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    ETHYLENETHIOUREA (ETU)

    EXPLANATION

         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

    BIOLOGICAL DATA

    Biochemical aspects

    Metabolism

    Monkeys

         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).

    Rats

         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.,
    1976).

         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).

    Mice

         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).

    Cats

         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).

    Toxicological Studies

    Special studies reproduction and teratology

    Rats

         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
    follows:

    Dose group                             Total rats per group
                                                                        

    Control 1 ml distilled water                 14
    T3 20 g/kg + T4 100 g/kg                   10
    Sodium iodide 333 g/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).

    Rats/Mice

         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
    250 ppm.

         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).

    Rabbits

         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).

    Cats

         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).

    Hamsters

         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

    Rats

         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

         (1)  alone;

         (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).

    Long-term studies

    Rats

         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 Ci 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
    study.

         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;
    1975).

    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).

    Short-term studies

    Rats

         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 Ci) 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).

    Mice

         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).

    Monkeys

         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.,
    1978b).

    COMMENTS

         The meeting recognized the concern regarding residues of ETU in
    processed and cooked foods resulting from the use of ethylene-
    bisdithiocarbamates.

         Although a monograph addendum was prepared, the Meeting could not
    re-evaluate the temporary ADI fully because the available data base
    was incomplete.

         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
    ongoing studies.

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    See Also:
       Toxicological Abbreviations
       Ethylenethiourea  (IARC Summary & Evaluation, Volume 7, 1974)
       Ethylenethiourea  (IARC Summary & Evaluation, Volume 79, 2001)