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    LIMONENE

    First draft prepared by
    Dr K.B. Ekelman and Dr D. Benz
    US Food and Drug Administration
    Washington, DC, USA

    1.  EXPLANATION

         Limonene has not been previously evaluated by the Committee.
    The active isomer is designated as  d-Limonene in this monograph.
    In the report (Annex 1, reference 101) it is designated as
    (+)-limonene.

          d-Limonene is a liquid with a pleasant, lemon-like odour and
    a fresh citrus taste. It is a natural constituent of a variety of
    foods and beverages and is especially prevalent in citrus fruits.

         Extracted  d-limonene is used primarily as a lemon fragrance
    in soaps, detergents, creams, lotions and perfumes, and as a
    flavouring agent in foods, beverages and chewing gum (NTP, 1990). It
    is found in non-alcoholic beverages (31 ppm), ice cream and ices (68
    ppm), candy (49 ppm), baked goods (120 ppm), gelatins and puddings
    (48-400 ppm), and chewing gum (2300 ppm) (NTP, 1990).

         Human exposure to  d-limonene is through consumption of foods
    and beverages, both those in which it naturally occurs and those to
    which it has been added.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

          d-Limonene was rapidly absorbed (43 min) through the intact,
    shaved abdominal skin of mice (Meyer & Meyer, 1959).

         Twelve Long-Evans male rats were administered single topical
    doses of 5 mg/kg bw 14C-limonene; the treated area was then
    occluded for 3 h (2 males) or 6 h (10 males). Following occlusion,
    the residual dose was removed and the treated area was re-occluded.
    Pairs of treated rats were killed at 3, 6, 24, 48, and 72 h; urine
    and faeces were collected from rats killed at 24, 48, and 72 h, and
    plasma and tissue samples were taken at all time points. Authors
    reported that peak concentrations of radioactivity in tissue samples
    were measured 3-6 h after dosing in the gastrointestinal tract
    (0.1-0.4% dose/g), livers and kidneys (0.08-0.2% dose/g), and
    thyroid and fat (0.02-0.06% dose/g); except for the gastrointestinal
    tract, concentrations of radioactivity in all tissues were
    appreciably lower at 24 h. After 6 h of exposure, 48% of the
    radioactivity was recovered in the skin; at the 24-72 h sampling
    times, 8-12% was excreted in urine, 1-3% was excreted in faeces, and
    14-18% was expired in air. Total mean recovery of radioactivity was
    reported to be approximately 76%.

         Following oral administration, the highest concentration of
     d-limonene or its metabolites in rats was found in the serum
    fraction of blood after 2 h. The other major organs containing
    metabolites of  d-limonene were the liver and kidney, with peaks
    1-2 h after ingestion. After 48 h, negligible amounts of
     d-limonene metabolites remained in the body. Approximately 60% of
     d-limonene was excreted in the urine, 5% in the faeces, and 2% was
    expired (Igimi  et al., 1974).

         Kodama  et al. (1974) reported that 72% of  d-limonene
    metabolites were excreted in male rabbit urine 72 h after oral
    administration, while 7% was found in the faeces.

    2.1.2  Biotransformation

         Kodama  et al. (1974) identified the metabolites of orally
    administered  d-limonene (also known as p-mentha-1,8-diene) in male
    rabbit urine as p-mentha-1,8-dien-10-ol (M-I),
    p-menth-1-ene-8,9-diol (M-II), perillic acid (M-III), perillic acid
    8,9-diol (M-IV), p-mentha-1,8-dien-10-yl-beta-D-glucopyranosiduronic
    acid (M-V) and 8-hydroxy-p-menth-1-en-9-yl-beta-D-
    glucopyranosiduronic acid (M-VI).

         The list of metabolites was expanded based on a study done with
    male rats, hamsters, guinea-pigs, rabbits, dogs and humans (Kodama
     et al., 1976b). Five additional metabolites of  d-limonene were
    identified: 2-hydroxy-p-menth-8-en-7-oic acid (M-VII),
    perillylglycine (M-VIII), perillyl-beta-D-glucopyrano-siduronic acid
    (M-IX), p-mentha-1,8-dien-6-ol (M-X) and p-menth-1-ene-6,8,9-triol
    (M-XI). The major metabolite in rats and rabbits was identified as
    M-IV, while the major metabolite in hamsters was found to be M-IX.
    In dogs, the major metabolite was M-II, but in guinea pigs and
    humans, it was M-VI. The possible metabolic pathways used by the
    various species are shown in Figure 1 below.

    FIGURE 1

         Wade  et al. (1966) reported that dietary limonene gives rise
    to uroterpenol (M-II: p-menth-1-ene-8,9-diol) in the urine of
    humans.

    2.1.3  Effects on enzymes and other biochemical parameters

         Groups of control (6 animals) and experimental (4 animals) male
    Wistar rats were administered single gavage doses of 0, 200, 400,
    600, 800, or 1200 mg/kg bw  d-limonene in 2% tragacanth solution in
    a total volume of 4 ml/kg. Authors reported that no effects were
    observed on liver triglycerides, microsomal proteins, cytochrome b5
    and drug metabolizing enzymes.

         In the same experiment, male Wistar rats were treated with
    gavage doses of 0 or 400 mg/kg bw  d-limonene in 2% tragacanth
    solution in a total volume of 4 ml/kg for 2, 3, 15 or 30 days;
    animals were killed 24 h following the last dose. Authors reported
    that, following repeated treatment for 30 days, relative liver
    weight and hepatic phospholipid content were slightly increased, and
    liver and serum cholesterol were decreased 49% and 8%, respectively.
    In addition, palmitic, linoleic and arachidonic acids were
    increased, and stearic acid was decreased in the liver; aminopyrine
    demethylase and aniline hydroxylase were increased 26% and 22%,
    respectively, and cytochrome P-450 and b5 were increased by 31% and
    30%, respectively (Ariyoshi  et al., 1975).

          d-Limonene was reported to enhance bile flow in Wistar rats
    and mongrel dogs in a dose-related manner, and to decrease the ratio
    of biliary bile salts and phospholipids to cholesterol (Kodama
     et al., 1976).  d-Limonene was reported to be an effective
    gallstone solubilizer in animals and humans (Igimi  et al., 1976;
    Schenk  et al., 1980), and to have no effect on liver weights nor
    levels of serum lipids when fed to male Wistar rats at 0.5 and 1% in
    the diet (Imaizumi  et al., 1985).

          d-Limonene was reported to significantly damage and increase
    permeability of membranes of human lung fibroblasts (Thelestam
     et al., 1980; Curvall  et al., 1984).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

        Table 1: Summary of acute toxicity studies with d-limonene
                                                                                  

    Species      Sex       Route           LD50                 Reference
                                                                                  

    Mouse        M&F       oral, in        6.3 ml/kg (M)        Tsuji et
                           juice, 7d       8.1 ml/kg (F)        al., 1974

    Mouse        M&F       ip, in          3.7 ml/kg (M)        Tsuji et
                           juice, 3d       3.6 ml/kg (F)        al., 1974

    Mouse        M&F       ip, in          0.7 ml/kg (M)        Tsuji et
                           juice, 10d      0.6 ml/kg (F)        al., 1974

    Mouse        M&F       sc, in          >25.6 ml/kg (M)      Tsuji et
                           juice, 7d       >25.6 ml/kg (F)      al., 1974

    Mouse        M&F       oral            5600 mg/kg (M)       Tsuji et
                                           6600 mg/kg (F)       al., 1975a

    Mouse        M&F       ip              1300 mg/kg (M)       Tsuji et
                                           1300 mg/kg (F)       al., 1975a

    Mouse        M&F       sc              >41500 mg/kg (M)     Tsuji et
                                           >41500 mg/kg (F)     al., 1975a

    Rat          M&F       oral            4400 mg/kg (M)       Tsuji et
                                           5100 mg/kg (F)       al., 1975a

    Rat          M&F       ip              3600 mg/kg (M)       Tsuji et
                                           4500 mg/kg (F)       al., 1975a

    Rat          M&F       sc              >20200 mg/kg (M)     Tsuji et
                                           >20200 mg/kg (F)     al., 1975a
                                                                                  
    
         An oral dose of 3 ml  d-limonene (in juice)/kg decreased
    spontaneous motor activities in rats and mice and potentiated
    hexobarbital-induced sleeping and hypothermia in mice. Authors also
    reported that nicotine-induced convulsion and death (but not maximum
    electroshock-, pentetrazol-, strychnine-, nor picrotoxin-induced
    convulsions) were inhibited by  d-limonene in mice. Intravenous
    administration of > 0.005 mg/kg  d-limonene lowered the blood

    pressure of rabbits and dogs, and > 0.1-0.3 mg/kg killed those
    animals. Oral administration of 3 ml/kg  d-limonene did not
    decrease the blood pressure of rats, however. Authors also reported
    that isolated smooth muscles of the intestine, vas deferens, uterus,
    and peripheral vessel were constricted by  d-limonene (Tsuji
     et al., 1974). Single high s.c. doses of  d-limonene produced
    scratch behaviour and single high i.v. doses of  d-limonene
    produced stretch behaviour in mice and rats (Tsuji  et al., 1975a).

    2.2.2  Short-term studies

    2.2.2.1  Mice

         Groups of 5 B6C3F1 male and female mice were given daily
    gavage doses of 0, 413, 825, 1650, 3300 or 6600 mg  d-limonene/kg
    bw in 10 ml/kg bw corn oil 5 days/week over a 16 day period (12
    total dosings). All mice (5/5) exposed to 6600 mg/kg bw/day, 4/5
    males and 5/5 females exposed to 3300 mg/kg bw/day, and 1/5 males
    and 1/5 females exposed to 1650 mg/kg bw/day died before the end of
    the study. The one female that died at the latter dose level was
    reported to have been killed by a gavage error. Authors concluded
    that no compound-related clinical signs were observed in mice that
    received 1650 mg  d-limonene/kg bw and lived to the end of the
    study, and that no compound-related histopathologic effects were
    seen (NTP, 1990).

         Groups of 10 B6C3F1 mice of each sex were given daily
    gavage exposures of 0, 125, 250, 500, 1000 or 2000 mg
     d-limonene/kg bw in 10 ml/kg bw corn oil 5 days/week for 13 weeks.
    One male exposed to 2000 mg/kg bw/day, two females exposed to
    2000 mg/kg bw/day, and one female exposed to 500 mg/kg bw/day, died
    during the course of the study. In addition, one female exposed to
    125 mg/kg bw/day and five males (one each from the groups exposed to
    250 and 1000 mg/kg bw/day, and three from the group exposed to
    500 mg/kg bw/day) died prior to the end of the study, but their
    deaths were attributed by the authors to accidents related to the
    gavage procedure used. At the two highest dose levels, mice were
    seen with rough hair coats and decreased activity. An alveolar cell
    adenoma was observed in the lung of one female mouse that had
    received 2000 mg  d-limonene/kg bw/day (NTP, 1990).

    2.2.2.2  Rats

         Groups of 5 F344/N rats of each sex were given daily gavage
    doses of 0, 413, 825, 1650, 3300 or 6600 mg  d-limonene/kg bw in
    10 ml/kg bw corn oil 5 days/week over a 16 day period (12 total
    doses). All rats receiving 6600 mg  d-limonene/kg bw/day as well as

    5/5 male and 3/5 female rats that were exposed to 3300 mg/bw/day
    died. The authors reported that no clinical signs were seen in rats
    receiving 1650 mg  d-limonene/kg bw/day or lower, and that no
    compound-related histopathologic effects were seen in any rats (NTP,
    1990).

         Groups of 5 male Fischer-344 rats were given single daily
    gavage doses containing 75, 150 or 300 mg  d-limonene/kg bw in corn
    oil (5 ml/kg bw) 5 days/week for 5 or 20 doses, primarily to follow
    the development of renal alterations. The authors reported that no
    signs of gross toxicity were observed nor was there any effect on
    weight gain or feed consumption over the course of this experiment.
    However, after only 5 days of exposure, liver and kidney weights
    showed a dose-related increase and there was a dose-related increase
    in the number and size of hyaline droplets and accumulation of
    alpha2u-globulin in the proximal convoluted tubule epithelial
    cells of the kidneys. In addition, after 20 doses, granular casts in
    the outer zone of the medulla and multiple cortical changes
    (classified as chronic nephrosis) were also seen in dose-related
    severity (Kanerva  et al., 1987a).

         In a subacute study, groups of 5 male and female rats were
    exposed daily to 0, 277, 554, 1385 or 2770 mg  d-limonene/kg bw
    orally for 30 days. A general decrease in food intake and a
    dose-related decrease in body weight were seen in groups of exposed
    males, but little or no effect on organ weights nor relative organ
    weights was observed. No significant changes were seen in
    urinalysis, haematology or biochemical values. The following tissues
    were examined histopathologically: adrenals, duodenum, heart,
    kidneys, liver, lungs, lymph nodes, pancreas, pituitary, spleen,
    stomach, testes/ovaries, thymus and thyroids. Authors reported that
    no significant changes were noted except that granular casts were
    seen in the kidneys of most exposed male rats (0/5, 3/5, 5/5, 5/5 or
    4/5 animals exposed to 0, 277, 554, 1385 or 2770 mg/kg bw/day
    respectively) (Tsuji  et al., 1975a).

         Groups of 10 F334/N rats of each sex were given daily gavage
    doses of 0, 150, 300, 600, 1200 or 2400 mg  d-limonene/kg bw in
    5 ml/kg bw corn oil 5 days/week for 13 weeks. Five male and nine
    female rats exposed to 2400 mg  d-limonene/kg bw/day died during
    the course of the study. In all rats receiving 1200 or 2400 mg/kg
    bw/day, rough hair coats, lethargy and excessive lacrimation were
    observed. Authors reported that nephropathy was seen in all groups
    of male rats, with a dose-related increase in severity. The
    nephropathy was reported to be characterized by degeneration of
    epithelium in the convoluted tubules, granular casts within tubular
    lumens (primarily in the outer stripe of the outer medulla), and
    regeneration of the tubular epithelium. In all groups of male rats
    (including control animals), hyaline droplets were observed in the

    epithelium of proximal convoluted tubules in at least some animals.
    An attempt to determine by "blind" evaluation if the incidence of
    these droplets was dose-related was reported to have resulted in an
    equivocal conclusion.

         The slides containing the renal sections made from male rats
    exposed to  d-limonene in this 13 week study were reviewed. They
    reported that hyaline droplet accumulation within the cytoplasm of
    proximal convoluted tubule (PCT) epithelial cells was uniform among
    all groups, including controls. However, chronic nephrosis, although
    seen in all animals (including control animals), appeared with a
    dose-related increase in severity. The authors attributed the
    uniformity of hyaline droplet accumulation to the fact that the
    animals were not exposed to  d-limonene for 4-5 days before
    necropsy and examination, giving the animals time to rid themselves
    of some of the droplets, although more extensive damage to the
    kidneys (chronic nephrosis) was not repaired (Kanerva & Alden 1987).

         Chronic nephrosis consisted of cytoplasmic basophilia of the
    PCT epithelial cells, tubular hyperplasia or atrophy, fibrosis of
    Bowman's capsule and an interstitial fibrolymphocytic response.
    Atrophic tubules were characterized by thickened basement membranes
    and a decrease in tubular cross-sectional diameter due to a decrease
    in cell size, whereas tubules considered to be hyperplastic showed
    increases in cell size and number and an increase in cross-sectional
    diameter compared with the surrounding unaffected tubules. In
    addition, occasional foci of PCT epithelial cell
    necrosis/degeneration were observed in kidneys from all
     d-limonene-treated animals but not in control animals. All treated
    animals, but not controls, also showed a dose-related increase in
    the number of granular casts found in the outer stripe of the
    medulla. Granular casts were markedly dilated and had attenuated or
    non-existent epithelium. In addition, animals exposed to the highest
    dose level (2400 mg/kg bw/day) were reported to show multifocal
    hyaline casts and tubular dilation in the cortex and medulla (NTP,
    1990).

    2.2.3  Long-term/carcinogenicity studies

    2.2.3.1  Mice

         To assess the pulmonary tumour carcinogenicity of  d-limonene,
    groups of 15 male or female A/He mice were injected i.p. with
     d-limonene 3 times weekly for 8 weeks (4.8 or 24 g/kg bw total
    dose) and then necropsied 24 weeks after the first injection. No
    significant induction of lung tumours was seen (Stoner  et al.,
    1973).

         Groups of 50 8-9 week old male B6C3F1 mice were given
    daily gavage doses of 0, 250, or 500 mg  d-limonene/kg bw and
    groups of 50 8-9 week old female B6C3F1 mice were given 0,
    500, or 1000 mg  d-limonene/kg bw in 10 ml/kg bw corn oil
    5 days/week for 103 weeks. Mice were observed twice a day and
    weighed weekly for the first 12 weeks, after which they were weighed
    monthly. Clinical signs were recorded at least monthly. Moribund
    animals were killed and a necropsy was performed on all available
    animals. The tissues of all mice that died during the study and
    animals with grossly visible lesions, as well as all animals in the
    control and high dose groups, were examined for histopathology.

         The authors reported that no clinical signs were associated
    with administration of the test chemical. The survival of male mice
    exposed to 250 mg  d-limonene/kg bw/day was lower than that of
    controls at the end of the study (controls: 33/50; 250 mg/kg bw/day:
    24/50; 500 mg/kg bw/day: 38/50). No other differences in survival
    were seen among exposed groups of either sex. Mean body weights of
    exposed and control male mice were generally comparable. Weights of
    female mice in the 1000 mg  d-limonene/kg bw/day group, however,
    were 5-15% lower than control animals after week 28.

         Multinucleated liver hepatocytes and cells with cytomegaly
    occurred at increased incidence in male mice exposed to 500 mg
     d-limonene/kg bw/day (multinucleated hepatocytes: controls - 8/49;
    250 mg/kg bw/day - 4/36 [incomplete sampling]; 500 mg/kg bw/day -
    32/50) (hepatocytes with cytomegaly: controls - 23/49; 250 mg/kg
    bw/day - 11/36 [incomplete sampling]; 500 mg/kg bw/day - 38/50). The
    combined incidence of hepatocellular adenomas and carcinomas,
    however, did not differ significantly from their incidence in
    control mice (controls: 22/49; 250 mg/kg bw/day: 14/36 [incomplete
    sampling]; 500 mg/kg bw/day: 15/50). No significant differences were
    observed in female mice for any of these endpoints.

         The incidence of adenomas or the combined incidence of adenomas
    and carcinomas in the anterior pituitary gland of female mice
    exposed to 1000 mg  d-limonene/kg bw/day were significantly lower
    than controls. The incidence of hyperplasia did not differ
    significantly from controls, however (hyperplasia: controls - 16/49;
    500 mg/kg bw/day - 0/8 [incomplete sampling]; 1000 mg/kg bw/day -
    17/48) (adenomas: controls - 12/49; 500 mg/kg bw/day - 5/8
    [incomplete sampling]; 1000 mg/kg bw/day - 1/48) (combined adenoma
    and carcinoma: controls - 12/49; 500 mg/kg bw/day - 5/8 [incomplete
    sampling]; 500 mg/kg - 2/48) (NTP, 1990).

    2.2.3.2  Rats

         Groups of fifty 7-8 week old male F344/N rats were given daily
    gavage doses of 0, 75, or 150 mg  d-limonene/kg bw and groups of 50
    7-8 week old female F344/N rats were given 0, 300, or 600 mg
     d-limonene/kg bw in 5 ml/kg bw corn oil 5 days/week for 103 weeks.

    Rats were observed twice a day; they were weighed weekly for the
    first 12 weeks, and monthly thereafter. Clinical signs were recorded
    at least monthly. Moribund animals were killed; necropsies were
    performed on all available animals. The tissues of all rats that
    died during the study, animals with grossly visible lesions, and all
    animals in the control and high dose groups were examined for
    histopathology.

         No compound-related clinical signs were reported. The survival
    of the female animals exposed to 600 mg/kg bw was lower than that of
    control animals after week 39, while the survival of male rats
    exposed. Mean body weights of male rats exposed to 150 mg/kg bw were
    generally 4%-7% lower than controls from week 28.

         Cataracts were observed at increased incidence in male rats
    exposed to 150 and female rats exposed to 300 or 600 mg
     d-limonene/kg bw/day, and retinal degeneration was seen in all
    dosed animals. The authors pointed out, however, that the rats of
    each sex that were exposed to their respective highest-dose levels
    were housed in the top rows of the cage racks, the animals exposed
    to the low-dose levels were below them, and the controls were housed
    in the lowest rows for all but the last 10 weeks of the study.
    Authors conclude that these effects were due to variations in the
    proximity of animals in different dose groups to the light source.

         Subcutaneous tissue fibromas in male rats occurred with a
    negative dose-response trend (controls: 8/50; 75 mg/kg bw/day: 2/50;
    150 mg/kg bw/day: 3/50). However, the combined incidence of fibromas
    and fibrosarcomas was not significantly different when dosed males
    were compared to controls (controls: 8/50; 75 mg/kg bw/day: 4/50;
    150 mg/kg bw/day: 3/50).

         Combined squamous cell papillomas or carcinomas in the skin of
    male rats showed a positive dose-response trend (controls: 0/50;
    75 mg/kg bw/day: 0/50; 150 mg/kg bw/day: 3/50). However, the
    incidence of these neoplasms in dosed male rats was within the range
    of incidences for NTP historical control rats, and did not differ
    significantly from the incidence in control animals. The authors
    concluded that the positive dose-response trend was not related to
     d-limonene exposure.

         Mononuclear cell leukaemia occurred in male rats with a
    positive dose-response trend by the incidental tumour test
    (controls: 10/50; 75 mg/kg bw/day: 10/50; 150 mg/kg bw/day: 19/50).
    The incidences in dosed male rats were not significantly different
    from the incidences in controls, however. The authors concluded that
    the positive dose-response trend was not related to  d-limonene
    administration.

         There was a significant positive dose-response trend in the
    incidence of interstitial cell tumours in the testes of male rats,
    and the incidences of these tumours in dosed groups were
    significantly higher than the incidences in control animals
    (control: 37/50; 75 mg/kg bw/day: 47/49; 150 mg/kg bw/day: 48/50).
    The authors argued, however, that since historically this type of
    tumour occurs in almost all aged F344 rats, this result should not
    be considered to be related to  d-limonene exposure, but rather was
    an artifact caused by the low survival of the control animals in
    this experiment (see above).

         The incidence of uterine endometrial stromal polyps in females
    exposed to 300 mg/kg bw/day was increased when compared to controls.
    However, the incidence in the control animals was well below the NTP
    historical incidence of this tumour. For this reason, and because
    there was no positive dose-response trend, the authors argued that
    this was not a compound-related effect.

          d-Limonene exposure of male rats was associated with
    dose-related increases in the incidences of mineralization and
    epithelial hyperplasia in the kidneys (mineralization: controls -
    7/50; 75 mg/kg bw/day - 43/50; 150 mg/kg bw/day - 48/50) (epithelial
    hyperplasia: controls - 0/50; 75 mg/kg bw/day - 35/50; 150 mg/kg
    bw/day - 43/50). The lesions consisted of linear deposits of mineral
    in the medulla (renal papilla) and focal hyperplasia in the
    transitional epithelium overlying the papilla. Hyperplasia was
    frequently located near the fornices of the renal pelvis and was
    occasionally bilateral. The severity of nephropathy, which occurs
    spontaneously in aged male rats, was also positively dose-related in
    this experiment (severity of nephropathy, rated on a scale from 0
    [not present] to 4 [marked]: control mean - 1.5; 75 mg/kg bw/day
    mean - 1.8; 150 mg/kg bw/day mean - 2.2). This nephropathy was
    characterized by degeneration and atrophy of the tubular epithelium,
    dilation of tubules with formation of hyaline droplets and granular
    casts, regeneration of tubular epithelium, glomerulosclerosis, and
    interstitial inflammation and fibrosis.

         Kidney tubular cell hyperplasia and neoplasms were also
    increased in dosed male rats (hyperplasia: controls - 0/50; 75 mg/kg
    bw/day - 4/50; 150 mg/kg bw/day - 7/50) (adenomas: controls - 0/50;
    75 mg/kg bw/day - 4/50; 150 mg/kg bw/day - 8/50) (adenocarcinomas:
    controls - 0/50; 75 mg/kg bw/day - 4/50; 150 mg/kg bw/day - 3/50)
    (combined adenomas and carcinomas: controls - 0/50; 75 mg/kg bw/day
    - 8/50; 150 mg/kg bw/day - 11/50). Incidences of tubular cell
    adenomas and the combined incidences of adenomas and
    adeno-carcinomas in male rats showed significant positive
    dose-response trends. High-dose males had a significantly higher
    incidence than control males of both categories of tumours, while
    low-dose males had a significantly higher incidence of the combined
    category than control males. These neoplasms are historically rare
    and none were seen in control males nor in any group of females.

         The authors stated that kidney tubular cell hyperplasia,
    adenomas, and adenocarcinomas were part of a morphologic spectrum.
    Proliferative lesions diagnosed as hyperplasia generally consisted
    of one to three adjacent cross-sections of enlarged tubules with
    stratification of the epithelium. In some lesions, the epithelial
    cells appeared to completely fill the lumen. The tubular cell
    neoplasms varied in diameter from less than 1 mm to 15 mm. They
    exhibited varied patterns of growth and were either solid, cystic,
    or papillary. The solid and cystic neoplasms showed little evidence
    of tubular structures and the cells were arranged in solid sheets or
    small solid nests separated by a delicate vascular stroma. Some
    neoplasms were reported to consist of layers of epithelium lining a
    fibrous connective tissue stroma and were arranged in complex
    branching papillary formations. The neoplasms were classified as
    adenocarcinomas primarily if they exhibited cellular pleomorphism
    and anaplasia, or if they were larger than 10 mm (NTP, 1990).

    2.2.3.3  Dogs

         Groups of 3 male or female Japanese beagle dogs were exposed
    orally to 0, 0.4, 1.2 or 3.6 ml  d-limonene/kg bw daily for 6
    months. Dose-related occurrences of frequent vomiting and nausea
    were seen in some animals. At doses of at least 1.2 ml/kg bw/day for
    females and 3.6 ml/kg bw/day for males, a decrease in body weight
    gain was noted compared to controls, but little or no effect on food
    intake, organ weights or relative organ weights was reported. No
    significant changes were seen in urinalysis, haematology nor
    biochemical values, except that in animals at the 3.6 ml/kg bw/day
    level, total cholesterol and blood sugar were lowered. The following
    tissues were examined histopathologically: adrenals, bile duct,
    duodenum, heart, kidneys, liver, lungs, pancreas, pituitary, rectum,
    spleen, stomach, testes/ovaries, thymus and thyroids. No significant
    changes were noted except that granular casts were seen in the
    kidneys of all male dogs exposed to 3.6 ml/kg bw/day and all females
    exposed to 0.4 ml/kg bw/day or more (1/3, 1/3, 2/3 or 3/3 for males;
    1/3, 2/2, 3/3 or 3/3 for females exposed to 0, 0.4, 1.2 or 3.6 ml/kg
    bw, respectively) (Tsuji  et al., 1975b).

         In a more recent study, groups of five male and female beagle
    dogs were gavaged twice daily for six months with 0, 0.12, or
    1.2 ml/kg bw/day (0, 10, or 1000 mg/kg bw/day)  d-limonene. The
    highest dose was reported to be near the maximum tolerated dose for
    emesis. The authors stated that food consumption and body weight
    were not affected by treatment. Although there was a positive
    dose-related trend for absolute and relative female kidney weights
    and for relative male kidney weight, authors reported that there
    were no histo-pathological findings in the kidneys that could be
    associated with the organ weight changes. In addition, there was no
    hyaline droplet accumulation nor other indication of nephropathy
    such as those associated with  d-limonene consumption in male rats
    (Webb  et al., 1990)

    2.2.3.4  Cocarcinogenicity/tumour promotion studies

         Two publications have reported the results of experiments in
    which 0.25 ml oil of sweet orange, composed of > 90%  d-limonene,
    was applied to the skin of 10 male and 10 female "101" mice weekly
    for 42 weeks, starting 3 weeks after an application of 300 g
    7,12-dimethyl-1,2-benzanthracene (DMBA) in 0.2 ml acetone. While no
    effects in the area of the treated skin were seen in mice exposed to
     d-limonene or to DMBA alone, mice exposed to DMBA followed by
    exposure to  d-limonene began to develop papillomas during the
    twelfth week of  d-limonene exposure. After 33 weeks of treatment,
    13/18 treated mice showed a total of 39 papillomas. No malignant
    tumours were seen in the exposed area, however.

         In a second study, mice were exposed to  d-limonene following
    exposure to: 1) 4 skin applications of 60 mg urethane in 0.3 ml
    acetone at 3 day intervals, or 2) 4 i.p. injections of 16 mg
    urethane in 0.1 ml distilled water at 3 day intervals. Papillomas
    appeared on 3/13 mice in group 1 and 2/13 mice in group 2 by the
    fourteenth week of exposure.

         In a final experiment, the terpene fraction of oil of sweet
    orange (containing  d-limonene) was isolated and tested with DMBA
    as described for the first experiment. Papillomas in mice exposed to
    both DMBA and the terpene fraction of oil of sweet orange began to
    appear in the eleventh week of treatment and were visible in 8/15
    mice by the thirty-third week of treatment. Although pure
     d-limonene was not tested in these experiments, the authors
    concluded that the promoting effects seen were probably caused by
    this chemical since it was the primary constituent of the test agent
    (Roe, 1959, Roe & Peirce, 1960).

         A group of 23 male and female albino mice were given single
    doses of 50 g benzo(a)pyrene (BAP) by stomach tube followed by 40
    weekly doses of 0.05 ml  d-limonene; seventeen male and female mice
    received BAP only, 15 male and female mice received  d-limonene
    only and 18 male and female mice served as untreated controls. While
    no tumours of the forestomach epithelium were seen in control
    animals, 2 animals treated with BAP alone (12%), 2 animals treated
    with  d-limonene alone (13%), and 5 animals treated with both BAP
    and d-limonene (22%), developed 0, 2, 3, and 8 tumours,
    respectively, although none was judged to be a carcinoma. The
    authors concluded that although  d-limonene was weakly carcinogenic
    for mouse forestomach epithelium it did not increase the tumour
    yield caused by pretreatment with BAP and, therefore, was not a
    promoter (Field & Roe, 1965).

         Cells of embryos taken from F344 rats were infected with
    Rauscher murine leukaemia virus and then treated with 0.05 g/ml
    3-methylcholanthrene (3-MCA) followed by 15 g/ml  d-limonene.

    Following treatment with 3-MCA and  d-limonene, colony formation in
    semisolid medium was observed and was considered to denote cell
    transformation. Authors reported that  d-limonene alone failed to
    transform cells and 3-MCA alone was only effective at concentrations
    exceeding 0.2 g/ml (Roe, 1959; Roe & Peirce, 1960; Traul  et al.,
    1981)).

         Elegbede and co-workers attempted to repeat the 1960 study of
    Roe and Peirce (see above) using purified  d-limonene as well as
    oil of sweet orange. The skin of groups of 24 female CD-1 mice were
    treated once with 51.2 g DMBA in 0.2 ml acetone followed 14 days
    later by twice weekly applications of 0.1 ml  d-limonene or oil of
    sweet orange mixed with 0.1 ml acetone. Other groups of mice were
    similarly initiated with DMBA, but beginning 7 days later were given
    a diet containing 1%  d-limonene or oil of sweet orange. In all
    cases, administration of  d-limonene or oil of sweet orange
    continued for 40 weeks after DMBA treatment. Topical (but not
    ingested)  d-limonene was found to slightly increase the number of
    tumours induced by DMBA alone, the increase becoming significant
    only after 34 weeks of treatment. When oil of sweet orange (95%
     d-limonene content) was applied to the skin, authors reported that
    a promoting effect was seen after 7 weeks of treatment; promotion
    was not reported, however, when oil of sweet orange was incorporated
    into the diet. The authors concluded that a component of orange oil
    other than  d-limonene was responsible for the promoting activity
    observed and that the very small promoting activity seen with
     d-limonene may be due to its contamination by this unidentified
    component (Elegbede  et al., 1986b).

    2.2.3.5  Anticarcinogenicity studies

         A group of 50 male C57Bl/6J mice were injected s.c. with 25 g
    dibenzpyrene (DBP) followed 24 h later by an injection of 0.15 ml
     d-limonene. After 34 weeks of observation, the authors reported
    that  d-limonene significantly delayed the appearance of lung
    adenomas induced by DBP and reduced the total number of tumours
    seen. At 30 weeks post-DBP exposure without subsequent exposure to
     d-limonene, lung adenomas were seen in approximately 70% of the
    animals; with exposure to  d-limonene, tumours were seen in
    approximately 30% of the animals.

         In a second experiment reported in the same paper, a group of
    50 female A/J mice were injected s.c. with DBP followed by 16 weekly
    injections into the tail vein of 1% v/v suspensions of 1 mg of
     d-limonene. The incidence of lung adenomas was reduced from 75% of
    animals exposed to DBP alone to 40% of animals exposed to DBP and
    injected with  d-limonene. Control incidence of lung adenomas in
    this experiment was 27%. Treatment of mice with  d-limonene alone
    reduced this background incidence to approximately 7% (Homburger
     et al., 1971).

         Simultaneously, 5 g of BAP and 10 mg  d-limonene were applied
    to the skin of groups of 50 female ICR/Ha Swiss mice three times a
    week for 440 days.  d-Limonene was found to partially inhibit BAP
    carcinogenicity (Van Duuren & Goldschmidt, 1976).

         Female Sprague-Dawley rats were fed diets containing 0, 1000,
    or 10 000 ppm  d-limonene for 1 wk, at which time they were given a
    single gavage dose of 65 mg DMBA/kg bw. The  d-limonene-containing
    diet was then continued for 27 weeks. Authors reported that
     d-limonene delayed the appearance of DMBA-induced mammary tumours
    in a dose-related manner, reduced the incidence of tumours, and
    caused increased regression of tumours that did appear (Elegbede
     et al., 1984a,b).

         Female (W/Fu x F344)F2 rats were fed a diet containing 10%
     d-limonene for 80 days beginning after the first appearance of
    tumours induced by intubation of rats with 130 mg DMBA/kg bw.
    Significant tumour regressive action by  d-limonene was observed,
    and the development of subsequent tumours was also reduced
    significantly (Elegbede  et al., 1986a).

         Following initiation with DMBA (single gavage dose of 65 mg/kg
    in sesame oil), groups of female Sprague-Dawley rats were (1) fed a
    basal cereal diet one week before and 24 weeks following initiation
    with DMBA (controls), (2) fed a diet containing 5%  d-limonene one
    week before and one week after treatment with DMBA, then fed the
    control diet for 24 weeks (initiation group), or (3) fed the control
    diet for one week before and one week following DMBA initiation,
    then fed the 5%  d-limonene diet for 24 weeks
    (promotion/progression group).  d-Limonene was reported to be
    effective in reducing the average number of rat mammary carcinomas
    in DMBA-initiated rats when fed during the initiation phase or
    during the initiation/progression phase of carcinogenesis. Time to
    appearance of the first tumour was extended only when  d-limonene
    was fed during the initiation phase, however. Authors concluded that
    these effects could not be attributed to changes in mammary-relevant
    endocrine functions (serum levels of prolactin and duration of
    estrus cycle) (Elson  et al., 1988).

         Female Wistar-Furth rats were (1) placed on diets containing 5%
     d-limonene or 5% orange oil (source of  d-limonene) for 2 weeks
    prior to initiation with nitrosomethylurea (NMU; single i.v. dose of
    50 mg/kg bw), then continued on these diets for 23 weeks; (2) fed
    diets containing 5%  d-limonene for two weeks before and one week
    following initiation with NMU, then fed a basal diet for 22 weeks
    (initiation group); or (3) fed basal diets prior to and for 1 week
    following initiation with NMU, then fed diets containing 5%
     d-limonene for 22 weeks (promotion/progression group). Both orange
    oil and  d-limonene decreased mammary tumour incidence (controls:

    80%;  d-limonene: 45%; orange oil: 47%) and the average number of
    mammary tumours per rat (controls: 1.8;  d-limonene: 0.4; orange
    oil: 0.8) when present in the diet for two weeks before and 23 weeks
    after NMU administration.  d-Limonene decreased tumour incidence
    and decreased the numbers of tumours per rat (by approximately 50%)
    when fed during the promotion/progression experiment but not the
    during the initiation experiment (Maltzman  et al., 1989).

    2.2.3.6  Studies on the mechanism of  d-limonene carcinogenicity

         This section discusses (1) spontaneously occurring nephropathy
    in mature male rats and (2) the current proposed mechanism of
    carcinogenicity of  d-limonene and other chemicals/mixtures that
    exacerbate this condition and lead to alpha2u-globulin-associated
    male rat nephropathy.

    (1)  Mature male rat nephropathy

         Normal male rats at least 30 days old exhibited marked
    increased protein excretion in their urine not seen in younger males
    nor in females of any age. This proteinuria reached a maximum level
    of approximately 2.5-3 times that seen in females at 90 days of age.
    Microscopically, the authors also observed intracellular droplets in
    the epithelial cells of the upper two-thirds of the proximal
    convoluted tubules of kidneys of male rats at least 60 days old. The
    occurrence of these hyaline droplets consisted of a few groups of
    small droplets in occasional cells in 60 day old animals. The number
    of cells having droplets and the number and size of droplets per
    cell increased with age; from 90 days of age most proximal tubule
    cells contained them. In contrast, at 120 and 180 days of age, only
    a small number of females showed occasional droplets in a few cells
    of the proximal tubules.

         These authors further demonstrated the sex-relatedness of this
    phenomenon by castrating male rats at 30 days of age and injecting
    female rats with 5 mg testosterone every other day from 50 days of
    age. When examined at 120 days of age, the castrated male rats
    showed proteinuria at a level equal to control females and hyaline
    droplets were only seen in 4/20 animals. Testosterone-treated
    females, on the other hand, had proteinuria at a level approximately
    midway between control males and females. No hyaline droplets were
    seen in the proximal tubule cells of these females, however, showing
    that the addition of testosterone alone was insufficient to cause
    them to appear (Logothetopoulos & Weinbren, 1955).

         Numerous authors have since reported detailed descriptions of
    spontaneously occurring nephropathy in mature male rats which is a
    consequence of this hyaline droplet accumulation. The sequence of
    events appears to be that: (1) protein accumulates in the lysosomes
    in the cytoplasm of epithelial cells of the P2 segment of the

    proximal convoluted tubules in the kidney cortex; (2) the
    accumulated material becomes so abundant that it crystallizes,
    forming the microscopically visible hyaline droplets; (3) the
    continued build-up of material eventually leads to the death of
    epithelial cells with a concomitant thinning of the epithelial layer
    (although some regeneration is usually seen); and (4) the dead cell
    debris becomes lodged in the outer strip of the outer medulla where
    the tubules narrow, forming granular casts and causing tubule
    dilation with pressure necrosis of the cells of the tubule walls.
    There is also an increase in the relative weight of the kidneys
    accompanying this phenomenon.

         A steadily increasing number of agents have been identified
    that appear to exacerbate this hyaline droplet formation and its
    consequences in mature male rats. With some, noticeable changes
    occur within days of exposure. These agents include complex mixtures
    of hydrocarbons such as mineral spirits (Carpenter  et al., 1975a;
    Phillips & Cockrell, 1984), "60" solvent (Carpenter  et al.,
    1975b), "high naphthenic solvent" (Carpenter  et al., 1977), the
    petroleum-derived or shale-derived jet fuel JP-5 (MacEwen & Vernot,
    1978a; Parker  et al., 1981; Bruner, 1984; MacNaughton & Uddin,
    1984), petroleum-derived and shale-derived diesel fuel marine (DFM),
    the jet fuels JP-4, JP-TS and JP-7, and the missile propellants
    JP-10 and RJ-5 (Bruner, 1984; MacNaughton & Uddin, 1984), C/10-C/11
    isoparaffin (Phillips & Egan, 1984; Phillips & Cockrell, 1984), five
    petroleum naphtha mixtures (Halder  et al., 1984) and unleaded
    gasoline (MacFarland  et al., 1984; Busey & Cockrell, 1984; Halder
     et al., 1984; Kitchen, 1984; Garg  et al., 1988a,b, 1989; Short &
    Swenberg, 1988; Short  et al., 1989a). Also included on the list
    are the individual chemicals decahydro-naphthalene (decalin, MacEwen
    & Vernot, 1978b; Gaworski  et al., 1980, 1981; Bruner, 1984; Alden
     et al., 1984; Olson  et al., 1986; Stone  et al., 1987a,b;
    Kanerva  et al., 1987b,c; Read, 1988), pentachloroethane (NTP,
    1983; Goldsworthy  et al., 1988), 1,4-dichlorobenzene (NTP, 1987a;
    Charbonneau  et al., 1989), perchloroethylene (Goldsworthy  et al.,
    1988), dimethyl methylphosphonate (NTP, 1987b),
    2,2,4-trimethylpentane (Stonard  et al., 1986; Short  et al.,
    1987; Charbonneau  et al., 1987; Lock  et al., 1987; Loury
     et al., 1987; Short  et al., 1989a), the chemically unrelated
    agents 3-methylamino-1-(3-trifluoromethyl-phenyl)-2-pyrazoline,
     cis/trans-2-(4'-t-butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone
    and 2,3,5,6-tetrahycro-6-phenylimidazo-(2,1-b) thiazole (Read
     et al., 1988) and  d-limonene (Kanerva  et al., 1987a; NTP,
    1990).

         There is no obvious common structure among the specifically
    identified chemicals that cause an increased accumulation of hyaline
    droplets in mature male rat kidneys. And there are examples of
    agents that do have similar mixtures/structures to those listed
    above that do not cause this phenomenon. These include "70" solvent

    (Carpenter  et al., 1975c), two other petroleum naphtha mixtures
    (Halder  et al., 1984), 1,2-dichlorobenzene (Charbonneau  et al.,
    1989) and an analog of  d-limonene, 4-vinylcyclohexene (NTP, 1986).
    Recently, however, Miller  et al. (1989) reported that they have
    identified correlations between structure and binding affinity to
    alpha2u-globulin, but no specific details were given in their
    published report.

         While exposure to unleaded gasoline for 72 h has been shown to
    cause effects that are reversible (Garg  et al., 1988a), with
    chronic exposure to several of these mixtures/chemicals the kidney
    nephrosis has been reported to progress to hyperplasia, adenoma and
    even adenocarcinoma (MacFarland  et al., 1984; Kitchen, 1984;
    MacNaughton & Uddin, 1984; NTP, 1987a,b,c; NTP, 1990).

    (2)  Mechanism for alpha2u-globulin-associated male rat
         nephropathy

         The mechanism proposed for alpha2u-globulin-associated male
    rat nephrosis has recently been thoroughly reviewed by the US
    Environmental Protection Agency (US EPA, 1991). Their review
    incorporated the results of many studies, including Charbonneau &
    Swenberg (1988), Swenberg  et al. (1989), Flamm & Lehman-McKeeman
    (1991), Lehman-McKeeman  et al. (1989), Lehman-McKeeman  et al.
    (1990a,b), Short  et al. (1987), Short  et al. (1989a,b), Dietrich
    & Swenberg (1991), Murty  et al. (1988), Ridder  et al. (1990),
    and Webb  et al. (1989). The review concluded that:

         "Of the eight model substances tested in chronic animal
         bioassays (decalin, dimethyl methyl phosphonate, JP-4 jet fuel,
         JP-5 shale-derived jet fuel,  d-limonene, methyl isobutyl
         ketone, pentachloroethane, and unleaded gasoline), all invoked
         a specific type of protein droplet nephropathy in male rats and
         also produced renal tumours in male rats but not in other
         species tested. It has been proposed that such renal tumours
         are the end product in the following sequence of functional
         changes in the epithelial cells of proximal tubules:

         *    Excessive accumulation of hyaline droplets in proximal
              tubules, representing lysosomal overload, cell loss, and
              regenerative cellular proliferation.

         *    Cell debris in the form of granular casts accumulates at
              the 'corticomedullary' junction with associated dilation
              of the affected tubule segment and more distally,
              mineralization of tubules within the renal medulla.

         *    Single-cell necrosis accompanied by compensatory cell
              proliferation and exacerbation of the chronic progressive
              nephropathy characteristically found in aging rats occurs.

         *    Renal tubule hyperplasia and neoplasia develop
              subsequently.

         According to this hypothesis, the increased proliferative
         response caused by the chemically induced cytotoxicity results
         in clonal expansion of spontaneously initiated renal tubule
         cells and increased incidence of renal tumour formation (Trump
          et al., 1984a; Alden, 1989; Swenberg  et al., 1989). This
         line of reasoning leads supporters of the hypothesis to
         conclude that the acute and chronic renal effects induced in
         male rats by such chemicals will be unlikely to occur in any
         species not producing alpha2u-globulin, or a very closely
         related protein, in the large quantities typically seen in the
         male rat (Alden, 1989; Borghoff  et al., 1990; Green  et al.,
         1990; Olson  et al., 1990; Flamm & Lehman-McKeeman, 1991;
         Swenberg, 1991)."

         The following paragraphs summarize some of the data that
    support this hypothesis:

    Contents and catabolism of hyaline droplets/
    phagolysosomes

         The composition of the hyaline droplet inclusions has been
    identified by Kanerva  et al. (1987c) using two-dimensional gel
    electrophoresis and immunological binding as being four species of
    the low molecular weight protein alpha2u-globulin. As expected,
    alpha2u-globulin was found to be absent in immature male rats and
    female rats of all ages. It was present, however, in female rats
    that had been ovariectomized and repeatedly injected with
    testosterone. More recently, groups have confirmed that
    alpha2u-globulin is the major constituent of the renal
    phagolysosomal inclusions in male rats exposed to unleaded gasoline
    (Garg  et al., 1988b), and perchloroethylene and penta-chloroethane
    (Goldsworthy  et al., 1988). Finally, Dietrich and Swenberg (1991)
    reported that male NCI-Black-Reiter rats, which do not produce
    alpha2u-globulin, are not susceptible to nephropathy produced by
    2,2,4-trimethylpentane, 1,4-dichloro-benzene, isophorone, PS-6
    unleaded gasoline, or  d-limonene.

         A sex-specific protein has been identified in human urine
    (Bernard  et al., 1989), but the concentration (up to 108 g/l) is
    about 10 000 times less than that of alpha2u-globulin in male rats
    (about 40 mg/24 h) (Roy & Neuhaus, 1967). Therefore assuming that
    the protein in human urine shows the same binding characteristics as
    alpha2u-globulin, it is reasonable to conclude that humans would
    be comparably less sensitive than the male rat.

         Alpha2u-globulin is synthesized in the liver of rats of both
    sexes. This protein is filtered by the kidney glomeruli and
    reabsorbed by the epithelial cells of the proximal convoluted
    tubules by the formation of phagosomes at the lumenal surface.
    Normally, the phagosomes then combine with cellular lysosomes and
    the protein is degraded. In mature male rats, however, this
    catabolism is slowed in some way under hormonal influence leading to
    the intracellular accumulation of the protein which becomes
    manifested as hyaline droplets. The degradation is slowed even more
    by exposure to at least some of the agents listed in the previous
    section (Stonard  et al., 1986; Short  et al., 1987; Kanerva
     et al., 1987c; Charbonneau  et al., 1987). A time- and
    dose-dependent progressive increase in the number and size, and
    alterations in the morphology of phagolysosomes in proximal
    convoluted tubule epithelial cells have been demonstrated with
    exposure to unleaded gasoline (Garg  et al,. 1989). The ability of
    unleaded gasoline to induce these effects declines with the age of
    the male rat, in proportion to the declining production of
    alpha2u-globulin (Murty  et al., 1988).

    Binding of chemicals to alpha2u-globulin

         1,4-Dichlorobenzene as well as its major metabolite,
    2,5-dichlorophenol, were both found to be reversibly bound (and
    1,4-dichlorobenzene also covalently bound) to alpha2u-globulin in
    the kidneys of treated male rats (Charbonneau  et al., 1989).

         Similarly, two groups have attempted to identify the specific
    binding between one of these agents (2,2,4-tri-methylpentane) and
    alpha2u-globulin. Loury  et al. (1987) reported that no covalent
    binding could be found, nor did there appear to be formation of a
    Schiff base. Lock  et al. (1987), however, detected reversible
    binding of the metabolite 2,4,4-trimethyl-2-pentanol to
    alpha2u-globulin. This specific metabolite was found in liver
    cells of male rats only.

         Likewise with decalin, an alcohol metabolite has been
    identified that is unique to male rats (Olson  et al., 1986). While
    both male and female rats synthesize  cis,cis-2-decalol and
     trans,cis-2-decalol from  cis-decalin in their livers,
     cis,cis-1-decalol was found only in males and
     cis,trans-1-decalol was produced in much higher concentrations
    than in females. In similar fashion, with  trans-decalin as the
    substrate  trans,trans-1-decalol was seen only in livers of male
    rats. When kidneys were examined, 2-decalone  (cis or  trans,
    depending on the original substrate) was found in the males while no
    decalin metabolites whatever were seen in female kidneys. Whether
    these unique male decalin metabolites react with alpha2u-globulin
    has not yet been tested.

         More recently, Lehman-McKeeman  et al. (1989) have reported
    that  d-limonene and  d-limonene-1,2-oxide, a major metabolite of
     d-limonene, were found reversibly bound to alpha2u-globulin
    (identified by amino acid sequencing) in the kidneys of treated
    male, but not female, rats.

    Inhibition of alpha2u-globulin catabolism

         Charbonneau  et al. (1988) reported that alpha2u-globulin
    bound in kidney cytosol was not digested by purified proteases or
    proteinase.

         Treating rats with leupeptin, an inhibitor of cathepsin B, a
    major lysosomal peptidase, mimics the ability of unleaded gasoline
    to cause the accumulation of alpha2u-globulin in kidney
    phagolysosomes. Authors concluded that the mechanism of
    nephrotoxicity involves inhibition of renal phagolysosomal
    proteolysis (Olsen  et al., 1988).

    Induction of cell proliferation

         Short  et al. (1989a) have shown that proliferation of
    epithelial cells in the P2 proximal tubule segment in male rats is
    associated with exposure of the animals to either
    2,2,4-trimethylpentane or unleaded gasoline. They noted that this
    proliferation closely paralleled the extent and severity of
    detectable alpha2u-globulin in the same cells. The authors
    speculated that this proliferation leads to the nephrocarcinogenic
    effects caused by these agents.

         Proliferation was also noted by Charbonneau  et al. (1989) in
    male rats treated with 1,4-dichlorobenzene. Short  et al. (1989b)
    observed increased numbers of atypical cell foci and renal cell
    tumours in male, but not female, rats initiated with
    N-ethyl-N-hydroxyethylnitrosamine and subsequently exposed to
    unleaded gasoline or 2,2,4-trimethylpentane. The extent of the
    promotion was reported to be dose-related and to parallel the
    effects of these compounds on chronic cell proliferation.

    2.2.4  Reproduction studies

         No information available.

    2.2.5  Special studies on embryotoxicity

          d-Limonene was reported to increase the number of abnormal
    chick embryos to approximately 50% when a single dose (25 M/embryo)
    in olive oil was injected suprablastodermically (Abramovici &
    Rachmuth-Roizman, 1983).

         Pregnant mice were given oral doses of 0, 591, or 2363 mg/kg bw
     d-limonene from days 7 through 12 of gestation. Authors reported
    significant decreases in body weight gain of dams in the high-dose
    group; fetuses of dams exposed to the high-dose showed increased
    incidences of lumbar rib, fused rib, delayed ossification, and
    decreased body weight gain relative to fetuses of control dams
    (Kodama  et al., 1977a).

         Pregnant Japanese white rabbits were given oral doses of 0,
    250, 500, or 1000 mg/kg bw  d-limonene from day 6 to day 18 of
    gestation. Significant decreases in body weight gain in dams given
    250 or 500 mg/kg bw/day  d-limonene were observed, and survival in
    dams given 1000 mg/kg bw/day was significantly reduced (40%
    survival). From examination of the fetuses, authors concluded that
     d-limonene was not teratogenic in rabbits (Kodama  et al.,
    1977b).

    2.2.6  Special studies on genotoxicity

        Table 2: Genotoxicity data on d-limonene
                                                                                  

    Test           Test                Concentration
    System         Object              of d-limonene       Results     Reference
                                                                                  

    Ames test      S. typhimurium      .03-3 mol/plate    neg         Florin et
    (+&-activ)     TA98, TA100,                                        al., 1980
                   TA1535, TA1537

    Yeast          S. cerevisiae       .1-100 mM/5x107     (1)         Fahrig,
    (-activ)       MP1 strain cells                                    1984

    Mammalian      Mouse embryo        0-215 mg/kg         (2)         Fahrig,
    (-activ)       system                                              1984

    Ames test      S. typhimurium      0-20 M/plate       neg         Watabe
    (+&-activ)     TA1535, TA100,                                      et al.,
                   TA1537, TA1538,                                     1980
                   TA98

    Ames test      S. typhimurium      0-3333 g/plate     neg         Haworth
    (+&-activ)     TA98, TA100,                                        et al.,
                   TA1535, TA1537                                      1983

    Ames test      S. typhimurium      0-3333 g/plate     neg         NTP,
    (+&-activ)     TA100, TA1535,                                      1990
                   TA1537, TA98

    Mammalian      Mouse L5178Y                            neg         NTP,
    (+&-activ)     cells in vitro                                      1990

    Sister         Chinese hamster     0-162 g/ml         neg         NTP,
    chromatid      ovary cells                                         1990
    exchange       in vitro
    (+&-activ)

    Chromosomal    Chinese hamster     0-500 g/ml (3)     neg         NTP,
    aberrations    ovary cells         0-100 g/ml (4)     neg         1990
    (+&-activ)     in vitro
                                                                                  

    Table 2 cont'd
                                                                                  

    Test           Test                Concentration
    System         Object              of d-limonene       Results     Reference
                                                                                  

    Ames test      S. typhimurium      150 mg/plate        neg         Heck et
    (+&-activ)     TA1535, TA1537,                                     al., 1989
                   TA1538, TA98,
                   TA100

    Mammalian      Mouse L5178Y        100 g/ml           neg         Heck et
    (+&-activ)     cells in vitro                                      al., 1989
                                                                                  

    (1)  Author reported that results indicated that  d-limonene was a
         co-recombinant and an anti-mutagen.

    (2)  Author reported that results indicated that  d-limonene is inactive
         when given alone, but reduces the mutagenic effect of ethylnitrosourea
         when the two are co-administered.

    (3)  Activated: addition of S9 from the livers of Aroclor 1254-induced
         male Sprague Dawley rats.

    (4)  Not activated
    
    2.2.7  Special studies on immune responses

         Limonene has been identified as a primary skin irritant in
    humans (Pirila  et al., 1964; Klecak  et al., 1977), rabbits
    (research Institute for the Development of Fragrance Materials,
    Inc., 1984; Research Institute for Fragrance Materials Inc., 1985),
    guinea-pigs (Klecak  et al., 1977), and mice (Gad  et al., 1986).
     d-Limonene was reported both to elicit (De Groot  et al., 1985)
    and not to elicit (Greif, 1967) allergic reactions in human skin and
    to elicit allergic reactions in mouse skin (Gad  et al., 1986).
     d-Limonene was reported to eliminate human skin sensitization to
    several aldehydes when they were co-administered with  d-limonene
    (Opdyke, 1975), and  d-limonene was reported to reduce the
    sensitivity and delayed hypersensitivity of guinea-pig skin to
    citral (Hanau  et al., 1983; Barbier & Benezra, 1983). Gozsy and
    Kato (1957) reported that local endothelial phagocytosis is induced
    in rat skin by  d-limonene, and that  d-limonene increases
    phagocytosis in guinea-pig skin  in vivo and in mouse skin
     in vitro (Gozsy & Kato, 1958) and increases capillary permeability
    in the guinea-pig (Kato & Gozsy, 1958).

         Male BALB/c mice were treated intragastrically with 0, 0.002,
    0.008, 0.033, 0.133, 0.531 or 2.125 mg/ml  d-limonene daily. After
    8 weeks, but not after 4 weeks, the mitogenic responses of spleen
    cells taken from the treated animals and exposed  in vitro to
    concanavalin A and phytohaemagglutinin, which stimulate T-cells, and
    lipopolysaccharide, which affects B-cells, were found to be
    suppressed by the  d-limonene treatment.

         Other animals in groups of 10 were given these same doses of
     d-limonene daily for 7 days prior to, or 8 days following,
    immunization with keyhole limpet haemocyanin (KLH). Primary effects
    on T- and B-cell responses were determined 10 days later.

         Secondary effects were determined by reinoculating the animals
    with KLH 21 days after the first inoculation and then assaying on
    day 24. The primary and secondary responses were reported to be
    suppressed by  d-limonene if the animals were exposed to KLH first,
    but stimulated in treated mice if exposed to KLH after  d-limonene
    exposure.

         Histopathological examination of secondary lymphoreticular
    tissue taken from animals exposed to  d-limonene showed significant
    secondary follicle development and prominent lymphoid nodules and
    aggregates in the pancreas and intestinal mucosa, which were evident
    in animals which had received the highest dose of  d-limonene
    (Evans  et al., 1987).

    2.3  Observations in humans

         Numerous prospective and retrospective cohort epidemiological
    studies have been done on populations of refinery, chemical plant,
    and service station workers, exposed to mixtures of chemicals that
    have been associated with male rat alpha2u-globulin nephropathy
    (Hanis  et al., 1979; Schottenfeld  et al., 1981; Hanis  et al.,
    1982; Thomas  et al., 1982; Austin & Schnatter, 1983; Higginson
     et al., 1984; Raabe, 1984; Wen  et al., 1984; Wong & Raabe, 1989;
    Page & Mehlman, 1989). No statistically significant evidence was
    found in any study to link incidence of cancer of the kidney to
    work-place exposure to mixtures of complex hydrocarbons.

         Igimi  et al. (1976) reported that  d-limonene was a safe and
    effective gallstone solubilizer in animals and humans; side effects
    in humans were reported to be pain and tenderness radiating from the
    upper abdomen to the anterior chest, nausea and vomiting, and
    diarrhoea.

    3.  COMMENTS

          d-Limonene has been shown to reduce body weights
    significantly in male and female mice and rats and in female
    rabbits. The NOEL for this effect was 150 mg of  d-limonene kg
    bw/day (administered by gavage) in a 2-year study in male rats.

         Oral administration of  d-limonene (400 mg/kg bw/day) to rats
    for 20 days slightly increased liver weight and phospholipid
    content, decreased liver and serum cholesterol levels, increased the
    concentration of cytochromes P450 and B5, and increased the
    activities of aminopyrine demethylase and aniline hydroxylase.
    Although liver lesions were not associated with the administration
    of  d-limonene in a 2-year study in rats (doses up to 150 mg/kg
    bw/day for males; doses up to 600 mg/kg bw/day for females), a daily
    gavage dose of 500 mg  d-limonene/kg bw/day for 2 years was
    associated with an increased incidence of multinucleated liver
    hepatocytes and cytomegaly in male mice. The NOEL for these effects
    was 250 mg/kg bw/day administered by gavage to male mice for 2
    years.

         Dermal application of  d-limonene has been reported to cause
    irritation and immune-mediated skin reactions in a number of
    species, including humans. Oral administration of  d-limonene to
    mice was reported to have immunological effects, including
    suppression of the  in vitro mitogenic response by mouse T-cells
    and B-cells and both suppression and stimulation of antigenic
    responses. However, the biological significance of these  in vitro
    changes is not known.

         Results of teratogenicity studies in mice suggest that maternal
    consumption of 2400 mg of  d-limonene/kg bw/day but not of
    600 mg/kg bw/day, affected the development of fetuses (decreased
    body weight gain, increased incidences of lumbar rib and fused rib,
    and delayed ossification). However,  d-limonene was reported to be
    non-teratogenic in rabbits at doses ranging from 250 to 1000 mg/kg
    bw/day (although the highest dose significantly reduced survival of
    the dams).

          d-Limonene consistently produced negative results in
    genotoxicity studies, has been reported to inhibit the activity of
    other mutagens and carcinogens, and gave mixed responses in
    tumour-promotion studies.

         Data clearly demonstrate that  d-limonene exacerbates the
    spontaneously occurring nephropathy in mature male rats which, after
    prolonged exposure, is associated with an increase in the incidence
    of renal tumours that are not commonly observed in control male
    rats. However,  d-limonene is not the only substance that has this

    effect: various hydrocarbon mixtures and several other chemicals
    have also been reported to exacerbate spontaneous nephropathy and
    cause renal tumours in male rats. Although, in studies designed to
    elucidate the mechanism of this carcinogenic process, chemicals
    other than  d-limonene were generally used as test substances, such
    studies have shown that the following mechanism is likely to be
    applicable to  d-limonene-induced nephropathy and renal tumours in
    male rats: (1)  d-limonene and  d-limonene-1,2-oxide (a metabolite
    of  d-limonene) bind reversibly to alpha2u-globulin in the
    kidneys of  d-limonene-treated male rats; (2) this binding further
    slows down the normally slow processing of reabsorbed
    alpha2u-globulin; and (3) as a result, alpha2u-globulin
    accumulates in the phagolysosomes (hyaline droplets) of epithelial
    cells of the proximal convoluted tubules of the kidney, leading to
    necrosis, accumulation of cell debris, and regenerative hyperplasia.
    The postulated mechanism of action for some non-genotoxic
    carcinogens suggests that regenerative hyperplasia may increase
    clonal expansion of spontaneously initiated cells and lead to the
    development of the renal tumours observed.

         A NOEL for the  d-limonene-associated increase in
    alpha2u-globulin levels in male rat kidney has not been
    identified, but the lowest-observed-effect level from a 21-day
    gavage study of  d-limonene in male rats was 75 mg/kg bw/day. A
    sex-specific protein with molecular features similar to
    alpha2u-globulin has been identified in human urine, but the
    concentration is at least four orders of magnitude less than that of
    alpha2u-globulin in male rats.

         The Committee concluded that the postulated mechanism for
     d-limonene-induced nephropathy and renal tumours in the male rat
    was probably not relevant to humans, and that toxic end points
    associated with this effect were not appropriate bases for the
    derivation of an ADI for  d-limonene.

    4.  EVALUATION

         Based on the significant decreases in body weight gain
    associated with administration of  d-limonene to male and female
    mice and rats and female rabbits, an ADI of 0-1.5 mg/kg bw was
    established for this substance. The Committee considered the known
    natural occurrence and food additive uses of  d-limonene, and
    concluded that only a small proportion of total intake is likely to
    be derived from direct additive use. The Committee therefore
    recommended that food additive intake be restricted to 75 g/kg
    bw/day, which represents 5% of the maximum ADI for  d-limonene.

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    See Also:
       Toxicological Abbreviations
       Limonene (CICADS 5, 1998)