ETU has been reviewed in conjunction with the ethylene
bisdithiocarbamates (EBDC) by the Joint Meeting in 1965, 1967, 1970,
1974, 1977, 1980 and 1986 (Annex I, FAO/WHO 1965b, 1968b, 1971b,
1975b, 1978b, 1981b and 1987a). The Joint Meeting in 1986 determined
that 0-0.002 mg/kg bw could be considered to be a TADI, but
recommended an evaluation of available data on ETU be scheduled for
1988. Additional data have been provided and are reviewed herein.
EVALUATION FOR ACCEPTABLE INTAKE
Male macaca mulatta (Rhesus) monkeys were given 2-3 mg/kg bw
14C-ETU equivalent to 100 microcuries 14C-ETU by oral gavage.
Whole blood and excreta (urine and faeces) were collected and
examined. Whole blood, measured over a 72-hour period, demonstrated
peak levels at 8 hours and relatively rapid decline at 24-48 hours.
Approximately 50% of the dose was excreted in urine in 24 hours. Less
than 1% of the dose was recovered in faeces during the first 24 hours,
and none thereafter (Emmerling, 1978a).
Approximately 80-82% of a single 4 mg/kg dose of 14C-ETU
(>99% purity) (p.o. in distilled water) was eliminated via the urine
within 24 hours by three male Sprague-Dawley rats. A half-life of 5.6
hours in rat blood was demonstrated. Unchanged ETU represented 62.6%
of the radioactivity in rat urine (Iverson et al., 1980).
2-14C-ETU or 4,5-14C-ETU (>98% purity) were administered to
four pregnant Wistar-Imamichi rats at 100 mg/kg via intra-gastric
intubation on the 12th day of gestation. Whole body radiography, TLC
and GC were used to analyze the uptake of radioactivity in tissues of
both foetus and dam. Radioactivity in the foetus reached maximal
activity within 2 hours and declined thereafter. Differences were
observed between 2-ETU and 4,5-ETU with respect to protein fraction
incorporation. Radioactivity was distributed homogeneously throughout
all tissues except for thyroid, where there was an increase in
activity during the first 24 hours. Thyroid hormones are reported to
play important roles in the development of the CNS and thyroidectomy
induces malformations in the rat. There was no significant difference
in the thyroxin (T4) levels between treated and control maternal
serum, whereas the appearance of malformed foetuses between controls
and treated rats was significant at 100 mg/kg (malformations were
observed in 100% of the foetuses from treated dams) (Kato et al.,
Two weeks prior to breeding, four female Fischer 344 rats were
administered ETU (96.7% purity) in the diet at dose levels of 0, 8,
25, 83 or 250 ppm. During the gestation period the amount of ETU
equivalents measured in maternal liver, amniotic fluid and foetal
carcass correlated with the maternal blood level, but the placental
levels did not. Trans-placental transport was demonstrated.
Post-partum, there was an apparent transfer of ETU to the nursing pups
via the milk. Levels of ETU equivalents in maternal liver, maternal
milk, neonatal blood and neonatal liver were increased compared to
maternal blood levels. There were no significant differences, however,
between ETU equivalents in maternal milk and levels in neonatal blood.
No accumulation of ETU in neonatal liver or maternal liver was
observed. The levels of ETU in neonatal liver correlated with the
levels in neonatal blood. Prior exposure of maternal animals to ETU
did not effect the pharmacokinetic behavior of ETU in post-partum
animals (dam and neonate) (Peters et al., 1982).
Two weeks prior to breeding, four female C57BL/6N mice were
administered ETU (96.7% purity) in the diet at dose levels of 0, 33,
100, 333, or 1000 ppm. During the gestation period the level of ETU
equivalents in amniotic fluid, placenta and foetal carcass correlated
with maternal blood levels; however, levels were increased in maternal
livers (3X). No differences between dosed dam and foetus were
observed. In the post-partum period, accumulation of ETU equivalents
was much more apparent, with ETU equivalents in maternal liver
approximately 10X greater than maternal blood. Levels of ETU
equivalents were also increased 2X in maternal milk compared to
maternal blood. However, neonatal blood levels were decreased (13-fold
less) in comparison to maternal milk. Neonatal liver and blood were
significantly correlated with regard to ETU equivalents. Pre-treatment
did not alter the pharmacokinetics of ETU in post-partum mouse dams
and their neonates (Peters et al., 1982).
Lewerenz and Plass (1984) noted possible qualitative differences
between rat and mouse metabolism of ETU based on urinary metabolites
and measurement of microsomal enzymes. The microsomal enzymes
(aminopyrine N-demethylase, aniline hydroxylase, cytochrome P-450)
were inhibited in rat whereas in mouse they were stimulated. This
suggests that ETU is metabolized by different enzymatic pathways in
the two species.
Ruddick, Newsome and Iverson (1977) also observed that the
metabolic pathway is somewhat different: in mouse, ETU comprised 40%
of labelled metabolites in urine versus 95% in rat. This suggests more
rapid metabolism in mouse than rat. A dose of 240 mg/kg of ETU
(>98% purity) was administered via stomach intubation to pregnant
rats and mice on day 15 of gestation. The major urinary metabolite
identified in mouse was 2-imidazolin-2-y1 from the oxidation of ETU
(Savolainen & Pyysalo, 1979).
Approximately 80-82% of a single 4 mg/kg dose of 14C-ETU
(>99% purity) (p.o. in distilled water) was eliminated via the urine
within 24 hours by 3 female cats. A half-life of 3.5 hours in cat
blood was demonstrated. Unchanged ETU represented 28% of the
radioactivity in urine. S-methyl ETU comprised 64% of the radioactivity
in urine (Iverson et al., 1980).
Special studies reproduction and teratology
Virgin Sprague-Dawley rats were mated one-to-two with males and,
after pregnancy was verified, were administered ETU (unknown purity),
T3/T4 and sodium iodide via oral gavage in varying concentrations,
either singly or in combination, as wall as a control solution of
water only, from day 7 to day 20 of gestation. Dosing regimen was as
Dose group Total rats per group
Control 1 ml distilled water 14
T3 20 µg/kg + T4 100 µg/kg 10
Sodium iodide 333 µg/kg 10
ETU 20 mg/kg 10
ETU 20 mg/kg + sodium iodide 16
ETU 20 mg/kg + T3/T4 16
ETU 40 mg/kg 11
ETU 40 mg/kg + sodium iodide 14
ETU 40 mg/kg + T3/T4 15
Each pregnant dam was killed on day 20 by chloroform asphyxiation
and the foetuses removed via hysterotomy. The number of resorptions,
live/dead foetuses and foetal birth weights were determined. Skeletal
analyses were performed on 1/3 and visceral analyses on 2/3 of the
foetuses. Results indicate a possible reduction in the teratogenic
response to ETU for some malformations when T3/T4 is administered
in conjunction with ETU. For example, 20 and 40 mg/kg ETU (alone)
produced 97.6 and 94.5% incidence of hydrocephaly, respectively. In
combination with T3/T4 these same levels produced 19.6 and 74.5%
incidence, respectively (Emmerling, 1978b).
ETU (100% purity) was administered via oral gavage at 40 mg/kg bw
from days 7 to 15 of gestation to pregnant CR rats (10-12 rats/group).
Rats were hypothyroid and euthyroid. There was a problem, however, in
maintaining the euthyroid state in rats given T4 supplement. Rats
were also given thyroxine to determine if ETU teratogenicity occurred
through alterations of maternal thyroid function. ETU was found to be
teratogenic in the rat but not through alteration of maternal thyroid
status. It was also demonstrated that ETU lowered serum [T4]; that
hypothyroidism per se increased the background level of
malformations in the rat; that T4 alone was embryotoxic but not
teratogenic; and that hypothyroidism altered the spectrum of
malformations in response to ETU both quantitatively and qualitatively
(Lu & Staples, 1978).
ETU (100% purity) was administered orally at doses of 0, 5, 10,
20, 40 and 80 mg/kg bw in distilled water to nulliparous rats (Wistar)
(10-17 pregnant dams per dose). Treatment was made from 21-42 days
before conception to pregnancy day 15, and on days 6-15 or 720 of
pregnancy. All pups were delivered via C-section and examined for
skeletal and visceral anomalies. Microscopic examinations were
performed on brains. Doses of 40 mg/kg were not toxic to rats;
however, 80 mg/kg was lethal to 9 of 11 female rats. Mean foetal
weight was reduced at 40 mg/kg compared to control. Measurements of
sterility, pre-implantation loss and post-implantation survival were
comparable to controls. The brain was the most commonly affected
organ. ETU induced meningoencephalocele, meningorrhagia, meningorrhea,
hydrocephalus, obliterated neural canal, abnormal pelvic limb posture
with equinovarus, and short or kinky tail at 10 mg/kg in all phases of
the rat studies. Although no abnormalities were reported in rats at
5 mg/kg, there was a higher frequency of delayed ossification of the
parietal bone, compared to controls (Khera, 1973).
In the first phase of a two-phase study, adult female rats and
mice were dosed with ETU (96.7% purity) and then bred to proven male
sires. Pregnant females delivered their pups via C-section for tissue
distribution analyses. Phase 2 consisted of weanling rats/mice dosed
for 9 weeks and then analyzed. Dose levels in the diet were: rats:
0, 8, 25, 83 or 250 ppm; mice: 0, 33, 100, 333 or 1000 ppm (rats:
Fischer 344, 30 per group; mice: C57BL/6N, 78 per group). Two weeks
after dosing began, breeding was initiated.
Results - Rats: No rat dams or weanlings died. There was a
trend toward decreased weight gain in dams in all groups and in
weanling males at >83 ppm. Food consumption was also reduced at
250 ppm for males only. No effects on females were observed. There
were no adverse effects on pregnancy, lactation, pup viability (except
at 250 ppm, where there was a noted decrease in pup survival to
postnatal day 4). Thyroid hyperplasia was observed in males at
>8 ppm and in females at >25 ppm, increasing in incidence and
severity with dose. Thyroid adenomas were reported in males at
>83 ppm. Vacuolization of pituitary glands in males was noted at
Results Mice: Ten non-dose-related deaths occurred in the 33,
333 and 1000 ppm dose group dams. There was a significant decrease in
body weight in high dose females during the period of lactation.
Weanling body weights were decreased in males and females at
<333 ppm. Initially, insufficient pregnancies were produced in all
dose groups. A re-breeding program, after 6-1/2 weeks on ETU diets,
produced sufficient numbers of litters for evaluation. However, no
pregnancies were achieved in the high dose group, and pregnancy rate
was reduced in other dose groups in comparison to control. There were
no differences in average foetal weight or gross anomalies between
groups. Survival of pups to day 12 was not affected by dose; however,
the number of pups surviving to day 28 was significantly decreased in
the high dose group. Thyroid hyperplasia and cellular alteration of
hepatocytes (cytomegaly, karyomegaly) were observed in both sexes at
1000 ppm. One male mouse at 333 ppm also had adverse effects in the
liver (Peters et al., 1982).
ETU (100% purity) was administered orally at doses of 0, 5, 10,
20, 40 and 80 mg/kg bw in distilled water to nulliparous rabbits (New
Zealand white). There were 5-7 pregnant does per group. Treatment was
made from days 7 to 20 of pregnancy. All pups were delivered via
C-section and examined for skeletal and visceral anomalies.
Microscopic examinations were performed on brains. No toxicity was
apparent in rabbits given 80 mg/kg bw. Foetal weights were not
affected. Measurements of sterility, pre-implantation loss and
post-implantation survival were comparable to controls. Rabbits
presented no evidence of malformations at the doses administered.
However, there was an increase in resorption sites, decreased brain
weight, and degeneration of the proximal convoluted tubules in the
kidneys of foetuses at 80 mg/kg bw (Khera, 1973).
ETU (purity not stated) was administered orally (in gelatin
capsules) to pregnant European and Persian breed cats (7-14 cats per
group) at doses of 0, 5, 10, 30 and 60 mg/kg on days 16-35 of
gestation, and 120 mg/kg from days 16 to 34 of gestation. All cats
were necropsied and their foetuses subjected to skeletal or visceral
examinations. No apparent effect was evident at 5 mg/kg. However, at
>10 mg/kg body weight was decreased, ataxia, tremors and hindlimb
paralysis were observed. No pregnant cats survived in the 30 and
60 mg/kg dose groups. The remaining cats showed no apparent
treatment-related effect on foetal viability or foetal weight.
Although this study was inconclusive in many respects, there was an
increased incidence of toxicity to the central nervous system at
10 mg/kg. Further, at 5 mg/kg and 120 mg/kg there were anomalous
foetuses in each group. Incidences of exencephaly, hydrocephaly, cleft
palate, kyphoscoliosis, umbilical hernia, coloboma, and spina bifida
were observed in these two treated groups. Similar anomalies were
observed in the rat (Khera & Iverson, 1978).
ETU (purity >99%) was administered orally to pregnant Syrian
hamsters at doses of 600, 1200, 1800 or 2400 mg/kg on day 11 of
gestation. All dams were killed on day 15 of gestation for necropsy
and foetal examination. There were only 5 pregnant dams in the control
group compared to 8-10 in treated groups.
Maternal toxicity was not reported at any dose. However, there
was an increased incidence of resorbed foetuses and foetuses dying
late in gestation with an associated decrease in the number of live
foetuses at the 2400 mg/kg dose level. Foetel body weights were
similarly reduced at 1800 mg/kg. Malformations were evident at
>1200 mg/kg, with no adverse effect reported at 600 mg/kg. Foetal
anomalies included cleft palate, ectrodactyly, hydrocephalus and
hypoplastic cerebellum. There was also increased incidence of delayed
ossification of the calcarium and sternebrae defects. As with other
species (i.e. rat, cat), the brain was particularly sensitive to ETU,
although at higher dose levels (Khera et al., 1983).
Special studies of effects of ETU on the thyroid
Reversibility of effects
Groups of four randomly selected weanling caesarian-delivered
Sprague-Dawley male littermate rats were divided among control, 75 and
150 ppm ETU (purity not stated) dose groups. There were 64 litters of
4 males in each treatment group and 32 litters of 4 males in the
control group. Within each treatment group dosing periods and control
diet periods were varied to examine the reversibility of compound-
related effects. General health, body weight, food consumption, thyroid
weight and thyroid histopathology were examined. Results suggest some
reversibility of thyroid effects which were related to time on test
and to the severity of effect on the thyroid (Arnold et al., 1982).
Groups of 50 male and 50 female Sprague-Dawley rats were fed
diets containing 0, 75, 100 or 150 ppm ETU (purity not stated) for 7
weeks. ETU was mixed in corn oil and added to the diet. Ten rats from
each group were killed at 7 weeks and at 2, 3 and 4 weeks post-dosing
on control diets in order to assess the extent of effect on the
thyroid and the subsequent reversibility of these effects. Body
weight, food consumption, thyroid weight, brain weight, serum T3 and
T4 levels (0 and 150 ppm groups only) were measured. All animals
were necropsied and thyroids examined histologically. At 7 weeks body
weights decreased with increasing dose, while thyroid weights
(absolute and relative) increased in both sexes. T3 levels were
somewhat variable, while T4 levels were significantly decreased at
150 ppm in both sexes at 7 weeks. These effects partially reversed
after 4 weeks on control diets. Histopathological findings included
reduced colloid content of thyroid acini in high dose rats. Acinar
epithelial cell size and height were not different from control. Two
rumours were identified in the high dose male group: a follicular cell
adenoma and a medullary carcinoma. The authors conclude that the
relationship between the duration of exposure to ETU and the possible
reversibility of various thyroid lesions requires further study
(Arnold et al., 1983).
An elaborate subchronic study (22 weeks) was conducted in
Sprague-Dawley rats (12 treatment groups with 55 males/55 females per
group) with the following dosing schedule:
ETU (972 purity) administered in the diet at levels of 125, 250
and 625 ppm
(2) plus 0.2 g triiodothyronine (T3) orally via gavage and
1.6 g thyroxine (T4) per 100 g rat;
(3) plus manganese and zinc.
Also included were treatment groups dosed with 650 and 1250 ppm
MANCOZEB alone and a control group. At 2-week intervals for 22 weeks,
5 males and 5 females were sacrificed. Rats in the 625 ppm ETU group
or ETU plus manganese and zinc were removed from test and placed on
control feed 4 weeks after study initiation. All surviving rats on
study were placed on control feed after study week 12. Reversibility
of response was examined as well as thyroid, pituitary and liver
histopathology; serum T3, T4, TSH, FSH, GH and prolaction (PRL);
and liver gluthathione activity (GSH).
Rats receiving 625 ppm ETU alone or in combination with manganese
and zinc were removed from test diet because of alopecia, weight loss,
dermatosis and mortality. Survivors received control diets for the
remainder of the study. Serum T4 decreased in both sexes at all
doses of ETU after 2 weeks of treatment. These levels returned to
normal when ETU was removed from the diet. Serum T3 decreased in
both sexes at 625 ppm ETU after 4 weeks of dosing. In males, serum
T3 decreased the first 4 weeks at 155 and 250 ppm ETU, but by week 8
returned to normal. In females at the same doses, T3 was normal
until week 16 when it decreased. The addition of T3/T4 by oral
gavage resulted in decreased T3 at week 8 in males and a decrease
during the first 6 weeks in females at all levels. T3 returned to
normal one month after removal of ETU. TSH increased in the ETU group
and less dramatically in ETU plus T3/T4 groups. These levels
returned to normal 2 weeks after ETU was removed from the diet. There
were no consistent changes in FSH, GH or PRL.
Body weights decreased in males and females after 4 weeks at
625 ppm ETU and in males after 8 weeks at 250 ppm ETU. Thyroid to body
weight ratio increased at >125 ppm ETU in males. When ETU was
removed from the diet, weights returned to normal. No effect was
observed on pituitary weights. Thyroid hyperplasia was increased at
>125 ppm ETU and reversed to normal 6-8 weeks after ETU was removed.
Approximately 1% (13/1300) of the rats developed hyperplasia of the
thyroid (focal areas of basophilic hyperplastic follicles and
follicular adenoma). A dose-related increase in liver weight was
observed at >125 ppm ETU for both sexes. Liver GSH levels were
inconclusive due to a non-specific substrate used for measuring the
liver enzyme levels.
Exposure to ETU resulted in a decrease in thyroid hormone
(T3/T4) levels and increased serum TSH levels in a dose-related
manner. Although TSH levels were reduced when ETU was supplemented
with T3/T4, the high dosage of ETU was apparently sufficient to
override these effects. The hormone imbalance induced by ETU
correlated with the histologic changes in the thyroid. Withdrawal of
ETU from the diet reversed the hypothyroid conditions induced to
euthyrotdy (Leber et al., 1978a).
Groups of 68 male/68 female Charles River rats were fed ETU
(purity not stated) in the diet at levels of 0, 5, 25, 125, 250, or
500 ppm for 2 years. Body weight and food consumption were measured
weekly. At week 66, 3 male/3 female rats from each test group were fed
control diet only for the remainder of the 2-year study. At 3, 11, 17
and 22 months blood samples were collected from the tail vein of 10
male and 10 female rats. At 6, 12, 18 and 24 months 10 male/10 female
rats from each group were administered 5 µCi 131I i.p., fasted for
24 hours, sacrificed and thyroid, heart, liver, kidneys and spleen
examined for radioactive uptake.
Body weights in both sexes were significantly decreased at
>25 ppm initially; at >500 ppm (males) and >125 ppm (females)
at 12 months; and at >500-ppm (both sexes) for the remainder of the
Liver to body weight ratios were significantly increased at
>125 ppm through 6 months in males, but comparable to control for
the remainder of the study. Relative liver weights in females were
significantly increased at >125 ppm at 2 months and >250 ppm
through 18 months; no differences between control and dose groups was
observed at 24 months. Thyroid to body weight ratio was significantly
increased in males at >250 ppm for 2, 6 and 18 months, and at
>125 ppm in females for the first 12 months. Thyroid weights were
significantly increased at >125 ppm in males at 12 and 24 months,
and at >250 ppm in females at 18 and 24 months.
Uptake of 131I, expressed as counts/min/mg tissue, was
significantly decreased in males at 500 ppm throughout the study.
Thyroids of females fed >125 ppm were hypofunctioning at 6 months
and hyperfunctioning at 12 months. At 24 months females had a
hypofunctioning thyroid at 500 ppm.
There were fewer rats surviving to 24 months in the 500 ppm dose
group compared to control and other dose groups. There was also a
significant increase in pneumonia in high dose group rats. This may
have been further complicated by obstruction of the trachea from
enlarged thyroids in high dose group animals. Effects in the thyroid
were evident at all doses (>5 ppm). However, histologic data were
summarized and not separated by sex. Increased vascularity and
hyperplasia in the thyroid were evident at 5 ppm and increased in
incidence and severity at >25 ppm. Thyroids of treated rats were
distinguishable from controls by lobulation, follicular size and
uniformity, height of follicular epithelium, colloid staining,
keratinization of follicles, and general size.
It is possible that ETU initially reduces thyroid activity, after
which compensation occurs by an increased release of TSH and that this
increase in TSH stimulated thyroid weight in an attempt to overcome
the blocking effect of ETU. The progression to neoplasia is believed a
result of excessive pharmacological stimulation. This is supported, in
part, by a lack of thyroid tumours at 1 year at 5 or 25 ppm, an
increase in tumour incidence after 1 year at 125 ppm, and confirmed
after 2 years in rats fed 250 and 500 ppm (Graham et al., 1973;
Rats and Hamsters
Groups of 20 male and 20 female rats and hamsters were
administered ETU (purity not stated) in the diet for 24 and 20 months,
respectively, at dose levels of 0, 5, 17, 60 and 200 ppm. (Strain of
animals not reported.) Body weight, food consumption, selected
clinical chemistry parameters and organ weights were measured.
Histopathological examinations of selected tissues were performed at
necropsy. SGPT, SAP and cholesterol were measured in the serum; GPT,
AP and G6PDH were determined in the liver. Organs weighed included
liver, thyroid, testes, kidneys and spleen.
In rats, food consumption was reduced at >60 ppm and body
weight decreased at >17 ppm. Effects on SAP and SGPT were not
clearly demonstrated due to fluctuations in control levels.
Cholesterol was increased at 5 ppm in both sexes. Some hepatic enzyme
levels were also affected: GPT increased in males at 60 ppm; AP
increased at 5 ppm (females) and 17 ppm (males); G6PDH did not change.
Thyroid weights were significantly increased in both sexes at 60 ppm.
No data were available on the histologic examination.
In hamsters, food consumption and body weight were reduced at
>60 ppm. SAP was increased in both sexes initially, then decreased
through 18 months. No effect was observed on SGPT. Cholesterol levels
were significantly increased in both sexes at all doses compared to
controls. Hepatic enzymes, GFT and AP, were significantly increased in
both sexes at all doses. G6PDH was significantly decreased in both
sexes at all dose levels. Relative thyroid weights were significantly
increased at >200 ppm in both sexes. No data were available on the
histologic examination (Gak et al., 1976).
Five groups of 20 male Osborne-Mendel rats were fed ETU (purity
not stated) in the diet at levels of 0, 50, 100, 500 and 750 ppm for
30, 60, 90 and 120 days. Body weight and food consumption were
measured weekly; 131I activity determined at 4 and 24 hours
post-injection (5 µCi) in 20 rats from each group at each sacrifice
period; thyroids were also weighed. Histologic examination was
conducted at 90 days.
Body weight was decreased at >500 ppm throughout the study.
Food consumption was reduced at 30 and 90 days by >100 ppm and at
60 and 120 days by >500 ppm. Relative thyroid weights were
increased at 30 days by >100 ppm, at 90 days by >500 ppm and at
60/120 days by >50 ppm.
Histologically there were no differences between control and
50 ppm groups. At 100 ppm there was slight hyperplasia evident in the
thyroid gland. At 500 ppm there was moderate to marked hyperplasia,
lack of colloid and heightened epithelial walls. There was an increase
in vascularization, demonstrating a response to increased blood level
TSH. At >500 ppm an increased incidence of follicular adenomas was
reported. The authors propose that one mechanism by which ETU acts on
the thyroid is via inhibition of iodide peroxidase, which oxidizes
iodide to iodine (Graham & Hansen, 1972).
Ten male and 10 female Fischer 344 rats were administered ETU
(purity not stated) in the diet for 13 weeks at levels of 0, 60, 125,
250, 500 and 750 ppm. No deaths were reported. Body weight gain
decreased between dose groups and control at >500 ppm. Food
consumption was also decreased at >500 ppm. Histologic changes were
evident in several tissues, including bone marrow, esophagus, liver,
pituitary, stomach, and thyroid. Hematopoietic depletion of the bone
marrow occurred in both sexes at >500 ppm; hyperkeratosis of the
esophagus was produced in both sexes at 750 ppm; hypertrophy of
hepatocytes with granular eosinophilic cytoplasm, multiple nuclei and
vacuolated cytoplasm at >60 ppm in males and in females at 750 ppm;
pituitary changes at >250 ppm in males and >500 ppm in females
consisted of increased size and vacuolation of the para distalis;
multifocal to diffuse hyperkeratosis of the nonglandular stomach in
both sexes at 750 ppm; follicular hyperplasia and congestion in the
thyroid of females at >250 ppm (a single incidence was reported at
125 ppm in males). Focal to multifocal thyroid adenomas were present
in males at >250 ppm and in females at >500 ppm. A NOEL was not
demonstrated in this study (Peters et al., 1980a).
Groups of Charles River CD-1 mice (15/sex/dose) were administered
ETU (100% purity) in diet at levels of 0, 1, 10, 100 and 1000 ppm for
3 consecutive months. Daily observations, weekly measurements of body
weight and food consumption were performed on all animals.
Hematological and clinical chemistry parameters were evaluated after 3
months in 10 mice/sex/dose. All animals were necropsied, selected
organs weighed and tissues stained for histopathological evaluation.
Liver samples were taken from 6 mice/sex/dose at 13 weeks for
determination of hepatic mixed function oxidase activity. These
included P-nitroaniline O-demethylation, aminopyrine, N-demethylation
and aniline hydroxylation.
There were no treatment-related deaths or effects on food
consumption and body weight. Mean compound intake for the 13-week
period was calculated to be 0, 0.16, 1.72, 18.18 and 168.2 mg/kg bw
for males, and 0, 0.22, 2.38, 24.09 and 231.1 mg/kg bw for females at
0, 1, 10, 100 and 1000 ppm ETU. There were no compound-related effects
on haematology on clinical chemistry parameters. Mixed function
oxidase activity was increased in both sexes at 1000 ppm, but only
statistically significant in males (aniline hydroxylase,
P-nitroanisole, O-demethylase). Absolute and relative thyroid weights
were increased statistically in both sexes at 1000 ppm. Absolute and
relative liver weights were significantly increased in males at
1000 ppm; relative liver weights only were significantly increased in
females at 100 and 1000 ppm ETU.
ETU produced thyroid follicular cell hyperplasia and decreased
colloid density in both sexes as >100 ppm, with increased
follicular epithelial cytoplasmic vacuolation and interstitial
congestion in both sexes at 1000 ppm. In the liver, ETU produced
centrilobular hypertrophy, nuclear pleomorphism and increased
intranuclear inclusions in both sexes at 1000 ppm. The pigment was
believed to be similar to lipofuscin. A NOEL of 10 ppm ETU was
demonstrated in both sexes (O'Hara & DiDonato, 1985).
Ten male and 10 female B6C3FI mice were administered ETU (97%
purity) in the diet for 13 weeks at doses of O, 125, 250, 500, 1000
and 2000 ppm. No compound-related deaths, changes in food consumption
or body weight gain were reported. Histologic changes were observed in
both sexes in the esophagus (hyperkeratosis at >2000 ppm); liver
(hepatocyte, hypertrophy, multiple nuclei, altered stain at
>250 ppm, more severe in males); and thyroid (follicular
hyperplasia, infolded follicular walls, and congestion at >500 ppm).
No adverse effects were evident at 125 ppm (Peters et al., 1980b).
In two separate studies, wild caught Rhesus monkeys (5 males, 5
females per group) were administered ETU (96.8-98% purity) in the diet
for 5-1/2 and 6 months at dose levels of O, 2, 10, 50, and 250, and 0,
50, 150 and 450 ppm respectively. Body weight and organ weights were
measured. Although food consumption values were not provided, each
monkey was given 200 grams of food per day, resulting in an estimated
0.1 mg/kg bw for 2 ppm and 0.5 mg/kg bw at 10 ppm. Clinical chemistry
and haematology parameters were assayed, as well as T3, T4, TSH,
GH, PRL and FSH levels. Radioactive 125I uptake was determined and
histopathology of high, low and control dose animals, with particular
emphasis on thyroid and pituitary glands. TBG and free thyroxine index
(FTI) were also measured in the first study. There were 5 males/5
females per dose, average weight of 6-9 pounds at initiation of
studies. The first 6-months study was terminated after 5-1/2 months
due to widespread tuberculosis infection in the colony at the
mid-point of the study. A positive control group was added to the
second study at 125/250 ppm (propylthiouracil). Baseline measurements
of serum T3, T4, TSH, TBG, and eleven clinical chemistry
parameters were determined during the 6-weeks quarantine period.
Results of Study 1: Body weights were not affected by ETU.
Thyroid weight was increased in both sexes at 250 ppm and in females
at >50 ppm, resulting from hyperplasia and/or hypertrophy. Females at
>50 ppm also had enlarged pituitary glands. Ovarian weights at
250 ppm were significantly decreased.
No changes in T3 or TBG were observed. Serum T4 was decreased
in both sexes at >50 ppm identified from FTI analyses. Serum TSH
was increased at 250 ppm. Iodine uptake (125I) also increased at
>50 ppm in both sexes.
Lesions reportedly associated with ETU were identified in the
pituitary and thyroid glands of animals at >50 ppm. These included
thyroid and pituitary hypertrophy, and thyroid follicular cell
hyperplasia (moderate to severe). A second study was conducted due to
the extent of tuberculosis in the first study which necessitated the
early termination at 5 to 5-1/2 months.
Results of Study 2: Body weights were not affected by RTU.
Thyroid and spleen weights were increased in males at >150 ppm and
at all doses in females. Serum T3 decreased in males at >150 ppm
and in females at 450 ppm. Serum T4 was decreased in both sexes at
>150 ppm. No changes were observed for GH, FSH or PRL. Radioactive
125I uptake was increased in all test groups. It is suggested that
the increased thyroid weight, thyroid iodine uptake, decrease in T3,
T4 and increase in TSH support the evidence for hypothyroidism
caused by ETU.
BUN was elevated in females at 450 ppm along with creatinine and
a decrease in calcium. Haemoglobin, haematocrit and RBC count were
decreased in both sexes at 450 ppm.
Histologic changes were identified in thyroid and pituitary
glands in both sexes, increasing in severity and incidence with
increase in dose. Thyroid follicular cell hyperplasia and pituitary
cytoplasmic vacuolation and swelling were the major changes observed.
Overall, a NOAEL of 10 ppm was demonstrated for 125I uptake and
a NOAEL of 50 ppm for changes in T3, T4 and TSH. In a separate
pathological examination, 10 ppm was considered to produce compound-
related changes in the thyroid gland in 1/7 monkeys. A NOAEL of 2 ppm
is considered a NOAEL for ETU in these combined studies (Leber et al.,
The meeting recognized the concern regarding residues of ETU in
processed and cooked foods resulting from the use of ethylene-
Although a monograph addendum was prepared, the Meeting could not
re-evaluate the temporary ADI fully because the available data base
The temporary ADI was extended.
STUDIES WITHOUT WHICH THE DETERMINATION OF A FULL ADI IS IMPRACTICABLE
(to be submitted to WHO by 1992)
All available data relating to the safety of ETU, including
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