, early implant loss in uteri with no gross evidence of implantation, and postimplantation loss. No examinations were conducted of fetuses for external,

visceral or skeletal abnormalities, body weight or sex ratio in either phase and cholinesterase

activity was not measured.

During the first phase of the study, all animals died or were sacrificed in a moribund

condition as a result of treatment at doses of ≥0.4mg/kgbw per day. The cause of death was

cardiorespiratory arrest or cerebral haemorrhage. Findings at necropsy included hepatic

congestion, renal and pulmonic hyperaemia, gastric congestion and loss of epithelium,

intestinal congestion and diarrhoea, and in some animals at doses of 1.4mg/kg per day and

above, haemorrhagic intestines. Deaths at 0.4mg/kgbw per day occurred between days 10

and 16 of gestation. At higher doses, deaths or early terminations occurred on days 7–9 of

gestation. Severe body-weight losses were observed in all groups treated with the test material. Clinical signs of toxicity, described as primarily yellow urogenital matting and dried

red matter around the eyes and nares were noted primarily in animals given doses of 0.4

and 0.8mg/kg per day, as survival was longest in these groups. Pregnancy rates in animals

in the vehicle control group and groups treated with terbufos ranged from 80% to 100%.

No abortions were reported. Uterine assessments were incomplete owing to mortality but,

relative to groups receiving the highest dose, an increase in early litter resorptions was noted

at necropsy at doses of 0.4mg/kgbw per day and, to a lesser extent, at 0.8mg/kgbw per

day; this effect may have been related to maternal toxicity and day of gestation. At 0.4mg/kg

bw per day and 0.8mg/kgbw per day, respectively, early resorptions were observed in five

out of five gravid females (maternal deaths on days 10–15 of gestation in this group) and

two out of four gravid females (maternal deaths on days 8 or 9 of gestation in this group),

while none were reported in pregnant animals at doses of ≥1.4mg/kgbw per day (maternal

deaths on day 7 of gestation in these groups).

365

TERBUFOS 333–385 JMPR 2003
In the second phase of the study, the results provided indicated that doses could have,

at least sometimes, been 10–15% below nominal levels. No mortality or clinical signs of

toxicity were reported at 0.2 or 0.05mg/kgbw per day. Groups treated with terbufos gained

slightly less weight than did the vehicle control group during dosing (days 6–16 of 

gestation) and overall gestation (days 0–20 of gestation), but a statistical analysis of the data

was not performed. Pregnancy rates were 100%, 80% and 100% in the vehicle control group

and groups receiving the lowest and intermediate doses, respectively. A delivery early in

the study on day 10 of gestation by one female at the lowest dose was attributed to an error

in the detection of mating, such that the day of parturition was assumed to be 22 on the

basis of pup size and development. The results of necropsy of this and other animals in the

second phase were not remarkable. There were no other premature deliveries nor were there

any abortions. Litter incidence (relative to that in the vehicle control group) of viable

fetuses, implantation sites and corpora lutea were similar for the three dams available for

evaluation at the lowest dose and the five available at the highest dose. There were no dead

fetuses or late resorptions in any group. A slightly higher postimplantation rate of 1.7 per

litter at 0.05mg/kgbw per day, associated with early resorptions, was noted compared with

a value of 1.0 per litter in the control group, but the value for the group receiving the lowest

dose was based on an evaluation of three animals only and the rate at 0.2mg/kgbw per day

(0.6 per litter) was lower than that for the control group. On the basis of the parameters

assessed in phases one and two, the NOAEL for maternal toxicity was 0.2mg/kgbw per

day, as doses administered above this were excessively toxic. A NOAEL for developmental toxicity could not be identified owing to the small number of animals evaluated and the

limited study design and assessments made (Rodwell, 1984). Statements of compliance with

QA and GLP were provided. Protocol deficiencies were noted, but the study was not performed to meet a specific guideline, being a preliminary study.

In a study of developmental toxicity, groups of 25 Charles River COBS CD rats 

were given technical-grade terbufos (AC 92,100; purity, 87.8%) at a dose of 0 (vehicle 

only), 0.05, 0.10, or 0.20mg/kgbw per day by gavage in corn oil once daily on days 6–15

of gestation, inclusive. Doses were adjusted for test material purity. The study was terminated on day 20 of gestation. Selection of doses for this main study was based on the 

results of a preliminary study of developmental toxicity in the same strain of rats; treatment-related mortality and clinical signs of toxicity had been reported after administration

of test material at daily doses of ≥0.4mg/kgbw per day by gavage on days 6–15 of gestation, inclusive (Rodwell, 1984). In the main study, evaluations in adult females included

observations for general condition and clinical signs of toxicity, mortality and moribundity,

body-weight changes, abortions, number of corpora lutea, and terminal necropsy. Litter and

fetal assessments included number of viable and dead fetuses, number of implantation sites,

early and late resorptions, early implant loss in uteri with no gross evidence of implantation, postimplantation loss, and fetal weight, sex, sex ratio, and uterine location. All 

fetuses were examined for external abnormalities. Approximately one-half of the fetuses

from each litter were examined for soft tissue findings using the Wilson sectioning method,

while the remainder were examined for skeletal defects using a modification of the Dawson

alizarin red S staining technique and low power magnification. Cholinesterase activity was

not measured.

In adult animals, no mortalities or treatment-related clinical signs of toxicity were

observed during the study. Slightly lower body-weight gain was noted in groups treated with

terbufos at the intermediate and highest doses, relative to the control group, during dosing

(days 12–16 of gestation and during days 6–16 of gestation), and after dosing (days 16–20

366

TERBUFOS 333–385 JMPR 2003
of gestation). During days 12–16 of gestation, body-weight gains at the intermediate and

highest doses were 7% and 10% less than that of the control group, respectively, while

during days 6–16 of gestation, body-weight gains in these groups were 7% less than that

of the control group. After dosing, body-weight gains at the intermediate and highest doses

were 5% less than that of the control group. In the study report, the relatively lower bodyweight gains at the intermediate and highest doses were considered to be related to administration of the test material. These decreases in body weight were not considered to be

toxicologically significant, being of similar magnitude at the intermediate and highest doses,

relatively small, and not statistically significant. In addition, there was no indication of an

increase in body-weight gain after cessation of dosing.

Pregnancy rates were similar among all groups, ranging from 96% to 100%, and there

were no abortions or premature deliveries.

There were no statistically significant differences in the number of corpora lutea or

implantation sites in groups treated with terbufos relative to values for these parameters in

the concurrent control group. Late resorptions, although not statistically significantly

increased, were observed only at 0.2mg/kgbw per day. All late resorptions (eight) and the

only dead fetus reported in the study were found in the litter of one dam at the highest dose,

along with one early resorption and one fetus with no remarkable findings out of a total of

11 conceptuses. At necropsy of the dam (which survived the study), abnormalities described

collectively in the study report as “severe pathology” but not treatment-related, were found

on gross examination of the liver (described as pale and soft with accentuation of lobular

markings), spleen (described as congested enlarged and indurated) and kidney (described

as pale, bilateral). Follow-up histopathology was not performed. Weight gain (50g) in this

animal during dosing (days 6–16 of gestation) was not dramatically different from the mean

for the control group (57g) or the mean for the group receiving the highest dose (53g);

however, it was the only animal at the highest dose to lose weight (-1g) after cessation of

dosing (mean weight gain at the highest dose after cessation of dosing was 60g). The relationship between litter/fetal findings and the pathology observed in this animal is not clear.

Early resorptions at the intermediate and highest doses of 1.2 and 1.0 per litter, respectively, were slightly elevated (not statistically significantly) relative to values for the concurrent control group (0.8 per litter) and the group receiving the lowest dose (0.5 per litter).

Fetal body weights were comparable in all groups. The number of viable fetuses at

the intermediate and highest doses (13.7 and 13.6 per litter, respectively) were slightly lower,

although not statistically significantly so, than those in the concurrent control group and the

group receiving the lowest dose (15.0 and14.8 per litter, respectively), and were within the

range of data for historical controls for the laboratory and rodent strain used (mean, 13.9

per litter; range, 11.9–15.4). These decreases in the number of viable fetuses were attributed to slight increases (not statistically significant) in postimplantation loss at the intermediate and highest doses (1.2 and 1.3 per litter, respectively) compared with those in the

respective concurrent control group and group receiving the lowest dose (0.8 and 0.5 per

litter, respectively). At the intermediate dose, the increased postimplantation loss (1.2 per

litter) was just inside the range for historical controls (mean, 0.6 per litter; range, 0.1–1.2)

and was associated with losses due to early resorption (no early resorption data for historical controls were provided). At the highest dose, postimplantation loss (1.3 per litter) was

just outside the range for historical controls and was associated with both early and late

resorptions and the one fetal death. If litter/fetal data from the dam with the reported macro367

TERBUFOS 333–385 JMPR 2003
scopic pathology at necropsy were not considered, the changes in litter or fetal parameters

at the highest dose relative to those in the control group appeared to be eliminated.

There was a statistically significant difference in the group mean fetal sex ratio in all

groups treated with terbufos relative to the value for concurrent controls. This was attributed in the study report to a skewed sex ratio in the concurrent control group (male:female,

about 1:1.4) relative to that based on the historical control data provided (calculated as 

male:female 1:1.006), although group means and ranges for historical controls were not

provided in the study report. External, visceral and skeletal fetal examinations did not reveal

any morphological findings associated with treatment.

The study authors did not consider any of the findings on fetal viability, resorptions,

or postimplantation loss at the intermediate and highest doses to be biologically meaningful, because none of the changes were statistically significant relative to the control group.

Available data for historical controls generally supported this contention at the intermediate dose and, in the case of fetal viability, at the highest dose. In addition, there was no

apparent effect on postimplantation loss, fetal viability or resorptions (early or late) at the

highest dose, if litter/fetal data from the female with reported pathology at the highest dose

are eliminated from consideration. Although no clear maternal or developmental toxicity

was considered to have occurred in this main study, mortality was seen at a dose of 

0.4mg/kgbw per day in a preliminary study (Rodwell, 1984), at just twice the highest dose

used in the main study (0.2mg/kgbw per day), thus providing support for dose selection in

the main study.

The NOAEL for maternal toxicity and developmental effects was 0.2mg/kgbw per

day, the highest dose tested (Rodwell, 1985). Statements of compliance with GLP and 

QA were provided and the protocol was consistent with US EPA Subdivision F (1982)

guidelines.

Rabbits

In a study of developmental toxicity, groups of 17 New Zealand white

(Hra:(NZW)SPF) rabbits were given technical-grade terbufos (AC 92,100; purity, 89.6%)

at a dose (adjusted for purity) of 0 (vehicle only), 0.05, 0.10, 0.25 or 0.50mg/kgbw per day

by gavage in corn oil, once daily during days 7–19 of gestation. The study was terminated

on day 29 of gestation. Data on dose selection were not provided. Upon analysis, the range

of received concentrations of test material was 87–98% of target concentrations. Evaluations in adult females included observations for general condition and clinical signs of 

toxicity, mortality and moribundity, body-weight changes, food consumption, abortions,

premature delivery, uterine weights, number of corpora lutea, and terminal necropsy. Litter

and fetal assessments included number of viable and dead fetuses, number of implantation

sites, early and late resorptions, postimplantation loss, and fetal weight, sex, sex ratio and

uterine location. All fetuses carried to study termination were examined for external, visceral and skeletal abnormalities using standard techniques (including the use of alizarin red

S staining). Cholinesterase activity was not measured.

There were no adult deaths and no premature deliveries. Two animals aborted, one in

the group receiving the highest dose on day 21 of gestation (9 conceptuses) and the other

in the group receiving a dose of 0.10mg/kgbw per day (6 conceptuses) on day 22 of 

gestation. Litters were not available for analysis, presumably due to cannibalization. The

368

TERBUFOS 333–385 JMPR 2003
study report did not attribute this litter loss to treatment, as there were no signs of toxicity

or apparent gross lesions at necropsy. There was some decrease in body weight and food

consumption in both animals in the days immediately before abortion. A possible relationship of abortion at the highest dose to treatment could not be discounted, as other maternal

treatment-related findings occurred at that dose.

The only clinical related to treatment was a statistically significant increase in the incidence of does with soft or liquid faeces at a dose of 0.5mg/kgbw per day. This effect, which

did not occur in the vehicle control group, was observed in four rabbits on 1 or 2 days and

was associated with some decrease in body weight and food consumption, but no gross

lesions at necropsy. Three rabbits (one not pregnant) had occurrences during the latter part

of or just outside of the dosing period during days 16–20 of gestation. Another pregnant

animal had one occurrence on day 29 of gestation. Instances of soft or liquid faeces associated with transient decreases in body weight and food consumption at other doses were

not considered to be related to treatment, as there was no statistically significant increase

and the incidence was low (e.g. found once, on day 18, in one animal at 0.05mg/kg per day

and twice, on days 24 and 25, in one animal at 0.10mg/kgbw per day).

Maternal body-weight gain at the two higher doses (+ 0.16 and +0.16kg, respectively)

was decreased relative to that in the vehicle control group and at the two lower doses 

(+0.26, +0.23 and +0.23kg, respectively) during the time interval encompassing the dosing

period (days 7–20 of gestation). Between days 16 and 20 of gestation, there was some loss

of body weight in animals at the highest dose, which was not seen in other groups. Although

none of the body-weight findings at the two higher doses was statistically significant, in the

study report they were considered to be related to treatment. Slight decreases in food consumption relative to that of controls were only noted at the two higher doses and then only

during the period after cessation of dosing (days 20–29 of gestation); the study report suggested that this could have been related to a delayed effect of treatment. The present reviewer

considered that his interpretation was possible but questionable. At necropsy, two animals

at the highest dose (not those with soft or liquid faeces) were found to have reddened areas

in the fundic area of the stomach that were considered to be treatment-related in the study

report. The results of necropsy in other animals were not remarkable.

The incidence of pregnancy in the control group and at 0.05, 0.10, 0.25 and 

0.50mg/kgbw per day was 94%, 71%, 82%, 76% and 88%, respectively. There was no

apparent effect of treatment with the test material on the number of corpora lutea, implantations, or live and dead fetuses (all fetuses were reported to be alive at study termination),

or on litter size, sex ratio, or uterine weights (not including data from the two females that

aborted). Although incidences of early or late resorptions were comparable between groups,

at the highest dose there was an increase in the incidence of does with any resorption site.

For the control group and at 0.05, 0.10, 0.25 and 0.50mg/kgbw per day, the total number

(and percentage) of does with resorptions of any type was 3 (18.8%), 5 (41.7%), 5 (38.5%),

4 (30.8%) and 10 (71.4%), respectively. The increase at the highest dose was not statistically significant and was not considered to be treatment-related in the study report. However,

the incidence was higher than that in the concurrent control and was outside of the range

for historical controls (data provided on a percentage basis, e.g. mean, 129 (39.2%) with a

range of 0 (0%) to 10 (64.3%) based on data from 329 control groups from 21 studies conducted at the test facility between 1986 and 1988; current study conducted in 1988), 

such that a relationship to treatment could not be dismissed. In addition, a slight decrease

in the group mean fetal body-weight at the highest dose (42.48g) relative to that of the 

369

TERBUFOS 333–385 JMPR 2003
concurrent control (44.8g) was considered by the study authors to be an effect of treatment,

although the difference was not statistically significant. Similar values were obtained when

male and female fetal body weights at the highest dose were separately compared with those

for the appropriate concurrent control group. No fetal external, visceral or skeletal findings

were considered to be related to treatment with the test material.

With regard to maternal toxicity, although the study report suggested that there was

a treatment-related effect at the intermediate dose, the evidence cited to support this contention (a relatively small body-weight decrease during the dosing period, which was not

dose-dependent and was not statistically significant in a species in which body-weight variability is common; and possibly a slight decrease in food consumption after dosing) is of

questionable toxicological significance in the absence of other findings. The NOAEL for

maternal toxicity was 0.25mg/kgbw per day on the basis of an increased number of does

with soft or liquid faeces, maternal body-weight loss during days 16–20 of gestation, occurrence of reddened areas in the fundic region of the stomach at necropsy, and one abortion

at the next highest dose. The NOAEL was 0.25mg/kgbw per day on the basis of decreased

fetal body weights and an increase in the incidence of does with any resorption sites at the

next highest dose (Hoberman, 1988a, 1988b). Statements of compliance with GLP and QA

were provided and the protocol was consistent with EPA Subdivision F Guidelines (1982

or 1984, revised).

2.6 Special studies

(a) Neurotoxicity

(i) Acute delayed neurotoxicity

Technical-grade terbufos (AC 92,100; purity, 96.7%) was tested for acute delayed neurotoxicity potential in sex-link hens (aged approximately 1 year). Confirmation of the

content of test material in the dosing solution was not provided in the study report. In a preliminary study to aid in dose selection for the main study, the acute oral LD50 in this strain

of hen for technical-grade terbufos in corn oil was estimated by probit analysis to be 

40mg/kgbw (95% confidence interval (CI), 31.8–48.2mg/kgbw). The test material was

administered by gavage as single doses at 10 (one animal), 20 (one animal), 28.3 (three

animals), 40 (four animals), 56.6 (four animals) or 80mg/kgbw (two animals). The incidence of mortality in these groups respectively, was 0/1, 0/1, 1/3, 1/4, 4/4 and 2/2. There

was some variability in the time to death. Mortality was observed at 22h after dosing at

28.3mg/kgbw, at 4.5h after dosing at 40mg/kgbw, at 1.5–22h after dosing at 56.6mg/kg

bw and within 2.5h of dosing at 80mg/kgbw. The only clinical findings reported were

observed in some animals at 28.3 and 40mg/kgbw and consisted of wobbly gait or unsteady

gait and stance at 20–24h after dosing, and resolved by 70–72h after treatment.

The main study consisted of two phases. In phase one, 10 hens received the test material in corn oil as a single dose of 40mg/kgbw by gavage. Included in this group of 10 were

the three surviving animals that had received a dose of 40mg/kgbw in the preliminary study

for determination of acute LD50. Other groups of hens received corn oil alone (four animals)

or tri-ortho-cresyl phosphate (TOCP) at 500mg/kgbw in corn oil as the positive control 

(10 animals). There was no pretreatment with atropine. Hens were observed for 21 days

after dosing for mortality, signs of neurotoxicity and locomotor abnormalities.

370

TERBUFOS 333–385 JMPR 2003
All animals surviving study phase one were entered into study phase two. This inadvertently included any animals exhibiting effects consistent with delayed neurotoxicity (e.g.

animals treated with TOCP) that were originally intended for sacrifice on day 22 after dosing

in phase one. In phase two (which appeared to commence about 26 days after phase one

dosing), the seven terbufos-treated hens surviving phase one received a second single dose

of the test material by gavage in corn oil, while the surviving three or seven hens in the

vehicle and positive control groups, respectively, were similarly dosed a second time with

either corn oil only or 500mg/kgbw of TOCP. Animals treated with TOCP were pretreated

with atropine. After a 21-day observation period, animals treated with TOCP (seven) and

vehicle control group animals (three) were terminated on day 22 after dosing. Surviving

hens (six) treated with TOCP were terminated three days after dosing in phase two (i.e. 29

days after dosing in phase one). The original study report indicated that the spinal cords of

only the three animals in the vehicle control group and the six animals treated with TOCP

(animals considered to display signs of (delayed) neurotoxicity) were subjected to

histopathological examination. Procedures used in preparation for the examination were

only described briefly in the study report. It was stated that spinal cords were fixed in situ

(perfusion was not indicated) in buffered formalin for 2 days before sectioning. Although

brain, spinal cord (cervical, thoracic and lumbosacral sections), and sciatic nerve tissue were

taken, only lumbosacral spinal cord sections (stained with haematoxylin and eosin) were

examined microscopically. The tibial nerve was not examined. The hens were not subjected

to a period of forced motor activity and there was no evaluation of cholinesterase or neuropathy target esterase activities in either study phase one or two.

During phase one of the main study, one out of four control animals died on day 19

and exhibited no signs or symptoms before death. Three out of 10 animals treated with terbufos and not pretreated with atropine died; two on day 0 and one on day 12 after dosing.

No clinical symptoms were reported for the animals that died on day 0. The animal that

died on day 12 exhibited two instances of wobbly gait and/or wobbly stance of low severity on days 1 and 7 after dosing. On day 1 after dosing, three additional animals treated

with terbufos displayed single occurrences of low severity wobbly gait and/or wobbly stance.

The deaths and clinical observations in the animals receiving terbufos were attributed to

cholinergic toxicity. None of the animals treated with terbufos or the vehicle control animals

dying during phase one were examined histopathologically. Based on the deaths in the terbufos animals in the preliminary study and in phase one of the main study, the acute oral

LD50 in hens was re-estimated to be 43.5mg/kgbw.

Animals treated with TOCP in phase one exhibited no signs of acute toxicity but did

have symptoms of locomotor impairment (wobbly gait and/or wobbly stance) that started

on day 11 after dosing in all animals and grew progressively worse, such that by day 21

after dosing most animals fell while walking, could only stand or walk in a squatted position or could not walk or stand at all. Three of the most severely affected hens died 26 days

after dosing in phase one and were not examined histopathologically.

After dosing in phase two, signs of acute toxicity were observed in all seven animals

treated with terbufos (and pretreated with atropine). The findings, which were attributed to

cholinergic toxicity, lasted up to 72h and were generally of low severity (wobbly gait and/or

wobbly stance) except in the case of one animal that could not walk or stand one day after

dosing only. There were no observational findings in these animals after day 3 after dosing.

According to the study report, these animals were not examined histopathologically after

371

TERBUFOS 333–385 JMPR 2003
termination on day 22 after dosing in phase two because they showed no indications of

delayed neuropathy.

Of the seven hens that inadvertently received a second dose of TOCP, three (which

already had severe symptoms of locomotor impairment subsequent to phase one) died on

day 2 or 3. The remaining hens in this group were terminated on day 3 after dosing in phase

two and the spinal cord of six of the seven hens was subjected to histopathological examination. Minimal (two hens), slight (two hens), and moderate (two hens) degrees of demyelination of spinal cord fibre tracts (white matter) were found. One animal had swollen axis

cylinders (white matter) and another had gliosis, both effects being of low severity. There

were no observational or histopathological findings in the three control animals carried over

into phase two.

Subsequently, the spinal cords of the seven animals treated with terbufos in phase two

were examined histopathologically; it is not clear how the tissues of these animals were prepared for examination. Two animals had a perivascular lymphocytic cell infiltrate of low

severity in the spinal cord, which was not considered to be of any concern. No demyelination or other adverse changes were apparent in the tissues examined. There was no evidence

to suggest that terbufos caused delayed neuropathy in hens under the conditions of the study

(Smith, 1972, 1973). No statements of compliance with QA or GLP were provided. The

protocol was generally consistent with EPA Subdivision F Guidelines (1982 and 1984,

revised).

(b) Acute neurotoxicity

In an study of acute oral neurotoxicity, groups of 20 male and 20 female SpragueDawley-derived (outbred) Crl:CD®(SD)IGS BR VAF/Plus® rats (fasted before dosing)

were given technical-grade terbufos (AC 92,100; purity, 89.7%) as a single dose (not

adjusted for purity) of 0 (vehicle only) 0.15, 0.30, or 0.90mg/kgbw by gavage in corn oil.

The study was terminated on day 15, following an observation period after dosing.

The selection of doses administered in the definitive study was based on the results

of a pilot study to determine time of peak effect in males and females of the same strain of

rat. In this study, groups of five males and five females were given single doses of the test

material at (0 (vehicle only), 0.025, 0.05, 0.15, 0.40, 1.2 or 1.6mg/kgbw in males and 0

(vehicle only), 0.025, 0.05, 0.15, 0.30, 0.90 and 1.2mg/kgbw in females by gavage in corn

oil. Five additional females were treated with terbufos at the highest dose, in case replacements were needed. All animals were monitored for clinical signs of toxicity, and at about

6h after dosing (designated as the time of peak effect), plasma, erythrocyte and brain

cholinesterase activities were measured in five animals of each sex per group. In the pilot

study, one female died from causes attributed to treatment with the test material within 5h

of treatment with a dose of 1.2mg/kgbw. Clinical signs of toxicity consistent with inhibition of cholinesterase activity were noted at the two higher doses in males and females about

6h after dosing; females were said to have been more severely affected. At 1.6mg/kgbw in

males, excessive salivation, irregular gait and tremors were observed in one male and irregular gait was observed in two males at 1.2mg/kgbw group. Seven females at 1.2mg/kgbw

group exhibited irregular gait and tremors. Other findings in this group were excessive salivation and lacrimation, moist rales and ventral surface and anogenital yellow staining in

two animals, and decreased activity in one animal. At a dose of 0.9mg/kgbw, the only 

clinical finding observed was ano-genital staining in one female. No clinical signs were

372

TERBUFOS 333–385 JMPR 2003
observed in other groups. Miosis was not reported for any animals in the study. Plasma

cholinesterase activity was statistically significantly inhibited in males by 45%, 90% and

88% at doses of 0.40, 1.2, and 1.6mg/kgbw per day, respectively, and in females by 17%,

87% and 96% at 0.3, 0.9, and 1.2mg/kgbw, respectively. Erythrocyte cholinesterase activity was inhibited at the two higher doses in both sexes: in males, by 92–94% (statistically

significant); and in females, by 88–93% (not statistically significant but considered to be

treatment-related). Brain cholinesterease activity was statistically significantly inhibited by

57% and 52% at 1.2 and 1.6mg/kgbw, respectively, in males. A statistically significant

decrease in brain cholinesterase activity of 67% was observed in females at 1.2mg/kgbw,

and a decrease of 41% at 0.9mg/kgbw was considered to be treatment-related, if not statistically significant. In the pilot study, no treatment-related effects were reported in either

sex at doses of <0.40mg/kgbw in males and <0.30mg/kgbw in females.

Before the start of the definitive study, survival was assessed in an additional group

of 10 females given the test material at a dose of 0.90mg/kgbw by gavage. The only data

reported for this study were the findings for animals manifesting clinical signs of toxicity

during daily observations. Signs of toxicity (first observed on the day of treatment and

apparently disappearing by day 5 after dosing) were reported for six of the 10 animals and

included bilateral tremors in fore and hind paws, slight to moderate anogenital staining,

decreased faecal volume, fasciculation, extreme lacrimation and excessive salivation. No

miosis was observed.

In the definitive study, all animals were assessed for physical condition, mortality,

clinical signs of toxicity, body-weight changes and food consumption. Neurobehavioural

evaluations (motor activity and functional observational battery) were conducted on 10

animals of each sex per dose pre-test, at approximately 6h after dosing (time of peak effect)

and on days 8 and 15 (study termination day). Blood was collected for plasma and erythrocyte cholinesterase activity measurements from 10 animals of each sex per dose at about

6h after dosing, and on day 8, and in five animals of each sex per dose on day 15. Brain

cholinesterase activity in one-half brain homogenates was also assessed in the same number

of animals at about 6h after dosing and on day 15. Cholinesterase inhibition was determined relative to the appropriate concurrent control group value. All animals received

macroscopic examinations and selected tissues from the central and peripheral nervous

system were evaluated histopathologically in five perfused animals of each sex per group.

In the definitive study, the only death attributed to treatment occurred in females at

the highest dose, 5–6h after dosing. There were no accompanying clinical findings reported

for this animal, which was replaced by another female for the remainder of the study. Clinical signs of toxicity, some commencing on the day of treatment, were noted in the daily

physical examinations in one surviving female at the highest dose; these included moderate to extreme ano-genital staining, lethargy, decreased faecal volume and food consumption, and oral/buccal staining. No clinical findings were reported after day 5 during daily

physical observations in this female. One male at the intermediate dose exhibited a red

ocular exudate on day 9, but this may have been related to orbital sinus bleeding for

cholinesterase measurements. Slight statistically significant decreases in body weights

observed in females at the highest dose may have been largely related to decreases noted

in the one animal exhibiting clinical signs of toxicity. There were no significant changes

noted in male body weights or food consumption in either sex, relative to values for the

respective control groups.

373

TERBUFOS 333–385 JMPR 2003
There were no statistically significant differences noted in the mean value for motor

activity for the group, relative to values for the respective control group, on days 1, 8 and

15. The study report mentioned that the one female at the highest dose that had clinical

signs of toxicity during the daily physical examinations also exhibited decreased motor

activity on days 1 and 8, but not on day 15.

Treatment-related abnormalities in the functional observational battery, attributed to

cholinergic toxicity, were apparent in males and females at the intermediate and highest

dose and only during the peak time assessment at day 1. Miosis, present in three males at

the intermediate dose, six males at the highest dose and one female at the intermediate dose,

was the only finding at these doses. A wider spectrum of findings was present in females

at the highest dose. In addition to miosis in all 10 rats, females in this group exhibited

tremors (seven animals), and muscle fasciculations (four animals) and, to a lesser extent,

ataxic gait and slightly impaired locomotion, tip-toe gait, moderate lacrimation and soiled

coat. In one animal, very low arousal state, flattened posture (in home cage), no approach

response, severe lacrimation and slight salivation, no open field movement, soiled coat, and

markedly decreased forelimb and hindlimb grip strength were reported. At the next two

time-points (days 8 and 15), no abnormal findings were evident in any group.

On day 1, around the time of peak effect, plasma cholinesterase activity was statistically significantly decreased in males by 31% and 69%, and in females by 20% and 90%

at the intermediate and highest doses, respectively. There was no statistically significant

inhibition at any dose in either sex on days 8 or 15. Erythrocyte cholinesterase activity was

statistically significantly inhibited on day 1 only in males (67%) and females (90%) at the

highest dose. Values continued to be decreased in females at the highest dose by about 45%

on days 8 and 15. Brain cholinesterase activity was statistically significantly inhibited on

day 1 only at the highest dose by 21% in males, and by 51% in females. Statistically significant depression of brain cholinesterase activity (of 13%) continued to be observed on

day 15 in females at the highest dose.

Macroscopic and microscopic examinations did not reveal any findings related to

treatment. Trauma related to retro-orbital sinus bleeding was considered to account for the

minimal to slight focal degeneration of optic nerve fibres accompanied by minimal to slight

gliosis observed in two females at the highest dose and minimal degeneration of a single

sciatic nerve fibre in only one male at the highest dose was considered to be incidental in

nature. It should be noted that miosis was observed during the functional observational

battery in both sexes at 0.3mg/kgbw, with no significant decrease in concurrently measured

brain and erythrocyte cholinesterase activity. Only marginal (but statistically significant)

concurrently measured plasma cholinesterase activity was observed at this dose in both

sexes. If inhibition of plasma cholinesterase activity is not considered to be an adverse

effect, the NOAEL was 0.15mg/kgbw in males and females on the basis of miosis in the

functional observational battery in both sexes at the next highest dose (Mandella, 1998).

Statements of compliance with QA and GLP were provided and the study protocol was consistent with EPA Subdivision F Guidelines (1984, revised and March 1991).

(c) Neurotoxicity after repeated doses

In a study of neurotoxicity, groups of 20 male and 20 female Sprague-Dawley derived

(outbred) Crl:CD®(SD)IGS BR VAF/Plus® rats were given diets containing technicalgrade terbufos (AC 92,100; purity, 89.7%; dissolved in acetone and mixed with a GRIT374

TERBUFOS 333–385 JMPR 2003
O’Cobs® carrier) at a dose (adjusted for purity) of 0 (diet mixed with acetone and carrier),

0.5, 0.8, or 5.0mg/kg in males (equal to 0, 0.036, 0.059 and 0.369mg/kgbw per day) and

of 0 (diet mixed with acetone and carrier), 0.5, 0.8 or 3.0mg/kg in females (equal to 0,

0.042, 0.064, and 0.251mg/kgbw per day) daily for approximately 13 weeks. Animals evaluated for potential neurobehavioural changes were treated for at least 13 weeks and animals

in which cholinesterase activities were measured were treated for at least 85 days. Selection of doses was based on a 22-day preliminary study of feeding in the same strain of 

rat (Mandella, 1999), in which treatment-related findings were noted at doses of ≥5mg/kg

(0.55mg/kgbw per day) in males and ≥3mg/kg (0.33mg/kgbw per day) in females. In the

13-week study, all animals were assessed for physical condition, mortality, clinical signs of

toxicity, body-weight changes, and food consumption and received ophthalmoscopic examinations. Neurobehavioural evaluations (motor activity and functional observational battery)

were conducted on 10 animals of each sex per group, before treatment and at weeks 4, 8,

and 13. In animals designated for cholinesterase activity determinations (9–10 animals of

each sex per group), blood samples for assessment of plasma, erythrocyte and brain (onehalf homogenate) cholinesterase activities were obtained at weeks 4, 8, and 13. In animals 

designated for neurobehavioural examinations, blood and brain samples for measurement

of cholinesterase activities were obtained from five animals of each sex per group during

weeks 13 or 14. Cholinesterase inhibition was determined relative to values for the appropriate concurrent control group. At study termination (week 13 or 14), macroscopic examinations were performed on all animals and selected tissues from the central and peripheral

nervous system were evaluated histopathologically in five animals of each sex per group.

One male at the lowest dose died in week 5 due to accidental causes. Otherwise, there

were no deaths in the study. There were no findings during physical examinations that were

considered to be treatment-related and miosis was not observed in any animal. The results

of ophthalmoscopic examinations were not remarkable. Over the 13-week period, males at

the highest dose gained only about 90% of the weight gained by animals in the control

group; a possible effect of treatment could not be dismissed. Weight gains in males at the

lowest and intermediate doses during the same period were slightly higher than those of

animals in the control group. Females at the highest dose gained about 13% more weight

over the duration of the study than did animals in the control group, but this was not considered to be an adverse effect. There were no changes in food consumption in either sex

that could definitively be ascribed to treatment and no obvious effect of treatment with the

test material on motor activity or on functional observational battery parameters. In the

macroscopic and microscopic examinations, there were no findings that were attributable

to treatment.

At the highest dose, plasma cholinesterase activity in animals designated for

cholinesterase measurements or neurobehavioural evaluations was statistically significantly

inhibited at all time-points by 70–74% in males and by 91–92% in females. In the same

groups, erythrocyte cholinesterase activity was virtually completely inhibited at all timepoints (decreases of 97–100% in males and 100% in females). At the intermediate dose,

erythrocyte cholinesterase activity was statistically significantly inhibited in males in the

group of animals designated for cholinesterase measurement at week 8 and 13 by 48% and

37%, respectively, and by 35% at week 13 in the group designated for neurobehavioural

evaluations. In both groups of females at the intermediate dose, inhibition of 33% (statistically significant) and 27% (not statistically significant) was observed at week 13. At study

termination, brain cholinesterase activity was decreased only at the highest dose in animals

375

TERBUFOS 333–385 JMPR 2003
designated for cholinesterase measurement and neurobehavioural evaluation, by 58% and

55%, respectively, in males and by 71% and 68%, respectively in females.

If inhibition of plasma cholinesterase activity is not considered to be an adverse effect

and statistically significant inhibition of erythrocyte cholinesterase activity of 33–43% is

not considered to be a relevant effect for terbufos, the NOAEL was 0.8mg/kg (equal to

0.059mg/kgbw per day in males and 0.064mg/kgbw per day in females) on the basis of

statistically significant inhibition of brain and erythrocyte (almost completely inhibited)

cholinesterase activity at the next highest dose (Mandella, 1999). Statements of compliance

with QA and GLP were provided. The protocol was consistent with EPA Subdivision F

Guidelines (November, 1984, revised, and March 1991).

(d) Studies on metabolites

In a study of short-tem toxicity, groups of four male beagle dogs were given gelatin

capsules containing technical-grade terbufos (AC 92,100; purity, 89.6%) or one of its

metabolites, terbufos sulfoxide (CL 94,301; purity, 90.0%) and terbufos sulfone (CL 94,320;

purity, 92.0%), in corn oil, administered orally once daily in the morning for at least 14

days. A similarly treated control group of six animals received gelatin capsules containing

corn oil only. The animals used were described as having physical impairments but not 

of sufficient magnitude to jeopardize study integrity. Doses administered (adjusted for

purity) were: terbufos, 2.5 or 250mg/kgbw per day; terbufos sulfoxide, 5, 15, 625, or 

250mg/kgbw per day; or terbufos sulfone, 15, 62.5, 250, and 1000mg/kgbw per day. A

limited number of parameters were assessed, including mortality, morbidity and clinical

signs of toxicity, measurement of body weight and food consumption, and a gross examination at the end of the study. Plasma and erythrocyte cholinesterase activity in fasted

376

TERBUFOS 333–385 JMPR 2003

Table 4. Acute toxicity of metabolites and degradation products of terbufos in female mice

Metabolite Strain Route Vehicle LD50 (mg/kg bw) Purity (%) Reference

Terbufoxon sulfoxidea CF1 albino Oral Corn oil 1.1 Not stated American Cyanamid Company

A72-35 (1972d)

Terbufos sulfoxideb CF1 albino Oral Corn oil 3.4 Not stated American Cyanamid Company

A72-37 (1972b)

Terbufoxon sulfonec CF1 albino Oral Corn oil 3.4 Not stated American Cyanamid Company

A72-38 (1972e)

Terbufos sulfoned CF1 albino Oral Corn oil 14i Not stated American Cyanamid Company

A72-34 (1972c)

Terbufoxone CF1 albino Oral Corn oil 2.2 Not stated American Cyanamid Company

A72-36 (1972f)

CL 202,135f CF1 albino Oral Corn oil 3670g Not stated American Cyanamid Company

A73-21 (1973a)

CL 202,474h CF1 albino Oral Corn oil >2500j Not stated American Cyanamid Company

A73-122 (1973b)

Although reports for these studies were not detailed and statements of compliance with GLP or QA were not provided, protocols

appeared to be generally consistent with EPA Subdivision F Guidelines (1982 or 1984, revised) a

Phosphorothioic acid, S-(t-butylsulfinyl) methyl O,O-diethyl ester (CL 94,365) bPhosphorodithioic acid, S-(t-butylsulfinyl) methyl O,O-diethyl ester (CL 94,301) c

Phosphorothioic acid, S-(t-butylsulfonyl) methyl O,O-diethyl ester (CL 94,302) dPhosphorodithioic acid, S-(t-butylsulfonyl) methyl O,O-diethyl ester (CL 94,320) e

Phosphorothioic acid, S-(t-butylthio) methyl O,O-diethyl ester (CL 94,221) f

Methane, bis(t-butylsulfonyl) (CL 202,135) gReport stated that acute oral LD50 was calculated assuming a mortality of 9 out of 10 animals at a dose of 10 000 mg/kg of feed hMethane, (t-butylsulfinyl)(methylsulfinyl) i

Report stated that acute oral LD50 ws calculated assuming a mortality of five out of five animals at a dose of 50 mg/kg of feed j

Report stated that animals were not fasted
animals was assessed twice before treatment, and on days 4, 8, 11, and 15. Brain

cholinesterase activity (in homogenates of cerebellum and cerebrum) was measured from

tissues taken at study termination (day 15 or 16) and stored frozen until analysis. Inhibition

of cholinesterase activity was determined relative to the value for the appropriate concurrent control group. Overall, the data on brain cholinesterase activity, especially those from

the cerebellum, were considered to be unreliable owing to inconsistencies, variability in the

data and poor dose–response relationships. Clinical chemistry, haematological, urine analysis, organ weight and histopathological evaluations were not conducted in this study.

There were no deaths in the control group. The only clinical observations reported

were occurrence of soft faeces (four animals) and one occurrence of alopecia. There were

no other adverse findings.

There were no deaths in the groups of animals treated with terbufos. Clinical findings

attributed to treatment at the highest dose (250mg/kgbw per day) were instances of emesis

and lacrimation, and an increased incidence of soft or soft sanguineous-looking faeces

(seven occurrences) compared with the control group; treatment-related decreases in body

weight and food consumption were also noted in some animals. At the highest dose, plasma

cholinesterase activity was statistically significantly inhibited by 57–76% at all time-points

during the study, and erythrocyte cholinesterase activity was statistically significantly inhibited by 42–79% on days 8, 11 and 15, and cerebellar and cerebral cholinesterase activities

were statistically significantly inhibited by 48% and 37%, respectively. At the lowest dose,

statistically significant decreases in plasma cholinesterase activity were noted on days 11

and 15, but they were of low magnitude (23–25%) and not clearly related to treatment. There

were no obvious effects of treatment on erythrocyte or cerebral cholinesterase activities at

the lowest dose. Cerebellar cholinesterase activity at the lowest dose was statistically significantly inhibited by 42%. As a finding of significant brain cholinesterase inhibition at this

dose was inconsistent with other studies of repeated dosing with terbufos, it was considered highly unlikely to be related to treatment. A finding of dark mesenteric lymph node(s)

at gross necropsy in one animal in the group treated with the lowest dose of terbufos was

not clearly related to treatment.

In the groups of animals treated with terbufos sulfoxide, there were no mortalities. At

the highest dose (250mg/kgbw per day), clinical findings ascribed to treatment included

ataxia, salivation, languid behaviour, salivation, miosis, no faeces, emesis, and an increased

incidence relative to control of soft faeces (11 occurrences). Decreases in body weight and

food consumption attributed to treatment were noted in some animals at the highest dose.

At all time-points during the study, plasma and erythrocyte cholinesterase activities

were statistically significantly inhibited by 60–71% and 30–93%, respectively, at the highest

dose. In groups given terbufos sulfoxide at 62.5 and 15mg/kgbw per day, there was no clear

treatment-related effect on body weights, food consumption or clinical findings. Single

occurrences of emesis or sanguineous-looking emesis were noted in each of these groups,

as was one instance of no faeces at 15mg/kgbw per day. Occurrences of soft stool in the

control group, and at the lowest, lower intermediate, higher intermediate and highest dose

were 4, 3, 13, 6, and 11, respectively. Increases relative to the control group at the two intermediate doses did not appear to be part of an obvious pattern that might be definitively

atributed to treatment. Although plasma cholinesterase activity was statistically significantly

inhibited at all time-points at both intermediate doses, erythrocyte enzyme activity was not

statistically significantly inhibited at any time-point at either dose. Inhibition of brain

377

TERBUFOS 333–385 JMPR 2003
cholinesterase activity was found at the two higher doses. Cerebral cholinesterase activity

was statistically significantly inhibited only at the highest dose (250mg/kgbw per day) by

45%. At the higher intermediate and highest doses, cerebellar cholinesterase activity was

statistically significantly inhibited by 52% and 31% (not statistically significant), respectively. At the lowest dose (5.0mg/kgbw per day), there were no obvious effects of treatment

on the parameters assessed. A statistically significant decrease in plasma cholinesterase

activity that was noted only on day 15 was of low magnitude (19%). Findings at gross

necropsy that were reported to be related to treatment were observed in one dog at the

highest dose. They were described as a dark red area in the mucosa of the jejunum mucosa,

associated with dark red mesenteric lymph node(s). This animal exhibited a number of 

clinical signs during the study and had decreases in plasma, erythrocyte cerebellar and 

cerebral cholinesterase activities. Dark mesenteric lymph node(s) were also reported in one

other animal at the highest dose.

In groups of animals treated with terbufos sulfone, there were three treatment-related

deaths preceded by clinical signs of toxicity. Two animals were found dead on day 9 and

14 and the third was terminated in a moribund condition on day 15. Clinical findings associated with treatment in this group included tremor, languid behaviour, prostration, ataxia,

emesis, salivation, dyspnoea, cold-to-touch, squint eye, miosis, no faeces, an increase in

soft faeces relative to the control group, prolapsed rectum, sanguineous-appearing material

in cage. Also at this dose, decreases in body weight and food consumption attributed to

treatment were noted in some animals. In groups given terbufos sulfone at 250, 62.5 or 

15mg/kgbw per day, there was no clear treatment-related effect on body weights, food consumption or clinical findings. Two occurrences of lacrimation and two occurrences of soft

faeces were reported at 250mg/kgbw per day, two instances of soft or sanguineous-looking

faeces and one instance of emesis were found at 62.5mg/kgbw per day and four occurrences

of soft faeces were found at the lowest dose. Plasma cholinesterase activity was statistically

significantly inhibited at all time-points: by 67–77% at the highest dose, by 39–56% at 

250mg/kgbw per day, and by 24–37% at 62.5mg/kgbw per day. At the lowest dose (15mg/kg

bw per day), statistically significant inhibition of plasma cholinesterase activity (of only

20%) was seen only on day 8 but not on day 15 and thus was not clearly related to treatment. Statistically significant decreases in erythrocyte cholinesterase activity were observed

only at the highest dose where inhibition was noted at all time-points ranging from 57% to

93%. Inhibition of brain cholinesterase activity was found at the two higher doses (statistically significantly only at the highest dose, by 62%). Cerebellar cholinesterase activity was

inhibited by 23% (not statistically significant) at 250mg/kgbw per day and by 46% (not 

statistically significant) at the highest dose.

At gross necropsy, findings related to treatment were observed in the group receiving

the highest dose: dark, red or dark red areas, intussusceptions, and/or a prolapsed anus with

associated redness in the mesenteric lymph nodes were observed in the gastrointestinal tract

of the animals that died. Some other gross findings at this dose were ascribed to the poor

condition of the animals.

If inhibition of plasma cholinesterase activity is not considered to be an adverse effect,

the NOAEL for terbufos was 2.5mg/kgbw per day in males on the basis of clinical findings

and statistically significant inhibition of erythrocyte cholinesterase activity.

If inhibition of plasma cholinesterase activity is not considered to be an adverse effect,

the NOAEL for terbufos sulfoxide was 15.0mg/kgbw per day in males on the basis of 

378

TERBUFOS 333–385 JMPR 2003
statistically significant inhibition of brain cholinesterase (cerebellum) at the dose above, for

which a relationship to treatment could not be dismissed.

If inhibition of plasma cholinesterase activity is not considered to be an adverse effect,

the NOAEL for terbufos sulfone was 62.5mg/kgbw per day on the basis of inhibition of

brain cholinesterase (cerebellar) activity at the dose above, for which a relationship to treatment could not be dismissed (Bailey, 1988). Statements of compliance with QA and GLP

were provided. This study was not conducted to fulfil a particular guideline.

3. Observations in humans

There have been a number of reports of occupational and non-occupational poisoning incidents associated with exposure to terbufos. With regard to possible effects from 

terbufos manufacturing facilities, no “reportable incidents” have been noted and no other

information was available.

Comments

In rats, absorption of single doses of [14C]terbufos was rapid and fairly complete. Most

of the radiolabel was excreted within 24–48h. Most (about 70–80%) of the administered

dose was excreted in the urine. Terbufos was extensively metabolized and little radioactivity was found in the tissues. There were no significant sex-specific differences in the

toxicokinetics of terbufos.

Sulfoxidation and desulfuration of terbufos is followed by hydrolysis of the 

thiolophosphorus bond (S–P), enzymatic S-methylation and then additional S-oxidation. 

On the basis of a 14-day study of repeated dosing, terbufos showed little potential for 

bioaccumulation.

By analogy with other phosphorodithioate compounds, it is likely that terbufos is

metabolically activated to terbufos oxon and other oxons, which cause inhibition of acetylcholinesterase activity.

Terbufos is of very high acute toxicity when administered by oral, dermal, and inhalation routes. Acute oral LD50 values in rodents and dogs were similar, ranging from 1.4 to

9.2mg/kgbw. Clinical signs observed were those typical of cholinergic toxicity.

The acute dermal LD50 for terbufos was about 1mg/kgbw in rabbits; a single application of undiluted terbufos to the shaved skin (0.25–0.5ml) or into the conjunctival sac

(0.1ml) killed all animals within 24h. The acute LC50 for terbufos administered by inhalation ranged from 0.0012 to 0.0061mg/l in rats.

In studies in rats and dogs, the critical effects of repeated doses of terbufos were inhibition of brain cholinesterase activity and associated clinical signs. NOAELs for inhibition

of brain cholinesterase activity in these studies ranged from about 0.04 to 0.11mg/kgbw

per day, and LOAELs ranged from about 0.085 to 0.55mg/kgbw per day. Inhibition of brain

cholinesterase activity of 7–12%, in the absence of clinical signs, was not considered to be

toxicologically relevant. NOAELs and LOAELs for inhibition of erythrocyte cholinesterasese activity were not substantially different from those for inhibition of brain cholinesterase

activity.

379

TERBUFOS 333–385 JMPR 2003
In a 1-year study in dogs given terbufos in capsules, the NOAEL was 0.060mg/kgbw

per day on the basis of inhibition of brain acetylcholinesterase activity at 0.090mg/kgbw

per day. The NOAELs for inhibition of brain acetylcholinesterase activity in other shortterm studies in dogs were consistent with that of the 1-year study.

In an 18-month study in mice fed with terbufos, there was no evidence of carcinogenicity at doses considered relevant for risk assessment. Cholinesterase activities were not

measured. The NOAEL was 3mg/kg (equivalent to 0.45mg/kgbw per day) on the basis of

significant decreases in body weights at the next highest dose of 6mg/kg (equivalent to 

0.9mg/kgbw per day).

A 2-year study of toxicity and carcinogenicity in rats had limitations that included

outstanding questions involving the etiology and/or relationship to treatment of certain nonneoplastic findings, confounding by non-treatment related illness in test animals and the

lack of supporting data to adequately quantify dietary intake, stability, and homogeneity.

However, on the basis of inhibition of brain cholinesterase activity in animals receiving the

highest dose, this study was considered to be adequate for testing for carcinogenicity. No

increase in tumour incidence was observed after a histopathological re-evaluation of tissues

for the assessment of carcinogenic potential. The NOAEL was 1mg/kg (equivalent to 

0.05mg/kgbw per day) on the basis of inhibition of brain acetylcholinesterase activity at 

4mg/kg (equivalent to 0.2mg/kgbw per day).

This conclusion was supported by a subsequent 1-year study of toxicity in rats; no

significant systemic or neoplastic effects were observed. The NOAEL was 1mg/kg (equal

to 0.055mg/kgbw per day) on the basis of the absence of significant inhibition of brain

acetylcholinesterase activity at all doses.

The Meeting concluded that terbufos was not carcinogenic in mice or rats.

The genotoxic potential of terbufos was assessed in an adequate range of in vitro and

in vivo tests. On the basis of the overall weight of evidence from the studies of genotoxicity, the Meeting concluded that terbufos is unlikely to pose a genotoxic risk to humans.

In view of the lack of significant genotoxicity and the absence of carcinogenicity

observed, the Meeting concluded that terbufos is unlikely to pose a carcinogenic risk to

humans.

In a study of reproductive toxicity in rats, mortality and clinical signs of toxicity in

females, some occurrences of excess salivation in males and decreases in body weight and

food consumption in both sexes were observed at 5mg/kg (equal to 0.42mg/kgbw per day),

a treatment that was terminated prematurely. At 2.5mg/kg, an increase in soft stools and

body-weight loss was noted in lactating females. Also observed were decreases in pregnancy rate, male fertility and significant decreases in the mean body weight of viable pups

on days 14 and 21 of lactation in F0 and F1 litters. The NOAEL for effects on reproduction

and offspring was 1.0mg/kg (equal to 0.086mg/kgbw per day). Inhibition of brain

cholinesterase activity was observed in both sexes, with a NOAEL of 0.5mg/kg (equal to

0.043mg/kgbw per day).

380

TERBUFOS 333–385 JMPR 2003
In a study of developmental toxicity in rats, the NOAEL for maternal and developmental effects was 0.2mg/kgbw per day, the highest dose tested. Mortality was seen at a

dose of 0.4mg/kgbw per day in a preliminary study.

In a study of developmental toxicity in rabbits, the NOAEL for maternal and developmental effects was 0.25mg/kgbw per day on the basis of clinical and systemic findings

in does, an increased number of does with resorption sites and decreased fetal body weights

at the next highest dose of 0.50mg/kgbw per day.

The potential of terbufos to induce delayed polyneuropathy in hens when given as a

single dose by gavage was assessed. The activity of neuropathy target esterase was not measured in this study. No significant changes in spinal cord and peripheral nerves were apparent in the group treated with terbufos. The Meeting concluded that at doses relevant to

dietary exposure in humans, there was no concern for the induction of delayed polyneuropathy by terbufos.

In a study of neurotoxicity in which terbufos was given to rats as a single dose by

gavage, mortality, clinical signs of toxicity, including miosis, and inhibition of brain and

erythrocyte cholinesterase activities were noted at the highest dose tested of 0.90mg/kgbw.

The only finding at the intermediate dose (0.3mg/kgbw) was miosis, which was observed

in the absence of inhibition of concurrently measured brain and erythrocyte cholinesterase

activities. No neurohistopathological lesions were found at any dose. The NOAEL was 

0.15mg/kgbw on the basis of findings of miosis in both sexes at the next highest dose of

0.30mg/kgbw.

A 13-week study of neurotoxicity was conducted in rats. Other than a slight decrease

in body weight and inhibition of brain and erythrocyte cholinesterase activities at the highest

dose of 3.0mg/kg (equal to 0.25mg/kgbw per day), no effects (including miosis) were

observed. The NOAEL was 0.8mg/kg (equal to 0.059mg/kgbw per day) on the basis of

inhibition of brain acetylcholinesterase activity at 0.25mg/kgbw per day.

The acute oral toxicity of a number of metabolites of terbufos was evaluated in female

mice. LD50s were as follows: 1.1mg/kgbw (terbufoxon sulfoxide), 3.4mg/kgbw (terbufos

sulfoxide), 3.4mg/kgbw (terbufoxon sulfone), 14mg/kgbw (terbufos sulfone), 2.2mg/kg

bw (terbufoxon), 3670mg/kgbw (methane, bis (tert-butylsulfonyl) and >2500mg/kgbw

(methane, (tert-butylsulfinyl)(methylsulfinyl)).

In a comparative 14-day study in dogs, terbufos given in capsules was found to be

more toxic than either terbufos sulfoxide or terbufos sulfone.

There have been a number of reports of occupational and non-occupational poisoning incidents associated with exposure to terbufos. No information was available regarding

possible effects from terbufos manufacturing facilities.

The Meeting concluded that the existing database on terbufos was adequate to characterize the potential hazard to fetuses, infants and children.

381

TERBUFOS 333–385 JMPR 2003
Toxicological evaluation

The Meeting established an ADI of 0–0.0006mg/kgbw based on an overall NOAEL

of 0.06mg/kgbw per day and a safety factor of 100 for inhibition of brain cholinesterase

activity, in the 1-year study of toxicity, the 13-week study of neurotoxicity and the twogeneration study of reproduction in rats, and the 1-year study in dogs.

The Meeting established an acute reference dose (RfD) of 0.002mg/kgbw based on

a NOAEL of 0.15mg/kgbw per day for miosis in the study of neurotoxicity in rats given a

single dose of terbufos, and a 100-fold safety factor. Since only in this study was miosis

observed in the absence of inhibition of cholinesterase activity, it might be possible to refine

the acute RfD after better characterization of this effect.

382

TERBUFOS 333–385 JMPR 2003

Levels relevant to risk assessment

Species Study Effect NOAEL LOAEL

Mouse 18-month study of toxicity Toxicity 3 mg/kg of feed, equivalent 6 mg/kg of feed, equivalent

and carcinogenicitya to 0.45 mg/kg bw per day to 0.90 mg/kgbw per day

Carcinogenicity 12 mg/kg of feed, equivalent —

to 1.8 mg/kg bw per dayd

Rat 2-year study of toxicity Carcinogenicity 4 mg/kg of feed, —

and carcinogenicitya nominally equivalent to

0.2 mg/kg bw per dayd

1-year study of toxicitya Toxicity 1 mg/kg of feed, equal to —

0.055 mg/kg bw per dayd

13-week study of Toxicity 0.8 mg/kg of feed, equal to 3.0 mg/kg of feed, equal to

neurotoxicitya 0.059 mg/kg bw per day 0.25 mg/kgbw per day

Single-dose study of Toxicity 0.15 mg/kg bw per day 0.30 mg/kgbw per day

neurotoxicityb

Multigeneration study of Parental toxicity 0.5 mg/kg of feed, equal to 1.0 mg/kg of feed, equal to

reproductive toxicitya 0.043 mg/kg bw per day 0.086 mg/kgbw per day

Offspring toxicity 1.0 mg/kg of feed, equal to 2.5 mg/kg of feed, equal to

0.086 mg/kg bw per day 0.21 mg/kgbw per day

Study of developmental Maternal toxicity 0.20 mg/kg bw per dayd —

toxicityb

Embryo- and fetotoxicity 0.20 mg/kg bw per dayd —

Rabbit Study of developmental Maternal toxicity 0.25 mg/kg bw per day 0.50 mg/kgbw per day

Embryo- and fetotoxicity 0.25 mg/kg bw per day 0.50 mg/kgbw per day

Dog 1-year study of toxicityc Toxicity 0.06 mg/kg bw per day 0.09 mg/kgbw per day

a

Diet

bGavage

c

Capsule

dHighest dose tested

Estimate of acceptable daily intake for humans

0–0.0006mg/kgbw

Estimate of acute reference dose

0.002mg/kgbw

Studies that would provide information useful for continued evaluation of the compound

• A study of delayed neurotoxicity with neuropathy target esterase measurements

(known to be ongoing)

• Further observations in humans

• Characterization of miosis
383

TERBUFOS 333–385 JMPR 2003

Summary of critical end-points for terbufos

Absorption, distribution, excretion and metabolism in mammals

Rate and extent of oral absorption Rapid and fairly complete

Dermal absorption No specific study; rapidly penetrating following dermal or ocular application

Distribution Relatively rapid and fairly complete

Potential for accumulation Little

Rate and extent of excretion Relatively rapid and complete; most eliminated in 24–48 h; elimination in urine

predominates

Metabolism in animals Sulfoxidation and desulfuration of terbufos is followed by hydrolysis of the

thiolophosphorus bond (S-P), enzymatic S-methylation and then additional

S-oxidation

Toxicologically significant compounds Terbufos

Terbufos oxon

Terbufos sulfoxide

Terbufos sulfone

Terbuoxon sulfoxide

Terbuoxon sulfone

Acute toxicity

Rat, LD50, oral 1.4–9.0mg/kgbw

Rabbit, LD50, dermal 0.81–0.93 mg/kgbw

Rat, LC50, inhalation Vapour, 4-h whole body exposure: 0.0012–0.0061 mg/l

Dermal irritation Could not be determined due to lethality, rabbit

Ocular irritation Could not be determined due to lethality, rabbit

Skin sensitization Not determined owing to potential for severe toxicity

Short-term studies of toxicity

Target/critical effect Inhibition of brain cholinesterase activity

Lowest relevant oral NOAEL 0.059 mg/kgbw per day (13-week study of neurotoxicity in rats)

Lowest relevant dermal NOAEL Data not available

Lowest relevant inhalation NOAEC No appropriate data available

Genotoxicity Unlikely to be genotoxic

Long-term studies of toxicity and carcinogenicity

Target/critical effect Inhibition of brain cholinesterase activity

Lowest relevant NOAEL 0.055 mg/kgbw per day (1-year study in rats)

Carcinogenicity No evidence of carcinogenicity;

Unlikely to pose a risk to humans

Reproductive toxicity

Reproduction target/critical effect Decreases in male fertility and female pregnancy rate

Lowest relevant reproductive NOAEL 0.086 mg/kgbw per day (rats)

Developmental target/critical effect Not teratogenic;

Reduced fetal body weight

Lowest relevant developmental NOAEL 0.25 mg/kgbw per day (rabbits)

Neurotoxicity/delayed neurotoxicity

Acute neurotoxicity

Target/critical effect Miosis

Relevant NOAEL 0.15 mg/kgbw (rats)

13-week study of neurotoxicity

Target/critical effect Inhibition of brain cholinesterase activity

Relevant NOAEL 0.059 mg/kgbw per day (rats)

Delayed neuropathy No evidence to suggest toxicity at dietary exposures

Medical data There have been a number of reports of occupational and non-occupational poisoning

incidents associated with exposure to terbufos. No information was available

regarding possible effects from terbufos manufacturing facilities.

Summary Value Study Safety factor

ADI 0–0.0006 mg/kgbw Rats and dogs, overall 100

NOAEL for studies of repeated doses

Acute RfD 0.002 mg/kgbw Rat, study of acute neurotoxicity 100
References

Allen, J.S. (1985) Bacterial/microsome reverse mutation (Ames) test on CL 92 100 COUNTER® terbufos.

Unpublished report GTOX Vol. 5 No.3 (BASF RDI No. TE-435-006) from American Cyanamid Company,

Princeton, NJ, USA.

Allen, J. & Johnson, E. (1983) Mutagenicity testing of AC 92100 in the in vitro CHO/HGPRT mutation assay.

Unpublished report GTOX Vol. 3 No.19 (BASF RDI No. TE-435-005) from American Cyanamid Company,

Princeton, NJ, USA.

American Cyanamid Company (1972b) Phosphorodithioic acid, S-(Tert-butylsulfinyl) methyl O,O-diethyl

ester (CL94 301): mouse oral LD50. Unpublished report No. A-72-37 (BASF RDI No. TE-470-008) from

American Cyanamid Company, Princeton, NJ, USA.

American Cyanamid Company (1972c) Phosphorodithioic acid, S-(Tert-butylsulfonyl) methyl O,O-diethyl

ester (CL94 320): mouse oral LD50. Unpublished report No. A-72-34 (BASF RDI No. TE-470-005) from

American Cyanamid Company, Princeton, NJ, USA.

American Cyanamid Company (1972d) Phosphorothioic acid, S-(Tert-butylsulfinyl) methyl O,O-diethyl ester

(CL94 365): mouse oral LD50. Unpublished report No. A-72-35 (BASF RDI No. TE-470-006) from American Cyanamid Company, Princeton, NJ, USA.

American Cyanamid Company (1972e) Phosphorothioic acid, S-(Tert-butylsulfonyl) methyl O,O-diethyl ester

(CL94 302): mouse oral LD50. Unpublished report No. A-72-38 (BASF RDI No. TE-470-009) from American Cyanamid Company, Princeton, NJ, USA.

American Cyanamid Company (1972f) Phosphorothioic acid, S-(Tert-butylthio) methyl O,O-diethyl ester (CL

94 221): mouse oral LD50. Unpublished report No. A-72-36 (BASF RDI No. TE-470-007) from American

Cyanamid Company, Princeton, NJ, USA.

American Cyanamid Company (1973a) Methane, Bis(Tert-butylsulfonyl) (CL 202 135): mouse oral LD50.

Unpublished report No. A-73-21 (BASF RDI No. TE-470-002) from American Cyanamid Company,

Princeton, NJ, USA.

American Cyanamid Company (1973b) Methane (Tert-butylsulfinyl)(methylsulfinyl) (CL 202 474): mouse

oral LD50. Unpublished report No. A-73-122 (BASF RDI No. TE-470-001) from American Cyanamid

Company, Princeton, NJ, USA.

Bailey, D.E. (1988) 14-Day oral toxicity study in the dog with AC 92 100 and its metabolites, CL 94 301 and

CL94 320. Unpublished report on HLA Study No. 362-190 (BASF RDI No. TE-420-007) from Hazleton

Laboratories America, Inc., Vienna, VA, USA.

BASF (1972a) AC 92 100 Technical: Acute rat and mouse oral, rabbit dermal, rabbit skin and eye irritation.

Unpublished report No. A-72-95 (BASF RDI No. TE-410-001) from BASF, Princeton, NJ, USA.

Berger, H. (1977) Experiments L-1680 and L-1680A: Cholinesterase activity of dogs receiving COUNTER*

soil insecticide in the diet for 28 days. Unpublished report No. A77-158 (BASF RDI No. TE-420-004)

from American Cyanamid Company, Princeton, NJ, USA.

Bradley, D. (1996) Oral LD50 study in albino rats with AC 92 100. Unpublished report No. A96-14; toxicology study T-0899 (BASF RDI No. TE-411-004) from American Cyanamid Company, Princeton, NJ, USA.

Cheng, T. (1992) Metabolism of 14C-terbufos (CL 92 100) in rats (preliminary and definitive phases). 

Unpublished report No. TE-440-004 from American Cyanamid, Princeton, NJ, USA.

Daly, I.W. & Knezevich, A.L. (1979) A three-month feeding study of COUNTER* terbufos insecticide in rats.

Unpublished report, project No. 78-2343 (BASF RDI No. TE-425-001) from Bio/dynamics Inc., East Millstone, NJ, USA.

Daly, I.W. & ? (1987) A one-year dietary toxicity study with AC 92100 in rats. Unpublished report, project

No. 85-2964 (BASF RDI No. TE-427-003) from Bio/dynamics Inc., East Millstone, NJ, USA.

Fischer, J.E. (1978) Experiment L-1728: 14-day rat feeding study with CL 92100 (COUNTER®). Unpublished report No. A78-129 (BASF RDI No. TE-412-003) from American Cyanamid Company, Princeton, NJ, USA.

Fischer, J.E. (1985) AC 92 100 Technical: acute dermal toxicity in rabbits. Unpublished report No. A85-54

(BASF RDI No. TE-420-006) from American Cyanamid Company, Princeton, NJ, USA.

Garces, T.R., Stryeski, V. & Clinton, J.M. (1977) Safety of COUNTER® terbufos insecticide when present in

the ration of sheep. Unpublished report (BASF RDI TE-411-003) from American Cyanamid Co., Agricultural Division, Princeton, NJ.

Gentile, J.M., Gentile, G.J., Bultman, J., Sechriest, R., Wagner, E.D. & Plewa, M.J. (1982) An evaluation of

the genotoxic properties of insecticides following plant and animal activation. Mutat. Res., 101: 19-29.

Godek, E.G. (1983) AC 92100: rat hepatocyte primary culture/DNA repair test. Unpublished report PH 311-

AC-001-83 (BASF RDI No. TE-435-004) from Pharmakon Research International, Inc., Waverly, PA, USA.

384

TERBUFOS 333–385 JMPR 2003
Haley, S. (1972) Teratogenic study with AC 92 100 technical in albino rats. IBT No. B1374-(B). Unpublished

report (BASF RDI TE-432-001) from Industrial Bio-Test Laboratories, Inc., Northbrook, IL, USA.

Hoberman, A.M. (1988a) A developmental toxicity (embryo-fetal toxicity/teratogenicity) study with AC 92

100 in rabbits. Unpublished report Argus protocol No. 101-003 (BASF RDI No. TE-432-006) from Argus

Research Laboratories, Inc., Horsham, PA, USA.

Hoberman, A.M. (1988b) Addendum to a developmental toxicity (embryo-fetal toxicity/teratogenicity) study

with AC 92, 100 in rabbits (sample identification and analysis of dosing solutions). Unpublished report

No. 101-003 from Argus Research Laboratories, Inc., Horsham, PA, USA.

Hoffman, G.M. (1987) An acute inhalation toxicity study with AC 92 100 in rats. Unpublished report,

Bio/dynamics Project No. 86-3128 (BASF RDI No. TE-430-002) from Bio/dynamics Inc., East Millstone,

NJ, USA.

Li, J-T.L., Sheng, S-J. & Du, X-L. (1999) Metabolism of terbufos in rat liver, J. Occup. Health, 41: 62-68.

MacKenzie, K.M. (1986) Dominant lethal study with AC 92 100 in rats. Unpublished report, Hazleton study

No. 6123-137 (BASF RDI No. TE-435-002) from Hazleton Laboratories America, Inc., Madison, WI, USA.

Mandella, R.C. (1998) An acute neurotoxicity study with AC 92 100 in the rat via oral garage administration.

Unpublished report, Huntingdon study No. 98-4525 (BASF RDI No. TE-451-002) from Huntingdon Life

Sciences, East Millstone, NJ, USA.

Mandella, R.C. (1999) 13-week dietary neurotoxicity study with AC 92 100 in the rat. Unpublished report,

Huntingdon study No. 98-4521 (BASF RDI No. TE-451-004) from Huntingdon Life Sciences, East Millstone, NJ, USA.

Morgareidge, K. (1973) Six-month feeding study in dogs on AC-92 100. Unpublished report, FDRL Lab. No.

1193 (BASF RDI No. TE-427-004) from Food Drug Research Laboratories Inc., Maspeth, NY, USA.

Morici, I.J. (1972) O,O-Diethyl-S(Tert-butylthiomethyl)phosphorodithioate: acute toxicity, and thirty-day

repeated feeding studies to albino rats, mice and beagle dogs. Unpublished report No. 72-3 (BASF RDI

No. TE-420-002) from American Cyanamid Company, Princeton, NJ, USA.

North, H. (1973) COUNTER® insecticide: rat metabolism of CL 92 100 (O,O-diethyl-S-I-butylthiomethylphosphorodithioate). Unpublished report (BASF RDI No. TE-440-001) from Princeton, NJ, USA.

Putnam, D.L. (1986) AC 92 100: the acute in vivo cytogenetics assay in rats. Unpublished report, MA study

No. T4277.105002 (BASF RDI No. TE-435-007) from Microbiological Associates, Inc., Bethesda, MD,

USA.

Rapp, W.R. (1974) A three and twenty-four month oral toxicity and carcinogenicity study of AC 92 100 in

rats. Unpublished report, Bio/dynamics project No. 71R-725 (BASF RDI No. TE-427-001), from

Bio/dynamics Inc., East Millstone, NJ, USA.

Rodwell, D.E. (1984) A range-finding teratology study with AC 92 100 in rats. Unpublished report, WIL study

No. WIL-35 013 (BASF RDI No. TE-432-003), from WIL Research Laboratories, Inc., Ashland, OH, USA.

Rodwell, D.E. (1985) A teratology study with AC 92 100 in rats. Unpublished report, WIL study No. WIL35 014 (BASF RDI No. TE-432-001), from WIL Research Laboratories, Inc., Ashland, OH, USA.

Schroeder, R.E. (1989) A two-generation (two-litters) reproduction study with AC 92 100 to rats. Unpublished

report, Bio/dynamics Project No. 86-3128 (BASF RDI No. TE-430-002) from Bio/dynamics Inc., East

Millstone, NJ, USA.

Shellenberger, T. (1987) 28-day oral toxicity study in the dog with AC92 100. Unpublished report No. 87019

(BASF RDI No. TE-420-003) from Tegeris Laboratories, Inc., Laurel, MD, USA.

Shellenberger, T. & Billups, L.H. (1986) One-year oral toxicity study in purebred beagle dogs with AC 

92 100. Unpublished report No. 8414 (BASF RDI No. TE-427-002) from Tegeris Laboratories, Inc., Laurel,

MD, USA.

Silverman, M.E.B., Shellenberger, T.E., Billups, L.H. & Tegeris, A.S. (1986) Chronic dietary toxicity and

oncogenicity study with AC 92 100 in mice. Unpublished report No. 8422 (BASF RDI No. TE-428-002)

from Tegeris Laboratories, Inc., Laurel, MD, USA.

Smith, J. (1972) A neurotoxicity study of AC 92 100, an organic phosphate cholinesterase inhibitor, in hens.

Unpublished report, Bio/dynamics Project No. 72S-788 (BASF RDI No. TE-451-001), from Bio/

dynamics Inc., East Millstone, NJ, USA.

Smith, J.M. (1973) A neurotoxicity study of AC 92 100, an organic phosphate cholinesterase inhibitor in hens:

addendum I (Project No. 72S-788). Unpublished report No. BASF RDI No. TE-451-001 from Bio/

dynamics, East Millstone, NJ, USA.

Thilager, A. (1983) AC 92 100: Chromosome aberrations in Chinese hamster ovary cells. Unpublished report,

MA study No. T1906.337 006 (BASF RDI No. TE-435-003), from Microbiological Associates, Inc.,

Rockville, MD, USA.

Whitney, W.K. (1980) A two week inhalation toxicity study of technical COUNTER® terbufos in the rat.

Bio/dynamics project 78-7168. Unpublished report No. TE-420-008.

385

TERBUFOS 333–385 JMPR 2003