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IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. Lyon (FR): International Agency for Research on Cancer; 1993. (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, No. 56.)

Cover of Some Naturally Occurring Substances

Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins.

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d-LIMONENE

1. Exposure Data

1.1. Chemical and physical data

Limonene is, with the possible exception of α-pinene, the most frequently occurring natural monoterpene. It is a major constituent of the oils of citrus fruit peel and is found at lower levels in many fruits and vegetables. It occurs naturally in the d (or R)- and l (or S) optically active forms and as dl mixtures including the optically inactive racemate (dipentene). For example, the d form comprises 98–100% of the limonene in most citrus oils (family Rustaceae), whereas that in oil of citronella and oil of lemongrass (family Gramineae) is 96–100% l-limonene (Furia & Bellanca, 1975; Clayton & Clayton, 1981; Sax & Lewis, 1987; Mosandl et al., 1990).

1.1.1. Synonyms, structural and molecular formulae

Chem. Abstr. Serv. Reg. No.: 5989-27-5

Deleted CAS Reg. Nos.: 7705-13-7; 95327-98-3

Chem. Abstr. Name: (R)-1-Methyl-4-(1-methylethenyl)cyclohexene

Synonyms: Cajaputene; carvene; cinene; ( + )-dipentene; d-( + )-limonene; D-( + )-limonene; ( + )-limonene; (R)-limonene; (R)-( + )-limonene; ( + )- para-mentha-1,8-diene; (R)-( + )-para-mentha-1,8-diene; 1-methyl-4-isopropenyl cyclohexene-1; Refchole

Image 006a-4_001

1.1.2. Chemical and physical properties

1.1.3. Trade names, technical products and impurities

d-Limonene is available commercially in an untreated technical grade (purity, 95%) as a clear liquid, which is variably colourless to yellow cast with a strong citrus odour; as a food grade (purity, 97%), a clear water-white liquid with a mild orange odour; and as a lemon-lime grade (purity, 70%), a clear water-white liquid with a lemon-lime odour (Florida Chemical Co., 1991a,b,c).

1.1.4. Analysis

Bertsch et al. (1974) described an analytical method in which trace quantities of organic materials, including limonene, in air are adsorbed on a porous polymer and separated by capillary gas chromatography (GC).

d-Limonene has been measured in a range of natural products, such as orange juice, by GC and head-space analysis (Massaldi & King, 1974; Marsili, 1986) and in packaging materials by thermal desorption (Lloyd, 1984). Searle (1989) described a procedure for monitoring airborne limonene vapour by GC (detection limit, 5 µg). GC with flame ionization detection has been used to analyse carrot volatiles collected on porous polymer traps. Samples were ground (blending), sliced or grated, and volatiles were collected on the polymer traps and eluted for analysis (Simon et al., 1980).

Oil recoverable by distillation from orange, tangerine and grapefruit juices is at least 98% d-limonene. The d-limonene content of such oils has been determined by co-distillation with isopropanol, acidification and titration with potassium bromide-potassium bromate solution (Boland, 1984). The distribution of optical isomers of limonene has been determined in various essential oils using multidimensional GC, by coupling chiral and nonchiral columns (Mosandl et al., 1990).

1.2. Production and use

1.2.1. Production

d-Limonene was first recovered as a commercial product during the 1941–42 Florida (USA) citrus season, from the steam evaporater condensate in the production of citrus molasses. By 1946, commercial production in Florida was common (Schulz, 1972).

The principal sources of d-limonene are the oils of orange, grapefruit and lemon (Verghese, 1968). It is the main volatile constituent of citrus peel oil, and the collected volatile portion of oil is usually referred to as d-limonene in the trade (Gerow, 1974). d-Limonene may be obtained by steam distillation of citrus peels and pulp resulting from the production of juice and cold-pressed oils or from deterpenation of citrus oils. It is sometimes redistilled Furia & Bellanca, 1975).

Citrus peel oil can contain up to 95% d-limonene and stripper oil over that amount. Stripper oil is the oil recovered during concentration of the liquor which separates from the peel during pressing. The press liquor is concentrated to give citrus molasses, and the vapour which separates during concentration is condensed to yield stripper oil. In commercial practice, the essential peel oil is extracted by mechanical rupturing of oil sacs in the subepidermal layer (flavedo) of the peel and expression of the oil as an aqueous emulsion, from which it is separated by centrifuging (Ranganna et al., 1983).

d-Limonene also occurs in other oils and essences obtained during the processing of citrus juice, including: juice oil, deoiler oil (oil separated from juice by centrifuging or decantation), essence oil (oil obtained from the recovery unit during concentration of fruit juices) and aroma oil (oil obtained by distillation of the aqueous discharge from the centrifuge used to separate pressed oil) (Ranganna et al., 1983).

Annual worldwide production of d-limonene and orange oil/essence oil (95% d-limonene) has recently been approximately 45 000 tonnes. Citrus plantings under way in southern Florida, Brazil, Venezuela, Mexico, the Caribbean basin and elsewhere are expected to increase that figure to 73 000 tonnes annually within a decade (Florida Chemical Co., 1991a). The production of d-limonene in Florida in 1989 and 1990 was estimated to be 8600 and 6800–7700 tonnes, respectively (Anon., 1989). Limonene production in Florida in 1971 was estimated at approximately 4500 tonnes; an additional 450 tonnes of terpenes were recovered from ‘folding’ cold pressed oils (Schulz, 1972). In 1990, 450 tonnes of d-limonene were produced in California (USA), and Brazilian production was estimated to have been between 4100 and 8200 tonnes. Brazilian d-limonene is used almost exclusively by resin producers (Topfer, 1990).

In 1990, Brazil was the largest producer of orange oil, while Florida led in production of tangerine and grapefruit oil (Anon., 1990); Mexico supplies ≥ 80% of the world’s lime oil (Anon., 1988a). In 1979, Italian production of essential oil from citrus fruit was as follows: oranges, 380 tonnes; lemon, 550 tonnes; bergamot, 100 tonnes; and mandarine, 30 tonnes (Anon., 1981).

Table 1 shows US imports of four citrus oils in 1981–90 and the major sources of the oils. In 1985–87, average US exports of orange oil were 1500 tonnes per year (Anon., 1988b). In 1983, Japan imported 200 tonnes of lemon oil (Anon., 1984).

1.2.2. Use

For nearly 50 years, d-limonene and orange oil/essence oil (95% d-limonene) have been used widely as flavour and fragrance additives in perfume, soap, food and beverages, d-Limonene has been used in non-alcoholic beverages, ice cream and ices, sweets, baked goods, gelatins and puddings, and chewing gum. It is also used as a chemical intermediate in the production of l-carvone, in terpene resin manufacture as a wetting and dispersing agent and in the preparation of sulfurized terpene lubricating oil additives. d-Limonene is also an important organic monomer in the synthesis of tackifying resins for adhesives. It has been used as a solvent, cleaner and odour in, e.g., the petroleum industry (Schulz, 1972; Furia & Bellanca, 1975; Sax & Lewis, 1987; Florida Chemical Co., 1991a,b).

Table 1. US imports of citrus oils.

Table 1

US imports of citrus oils.

Because d-limonene is a natural product with low toxicity for mammals and high acute toxicity for bark beetles, fruit flies and cat fleas, it has been proposed as an alternative to synthetic insecticides. Karr and Coats (1988) found that d-limonene had limited insecticidal properties against German cockroaches, house flies, rice weevils and corn rootworms. It has been used in shampoos and sprays for the control of fleas on dogs and cats; one such product reportedly contained 78.2% d-limonene (Hooser et al., 1986; Hooser, 1990).

d-Limonene has been used to dissolve retained cholesterol gallstones postoperatively (Igimi et al., 1991).

1.3. Occurrence

1.3.1. Foods and botanical species

Limonene is widely distributed among citrus and other plant species. It has been reported in more than 300 essential oils, at concentrations up to 90–95%, and at lesser, although still appreciable, concentrations in foods (e.g., 800 mg/1 in non-alcoholic beverages, 3000 mg/kg in chewing gum) (Flavor and Extract Manufacturers’ Association, 1991) (Table 2). Botanical species in which limonene occurs and which are used in pharmaceutical and para-pharmaceutical products, cosmetics, foods and beverages are presented in Table 3.

Table 2. Some food products containing d-limonene.

Table 2

Some food products containing d-limonene.

Table 3. Occurrence of limonene in various botanical species.

Table 3

Occurrence of limonene in various botanical species.

Frozen reconstituted orange juice samples contained 219 ppm (mg/1) limonene [isomer unspecified]. Oxidation did not occur to a significant extent over four weeks of storage in glass (Marsili, 1986). When citrus juices are packed aseptically into laminated cartons, the d-limonene content of the juice is reduced by about 25% within 14 days’ storage owing to absorption by the polyethylene (Mannheim et al., 1987). Limonene has been found in packaging material at 25 ppm (µg/g) (Lloyd, 1984).

Limonene [isomers unspecified] was found to represent 32.4% of total terpenes in Pinus greggii and 0.5% in P. pringlei, two pine species indigenous to Mexico (Lockhart, 1990).

Daily US per-caput consumption of d-limonene, as a result both of its natural occurrence in food and of its presence as a flavour, was estimated to be 0.27 mg/kg bw per day for a 60-kg individual (Flavor and Extract Manufacturers’ Association, 1991). Intake of d-limonene can vary considerably, however, depending on the types of food consumed. Citrus juice products are among the richest sources of d-limonene: intake owing to consumption of these products may approach 1 mg/kg bw per day for adults and 2 mg/kg bw per day for young children (US Department of Agriculture, 1982).

Annual US consumption of d-limonene from a variety of foods was calculated as follows: carrots, 5879 kg; celery leaves, 165 379 kg; heated chicken, 3.5 kg; roasted coffee, 1896 kg; cranberries, 3.0 kg; muscat grapes, 1.2 kg; grapefruit juice, 254 320 kg; lemon oil, 465 750 kg; mango, 736 kg; nutmeg, 17 250 kg; orange juice, 154 560 kg; oregano, 508 kg; peaches, 209 kg); pepper, 234 312 kg; raspberries, 2.5 kg; and green tea, 184 kg. Total annual US consumption of d-limonene as a result of its natural occurrence in these foods was 1300 tonnes. Asurvey in 1982 indicated that annual US consumption of d-limonene as a flavouring additive was 68 tonnes (Stofberg & Grundschober, 1987).

1.3.2. Air

d-Limonene was detected in the air of 81% of the mobile homes surveyed in Texas (USA) during a survey of air quality. Levels of d-limonene analysed by GC-mass spectrometry were 0.01–29 ppb [0.06–162 µg/m3], with a mean of 2.2 ppb [13 µg/m3] (Connor et al., 1985). Limonene [isomer unspecified] was detected at 0–5.7 ppb [32 µg/m3] in air samples taken at various locations around Houston, Texas. It was present in all of the more than 150 samples analysed by GC-mass spectrometry over a 15-month period (Bertsch et al., 1974).

1.3.3. Biological fluids

Limonene [isomer unspecified] has been detected in human urine (Zlatkis & Liebich, 1971), as have its metabolites (Kodama et al., 1974).

1.4. Regulations and guidelines

d-Limonene is generally recognized as safe for human consumption as a synthetic flavouring substance by the US Food and Drug Administration (1991).

2. Studies of Cancer in Humans

No data were available to the Working Group.

3. Studies of Cancer in Experimental Animals

3.1. Oral administration

3.1.1. Mouse

Groups of 50 male and 50 female B6C3F1 mice, eight to nine weeks of age, received 0, 250 or 500 (males) and 0,500 or 1000 (females) mg/kg bw d-limonene ( > 99% pure) in corn oil by gavage on five days a week for 103 weeks. The experiment was terminated after 105 weeks. No significant increase in the incidence of neoplasms was observed. The incidence of neoplasms (adenomas and carcinomas combined) of the anterior pituitary was lower in high-dose females than in controls (2/48 versus 12/49) (US National Toxicology Program, 1990).

3.1.2. Rat

Groups of 50 male and 50 female Fischer 344/N rats, seven to eight weeks of age, received 0, 75 or 150 (males) and 0, 300 or 600 (females) mg/kg bw d-limonene (> 99% pure) in corn oil by gavage on five days a week for 103 weeks. The experiment was terminated after 105 weeks. In males, treatment-related increases were observed in the incidences of renal tubular hyperplasia (vehicle control, 0/50; low-dose, 4/50; high-dose, 7/50), renal tubular-cell adenoma (vehicle control, 0/50; low-dose, 4/50; high-dose, 8/50; p > 0.01, trend test) and renal tubular-cell adenocarcinoma (vehicle control, 0/50; low-dose, 4/50; high-dose, 3/50). The incidence of lesions of the kidney was not increased in female rats (US National Toxicology Program, 1990).

3.2. Intraperitoneal administration

Mouse

In a screening assay based on the accelerated induction of lung tumours in a strain highly susceptible to development of this neoplasm, groups of 15 male and 15 female A/He mice, six to eight weeks old, received intraperitoneal injections of 0.2 g/kg bw or 1 g/kg bw (maximal tolerated dose) d-limonene in tricaprylin [purity 85–99%] three times per week for eight weeks (total doses, 4.8 and 24 g/kg bw). Vehicle control groups of 80 males and 80 females received intraperitoneal injections of 0.1 ml tricaprylin on the same schedule. The experiment was terminated 24 weeks after the first injection, and the lungs were removed and surface nodules counted. Survival was comparable between the groups. Lung tumour incidence was not increased; males—control, 22/77 (28%), low-dose, 1/15 (7%); and high-dose, 3/15 (20%); females-control, 15/77 (20%); low-dose, 2/15 (13%); and high-dose, 2/13 (15%) (Stoner et al., 1973).

3.3. Administration with known carcinogens

3.3.1. Oral administration

(a) Mouse

Groups of 20–30 [exact number unspecified] male and female (both sexes being represented almost equally) stock albino mice [strain unspecified], < 6–8 weeks of age, received a single dose of 50 ug benzo[ a]pyrene in 0.2 ml polyethylene glycol or polyethylene glycol alone by stomach tube and no further treatment, or they subsequently received 40 weekly intubations of 0.05 ml d-limonene [concentration unspecified] contaminated with < 0.1% para-cymene . A further group of 20–30 male and female mice served as untreated controls. In the group that received benzo[ a]pyrene plus d-limonene, 5/23 mice that survived > 60 days and were autopsied within 24 h after death had a total of eight forestomach papillomas; in the group that received benzo[ a]pyrene alone, 2/17 mice had a total of two forestomach papillomas; and in the group that received polyethylene glycol and d-limonene, 2/15 mice had a total of three forestomach papillomas. None of the 18 mice that survived more than 60 days in the untreated control group had a forestomach tumour (Field & Roe, 1965). [The Working Group noted the limited reporting.]

Two groups of 15 female A/J mice, nine weeks of age, were administered 0.2 mmol [27.3 mg] d-limonene (99% pure) in 0.2 ml cottonseed oil or cottonseed oil alone by oral gavage once a week for eight weeks; 1 h later they received a gavage of 20 mg/kg bw N-nitrosodiethylamine (NDEA) in 0.2 ml water. Mice were autopsied 26 weeks after the first dose of NDEA, but only forestomachs and lungs were examined for tumours. All of the animals that received cottonseed oil plus NDEA had forestomach papillomas; 11/15 (73%) had more than 30 papillomas/stomach, and 4/15 (27%) also had carcinomas of the fore-stomach. In addition, the average number of pulmonary adenomas/mouse in this group was 10.4. Of the animals that received d-limonene prior to NDEA, only 10/15 (67%) had stomach papillomas ( p > 0.05, two-sided U-test of Wilcoxon, Mann and Whitney); no mouse had more than 30 papillomas/stomach ( p < 0.001), and none was shown to have a carcinoma of the forestomach ( p < 0.05). The average number of pulmonary adenomas/mouse in this group was 6.5 ( p < 0.05) (all p values are versus vehicle controls) (Wattenberg et al., 1989). [The Working Group noted the limited number of tissues examined.]

Two groups of female A/J mice, nine weeks of age, received oral intubations of 0.5 mg/mouse 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK), in 0.1 ml tricaprylin twice a week for eight weeks. One hour prior to each administration of NNK, one group of 15 mice each received 25 mg d-limonene (99% pure) in 0.2 ml cottonseed oil by oral gavage and one group of 20 mice received cottonseed oil alone. Mice were autopsied 28 weeks after the initial administration of NNK, but only stomachs and lungs were examined for tumours. Of the animals that received cottonseed oil plus NNK, 90% (18/20) had forestomach papillomas, with an average of 2.8 papillomas/mouse; one mouse also had a stomach carcinoma. In the group that received d-limonene plus NNK, no forestomach papilloma or carcinoma was observed ( p > 0.001). Mice that received cottonseed vehicle plus NNK had 50.8 ± 2.6 (standard error) pulmonary adenomas/mouse, while those that received d-limonene plus NNK had only 15.3 ±2.1 pulmonary adenomas/mouse ( p<0.001) (Wattenberg & Coccia, 1991). [The Working Group noted the limited number of tissues examined.]

Two groups of female A/J mice, nine weeks of age, each received an intraperitoneal injection of 2 mg NNK in 0.1 ml saline. One hour prior to the injection of NNK, one group of 14 mice each received 25 mg d-limonene (99% pure) in 0.2 ml cottonseed oil by oral gavage and one group of 15 mice received cottonseed oil alone. Mice were autopsied 28 weeks after the initial injection of NNK, but only stomachs and lungs were examined. Neither group of mice developed stomach papillomas, but mice that received cottonseed oil plus NNK had an average of 11.2 ± 1.1 pulmonary adenomas/mouse, while those that received d-limonene plus NNK had an average of 2.5 ± 0.7 pulmonary adenomas/mouse ( p < 0.001) (Wattenberg & Coccia, 1991). [The Working Group noted the limited number of tissues examined.]

(b) Rat

Groups of 25 female Sprague-Dawley rats, 47 days old, were fed diets containing 0,1000 or 10 000 mg/kg d-limonene ( > 99% pure) one week prior to a single oral administration by gavage of 65 mg/kg bw 7,12-dimethylbenz[ a]anthracene (DMBA) in sesame oil [volume unspecified]. The diets were continued for a further 27 weeks, during which time the animals were weighed weekly and palpated for tumours. The experiment was terminated 27 weeks after DMBA administration. In animals fed d-limonene, the time to development of the first mammary tumour was reported to have been longer than that in controls (Elegbede et al., 1984). [The Working Group noted that the number of tumour-bearing animals was not given.]

Groups of 30 six-week-old female Sprague-Dawley rats were fed diets containing 5% d-limonene [purity unspecified] for one week before administration by gastric intubation of a single dose of 65 mg/kg bw DMB A. The d-limonene diet was continued for a further week, after which time animals were returned to basal diet. A second group of 30 females received DMBA by gastric intubation, followed one week later by administration of 5% d-limonene in the diet for 25 weeks. A third group of 30 females received DMBA and was maintained on basal diet, thus serving as controls. The experiment was terminated 25 weeks after the DMBA treatment. Tumours larger than 350 mm3 were surgically resected, and all resected tumours and those found at necropsy were examined histologically. More than 95% of the mammary tumours were carcinomas. In animals that received d-limonene one week prior to and one week after DMBA, tumour latency was significantly increased ( p < 0.005); no effect on latency was seen in animals fed d-limonene one week after DMBA for 25 weeks. The number of tumours per rat was reduced in both groups fed d-limonene ( p<0.05) (Elson et al., 1988). [The Working Group noted the lack of detailed reporting.]

In a study reported as a short communication, groups of female Wistar Furth rats [number per group unspecified], 64–69 days old, were fed diets containing 5% d-limonene [purity unspecified], 5% orange oil [percentage of d-limonene unspecified] or basal diet throughout the study. After two weeks, the animals received a single intravenous injection of 50 mg/kg bw N-methyl- N-nitrosourea (MNU). All animals survived to the end of the experiment at 23 weeks. Feeding of both orange oil and d-limonene decreased tumour incidence ( p > 0.001): At the end of the experiment, 80% of controls had mammary carcinomas, while the incidence in orange oil-treated animals was 47% and that in d-limo-nene-treated animals was 45% (Maltzman et al., 1989). [The Working Group noted the lack of detailed reporting.]

In a study reported in a brief communication, groups of female Wistar Furth rats [numbers unspecified], 64–69 days old, were fed diets containing 5% d-limonene [purity unspecified] or basal diet. After two weeks, the animals received a single intravenous injection of 50 mg/kg bw MNU, and the d-limonene diet was continued for a further week, after which time the rats were returned to basal diet. A further group of females [number unspecified] received a single intravenous injection of MNU and one week later were fed diets containing 5% d-limonene until the end of the experiment. All animals survived to the end of the experiment at 23 weeks. Administration of d-limonene two weeks before and one week after MNU treatment did not affect the incidence or number of mammary tumours. Rats given d-limonene from one week after MNU treatment until the end of the experiment had about half the average number of tumours per rat as MNU-treated controls (Maltzman et al., 1989). [The Working Group noted the lack of detailed reporting.]

Two groups, of 31 and 32 female Sprague-Dawley rats, six weeks old, were fed diets containing 0 and 1% (w/w) d-limonene (99.9% pure), respectively, for two weeks and then received 65 mg/kg bw DMBA ( < 95% pure) once by gastric intubation; the two groups were continued on their respective diets for 20 weeks. d-Limonene caused a significant reduction in the incidence of mammary tumours: 58 in rats fed d-limonene plus DMBA (1.8 tumours/rat; median latency, 84 days) and 81 in rats fed DMBA alone (2.6 tumours/rat; median latency, 70 days). Two further groups of 52 female Sprague-Dawley rats, six weeks old, were fed diets containing 0 or 0.5% (w/w) d-limonene (99.9% pure) for two weeks and then received 65 mg/kg bw DMBA once by gastric intubation; the diets were continued for a further week, after which time both groups were continued on basal diet until 20 weeks. Administration of d-limonene for two weeks prior to and one week after DMBA did not significantly reduce the incidence of mammary tumours: 156 in the d-limonene plus DMBA-treated rats (3.0 tumours/rat; median latency, 61 days) compared to 129 in rats fed DMBA alone (2.5 tumours/rat; median latency, 58 days) (Russin et al., 1989).

Groups of 31–38 male Fischer 344 and 31–37 male NBR rats, eight weeks of age, were given 0 or 0.05% (500 mg/1) N-nitrosoethylhydroxyethylamine (NEHEA) in the drinking-water for two weeks; they were then given tap-water and treated by oral gavage with 150 mg/kg bw d-limonene ( < 99% pure) in 3 ml/kg corn oil daily on five days a week for 30 weeks. The livers and kidneys were examined, and other tumours were noted grossly. In Fischer 344 rats, 9/31 treated with NEHEA and d-limonene had renal adenomas, compared with 1/30 treated with NEHEA alone and none of 31 rats given d-limonene alone and none of 31 untreated controls ( p<0.05). The numbers of atypical hyperplasias in the kidney were 15.5 ± 1.5/rat treated with NEHEA and d-limonene, 1.2 ± 0.2/rat treated with NEHEA alone, 0.4 ± 0.1/rat treated with d-limonene alone and none in untreated controls. NBR rats did not develop renal adenomas, and no difference in the number of atypical hyperplasias was seen after subsequent administration of d-limonene (0.2 ± 0.1/rat in both groups), after feeding of d-limonene alone (0.1 ± 0.0/rat) or after no treatment (0.1 ± 0.1/rat). In Fischer rats, but not in NBR rats, the number of liver tumorus was reduced by administration of d-limonene (Dietrich & Swenberg, 1991).

3.3.2. Skin application

The Working Group was aware of a series of studies on orange oil (which contains d-limonene) in which a promoting effect on mouse skin carcinogenesis initiated by DMBA was reported (Roe, 1959; Roe & Peirce, 1960). Because of limitations in the conduct and reporting of these studies, they were not reviewed.

Mouse

Groups of 50 female ICR/Ha Swiss mice, six to eight weeks of age, each received topical applications on shaved back skin three times a week for 440 days of 10 mg limonene [stereochemistry and purity unspecified] in 0.1 ml acetone, 10 mg limonene simultaneously with 5 µg benzo[ a]pyrene in 0.1 ml acetone, 5 µg benzo[ a]pyrene alone or 0.1 ml acetone alone. A group of 100 females served as untreated controls. Only tumours that persisted 30 days or more were counted in the cumulative totals. Animals that developed skin carcinomas were killed approximately two months after the tumours had been classified as malignant or when they were moribund. No skin tumour was seen at 440 days in untreated controls, vehicle controls or mice that received limonene alone. In the benzo[ a]pyrene-treated group, 16/50 mice [number of survivors not specified] had a total of 26 papillomas and 12 carcinomas (first papilloma seen at 210 days). Of mice that received limonene plus benzo[ a]pyrene, 13/50 [number of survivors unspecified] had a total of 13 papillomas and four carcinomas (first papilloma seen at 295 days). The authors concluded that limonene had slightly inhibited benzo[ a]pyrene carcinogenesis (Van Duuren & Goldschmidt, 1976). [The Working Group noted that no statistical analysis was performed and that this conclusion appears to be based on the lowered multiplicity of tumours and decreased number of malignant tumours in those given limonene.]

3.3.3. Topical application and feeding

Mouse

Groups of 24 female CD-1 mice, eight weeks of age, each received a single topical application of 0.2 µmol (51.2 µg) DMBA in 0.2 ml acetone or acetone alone on the shaved back. On day 7 after DMBA treatment, one DMBA-treated and one acetone-treated group were fed a diet containing 1% d-limonene ( > 99% pure). On day 14 after DMBA treatment, three other groups received topical applications of 0.2 ml d-limonene (630 mmol, 1:1) in acetone, 10 nmol 12- O-tetradecanoylphorbol 13-acetate (TPA) in acetone or acetone alone twice a week. Beginning seven weeks after DMBA treatment, the number of mice bearing papillomas and the number of papillomas were recorded weekly. The experiment was terminated 40 weeks after DMBA treatment. All mice that received DMBA plus TEA rapidly developed skin tumours, whereas mice that received TPA alone or DMBA alone did not. DMBA-treated mice fed 1% d-limonene in the diet did not develop skin tumours, nor did untreated mice that received an application of d-limonene alone on the skin. In DMBA-treated mice that received skin applications of d-limonene, the incidence of skin papillomas/ mouse was increased slightly (Elegbede et al., 1986). [The Working Group noted the lack of detailed reporting.]

3.3.4. Subcutaneous injection

Mouse

Three groups of 50 male C57B1/6 Jax mice [age unspecified] each received a subcutaneous injection of 25 µg benzo[ rst]pentaphene (dibenzo[ a,i]pyrene; DBP) in 0.1 ml tricaprylin and, 24 h later, a subcutaneous injection (at about the same site) of 0.2 ml 75% (v/v) orange oil (containing about 45–50% d-limonene) or 75% (v/v) orange oil (containing about 85–90% d-limonene) or no additional treatment. A vehicle-control group of 50 males received an initial subcutaneous injection of 0.1 tricaprylin only. Two further groups of 50 male C57B1/6 Jax mice [age unspecified] received a single subcutaneous injection of 0.2 ml 75% (v/v) orange oil (containing about 45–50% limonene) or 75% (v/v) orange oil (containing about 85–90%) with no DBP pretreatment. The animals were observed for up to two years. Neither tricaprylin alone nor either of the two orange oils alone caused subcutaneous tumours (survival at two years: tricaprylin, 33/50; 45% limonene, 26/50; 84% limonene, 30/50). Mice that received DBP alone had a higher incidence of subcutaneous tumours than did the group that also received orange oil containing 45–50% limonene, and the group that also received orange oil containing 85–90% limonene had an even lower incidence (Homburger et al., 1971).

Groups of 50 male C57B1/6 Jax mice [age unspecified] each received a subcutaneous injection of 25 µg DBP in 0.1 ml tricaprylin and, 24, 48, 72 and 96 h later, subcutaneous injections (at about the same site) of either 0.05 ml 75% (v/v) d-limonene in tricaprylin, 0.05 ml 75% (v/v) autoxidized d-limonene (containing 6% hydroperoxides) in tricaprylin, 0.05 ml hydroperoxides alone [75% solution in tricaprylin unspecified], 0.05 ml orange oil (containing 85–90% limonene; control 1), 0.05 ml tricaprylin alone (control 2) or no further treatment (positive control). Tumour growth was reported to be reduced by d-limonene and by the hydroperoxides of d-limonene (Homburger et al., 1971). [The Working Group noted the inadequate reporting.]

3.3.5. Intravenous and intraperitoneal injection

Mouse

Groups of 50 female A/Jax mice, two to three months old, each received a single subcutaneous injection of 500 µg DBP in 0.1 ml peanut oil or 0.1 ml peanut oil alone, followed after 24 h by weekly intravenous injections into the tail vein (later, intraperitoneal injections) of either 1% (v/v) orange oil (containing 45–50% limonene [stereoisomer, volume and diluent unspecified]), 1% (v/v) orange oil (containing 85–90% limonene) [volume and diluent unspecified] or 0.1 ml diluent. At the end of 13 weeks, the incidence of lung tumours was 21% in mice receiving peanut oil and diluent, 74% in mice receiving DBP plus diluent and 43 and 44% in mice receiving DBP plus either of the orange oils. In a second experiment, groups of 50 female A/Jax mice, seven weeks old, received a single subcutaneous injection of 500 µg/mouse DBP in 0.1 ml peanut oil or 0.1 ml peanut oil alone, followed after 24 h by weekly intravenous injections into the tail vein of either 1% (v/v) d-limonene, 1% (v/v) of its hydroperoxide or 0.1 ml diluent. At the end of 16 weeks, the incidences of lung adenomas were 27% in mice receiving peanut oil and diluent, 75% in mice receiving DBP and diluent, 40% in mice receiving DBP plus d-limonene and 50% in mice receiving DBP plus d-limonene hydroperoxide. The two orange oils and d-limonene hydroperoxide given alone without DBP pretreatment had no significant effect on the incidence of lung adenomas, whereas d-limonene alone reduced the incidence of lung adenomas from 27% (diluent controls) to 7% ( p < 0.05) (Homburger et al., 1971).

4. Other Relevant Data

4.1. Absorption, distribution, metabolism and excretion

4.1.1. Humans

d-Limonene is absorbed in the gastrointestinal tract. Two male volunteers administered 14C- d-limonene at 1.6 g orally excreted 55–83% of the dose in their urine within 48 h. The major urinary metabolite isolated was 8-hydroxy- para-menth-l-en-9-yl-β-D-glucopyranosi-duronic acid (M-VI, Fig. 1) (Kodama et al., 1976).

4.1.2. Experimental systems

14C- d-Limonene was absorbed rapidly following administration (800 mg/kg; 4.15 µCi/animal) by stomach tube to male Wistar rats. Radiolabel levels were maximal in the blood after 2 h; large amounts of radiolabel were also observed in the liver (maximal after 1 h) and the kidneys (maximal after 2 h). Negligible concentrations were found in blood and organs after 48 h (Igimi et al., 1974).

Figure 1

Figure 1

Possible metabolic pathways of d-limonene

From Kodama et al. (1976)

M-I, p-Mentha-l,8-dien-10-ol; M-II, p-menth-l-ene-8,9-diol; M-IV perillic acid-8,9-diol; M-V, p-mentha-l,8-dien-10-yl-β-D-glucopyranosiduronic acid; M-VI, 8-hydroxy- p-menth-l-en-9-yl-β-D-glucopyranosiduronic acid; M-VII, 2-hydroxy- p-menth-8-en-7-oic acid; M-VIII, perillylglycine; M-IX, perillyl-β-D-glucopyranosiduronic acid; M-X, p-mentha-l,8-dien-6-ol; M-XI, p-menth-l-ene-6,8,9-triol

Urinary recovery of 14C- d-limonene was 77–96% within three days in rats, guinea-pigs, hamsters and dogs. Faecal recovery was 2–9% within three days (Kodama et al, 1976). Bile-duct cannulated rats administered d-limonene orally excreted 25% of the dose in the bile within 24 h (Igimi et al., 1974) .

Following oral administration of d-limonene to rabbits, the urinary metabolites isolated were para-mentha-l,8-dien-10-ol (M-I), para-menth-l-ene-8,9-diol (M-II), perillic acid (M-III), perillic acid-8,9-diol (M-IV), para-mentha-l,8-dien-10-yl-β-D-glucopyranosi-duronic acid (M-V) and 8-hydroxy- para-menth-l-en-9-yl-β-D-glucopyranosiduronic acid (M-VI) (Kodama et al., 1974) (see Fig. 1). Following oral administration of d-limonene to dogs and rats, a further five urinary metabolites were isolated: 2-hydroxy- para-menth-8-en-7-oic acid (M-VII), perillylglycine (M-VIII), perillyl-β-D-glucopyranosiduronic acid (M-DC), para-mentha-l,8-dien-6-ol (M-X) and probably para-menth-l-ene-6,8,9-triol (M-XI). The major urinary metabolite was M-IV in rats and rabbits, M-IX in hamsters, M-II in dogs and M-VI in guinea-pigs (Kodama et al., 1976).

d-Limonene was metabolized by rat liver microsomes in vitro to the glycols d-limonene 8,9-diol and d-limonene 1,2-diol via the 8,9- and 1,2-epoxides (Watabe et al., 1980, 1981) (See Fig. 2).

Figure 2

Figure 2

Oxidation of d-limonene double bonds by rat liver microsomes

From Watabe et al. (1980)

4.2. Toxic effects

4.2.1. Humans

Five healthy male adult volunteers who received a single oral dose of 20 g d-limonene all developed transient proteinuria, non-bloody diarrhoea and tenesmus. The results of other functional tests of the liver, kidney and pancreas were normal (Igimi et al., 1976).

4.2.2. Experimental systems

LD50 values for d-limonene were reported in male and female mice to be 5.6 and 6.6 (oral), 1.3 and 1.3 (intraperitoneal) and > 41.5 and > 41.5 (subcutaneous) g/kg bw, respectively; those in male and female rats were reported to be 4.4 and 5.1 (oral), 3.6 and 4.5 (intraperitoneal), > 20.2 and > 20.2 (subcutaneous) and 0.125 and 0.11 (intravenous) g/kg bw, respectively (Tsuji et al., 1975a). The acute oral LD50 in rats and the acute dermal LD50 in rabbits were reported to exceed 5 g/kg bw (Opdyke, 1975).

After daily oral administration of d-limonene at 277–2770 mg/kg bw to male and female Sprague-Dawley rats for one month, the highest dose was found to have caused a slight decrease in body weight and food consumption. On histological examination, granular casts were observed in the kidney of males, but no significant change was found in the other organs (Tsuji et al., 1975a).

d-Limonene did not cause renal disease in NCI Black Reiter (NBR) male rats. These rats do not synthesize α2µ-globulin, which is normally present in the hyaline droplets that are found in male Fischer 344 rats with d-limonene-induced nephrotoxicity (Dietrich & Swenberg, 1991).

A dose-related increase in relative liver and kidney weights was observed in young adult male Fischer 344 rats administered 75,150 or 300 mg/kg bw d-limonene daily by gavage on five days per week and killed on study days 6 or 27. Dose-related formation of hyaline droplets was also observed in the kidneys. α2µ-Globulin was detected in larger amounts in the renal cortical tissue of animals treated with d-limonene than in controls. Alterations considered to be sequelae of the hyaline droplet response, including granular casts in the outer zone of the medulla and multiple cortical changes collectively classified as chronic nephrosis, were observed in the kidneys of all rats killed on day 27 (Kanerva et al., 1987).

Chronic oral administration of 75 or 150 mg d-limonene to male Fischer 344/N rats on five days per week for two years was associated with dose-related alterations to the kidney, such as increased incidences of mineralization and epithelial hyperplasia and increased severity of spontaneous nephropathy (US National Toxicology Program, 1990).

After single administration of 14, 41, 136 or 409 mg/kg bw d-limonene by gavage to male and female Sprague-Dawley rats, a dose-related accumulation of hyaline droplets was observed in proximal renal tubules only in male rats. After administration of 14C-labelled d-limonene at 409 mg/kg bw, 2.5 times more radiolabel was accumulated in renal tissue of males than in females; the label was found to be reversibly bound to protein (Lehman-McKeeman et al., 1989). In contrast, adult male and female beagle dogs administered d-limonene at 100 or 1000 mg/kg bw (maximal tolerated dose for emesis) per day by gavage twice daily for six months had increased kidney weights but showed no histopathological change, hyaline droplet accumulation or nephropathy (Webb et al., 1990).

In a chronic toxicity study in beagle dogs, oral doses of more than 340 mg/kg bw (females) and 1000 mg/kg bw (males) per day for six months resulted in protein casts in the renal tubules. Daily doses of more than 1000 mg/kg bw (females) and 3024 mg/kg bw (males) resulted in slight weight loss in some animals due to frequent vomiting (Tsuji et al., 1975b).

d-Limonene is a mildly toxic skin irritant when applied at full strength to intact or scratched rabbit skin for 24 h under occlusion (Opdyke, 1975). Continuous perfusion of 0.5% d-limonene (0.5–0.6 ml [420–504 mg]/min) into the gall-bladder of rabbits for 6 h irritated particularly the mucous membranes and the common bile duct (Tsuji et al., 1975c).

4.2.3. Mechanisms of toxicity

Treatment of male rats with d-limonene leads to a characteristic nephrotoxicity, a key feature of which is the accumulation in proximal tubule cells of hyaline droplets containing α2µ-globulin. α2µ-Globulin is the major low-molecular-weight protein excreted in male rat urine; it is present at much lower levels in females.

A number of studies have shown that d-limonene and d-limonene-l,2-oxide bind specifically, but reversibly, to α2µ-globulin; the binding of d-limonene-l,2-oxide resulted in reduced degradation of the protein by lysosomal proteinases in vitro (Lehman-McKeeman et al., 1990). Cell death and proliferation were enhanced in renal tubules of Fischer 344 rats treated with d-limonene; no enhancement of cell proliferation occurred in NBR rats, which do not synthesize α2µ-globulin (Dietrich & Swenberg, 1991).

In mature male rats, approximately 50 mg of α2µ-globulin are filtered per day, about 40% being excreted in urine and 60% being reabsorbed and catabolized (Neuhaus et al., 1981; Baetcke et al., 1991). Female rats excrete 100–300 times less α2µ-globulin in urine (Borghoff et al., 1990; Baetcke et al., 1991; Dietrich & Swenberg, 1991). Mice excrete large amounts of a structurally similar protein in a sex-dependent manner; however, this protein does not bind to d-limonene, nor is it reabsorbed in the kidney (Lehman-McKeeman & Caudill, 1992a).

α2µ-Globulin belongs to a superfamily of proteins called lipocalins, which are widely distributed among mammalian species and bind, stabilize and transport hydrophobic ligands such as retinol and steroid hormones (Lehman-McKeeman & Caudill, 1992b). Normal human urine contains very little of this class of proteins, although a sex-dependent protein (urine protein 1) has been identified, which occurs in male urine at concentrations five times higher than in female urine. The concentration in male human urine is four to five times lower than that of α2µ-globulin in male rat urine (Bernard et al., 1989; Baetcke et al., 1991). It is structurally related to rabbit uteroglobulin and not to the rat protein (Jackson et al., 1988). It does not bind to d-limonene. Of the lipocalins studied, only that in male rat kidney binds to limonene, whereas those of mice, hamsters, guinea-pigs, dogs and humans do not (Lehmann-McKeeman & Lehmann-McKeeman & Caudill, 1992b).

4.3. Reproductive and developmental toxicity

4.3.1. Humans

No data were available to the Working Group.

4.3.2. Experimental systems

Mice, rats and rabbits were treated orally during the period of organogenesis with daily doses of d-limonene up to 2363 mg/kg bw (mice), 2869 mg/kg bw (rats) and 1000 mg/kg bw (rabbits). The highest dose was lethal to<40% of pregnant rabbits, and several rat dams died. The studies consistently showed impaired weight gain in the dams and delayed prenatal development, which was restored to normal during the postnatal period. In mice and rabbits, anomalies of the ribs were observed in the offspring (Tsuiji et al., 1975d; Kodama et al., 1977a, b).

4.4. Genetic and related effects

4.4.1. Humans

No data were available to the Working Group.

4.4.2. Experimental systems (see also Table 4 and Appendices 1 and 2)

Table 4. Genetic and related effects of d-limonene and its metabolites.

Table 4

Genetic and related effects of d-limonene and its metabolites.

d-Limonene was not mutagenic to Salmonella typhimurium. In single studies, it did not induce differential toxicity in Bacillus subtilis strains, sister chromatid exchange or chromosomal aberrations in Chinese hamster ovary cells, trifluorothymidine resistance in mouse lymphoma L5178Y cells or transformation in rat tracheal cells in vitro. d-Limonene-1,2-oxide did not induce unscheduled DNA synthesis in primary cultures of rat hepatocytes.

5. Summary of Data Reported and Evaluation

5.1. Exposure data

d-Limonene is found widely in citrus and many other plant species and is a major constituent of many essential oils. It is used extensively as a component of flavourings and fragrances, as a chemical intermediate and as an insect repellant. Widespread exposures occur through consumption of fruits, vegetables and products containing essential oils. Consumption of d-limonene has been estimated to be 0.2-2 mg/kg bw per day.

5.2. Human carcinogenicity data

No data were available to the Working Group.

5.3. Animal carcinogenicity data

d-Limonene has been tested for carcinogenicity by oral gavage in one study in mice and one study in rats. In mice, no treatment-related tumour was observed. It significantly increased the combined incidence of renal-cell adenomas and carcinomas and induced renal tubular hyperplasia in male rats.

In a two-stage experiment, oral treatment with d-limonene after administration of N-nitrosoethylhydroxyethylamine enhanced the development of renal adenomas and renal tubular hyperplasia in male Fischer 344 rats, which synthesize α2µ-globulin, but not in male NBR rats, in which there is no evidence that α2µ-globulin is synthesized in measurable quantities.

5.4. Other relevant data

In men, oral intake of d-limonene induced transient proteinuria. d-Limonene induced nephrotoxicity in male Fischer 344 but not NBR rats.

No data were available on the genetic and related effects of d-limonene in humans. In a small number of studies with a variety of end-points, d-limonene showed no evidence of genotoxic activity.

5.5. Evaluation1

No data were available on the carcinogenicity of d-limonene to humans.

There is limited evidence in experimental animals for the carcinogenicity of d-limonene.

Overall evaluation

d-Limonene is not classifiable as to its carcinogenicity to humans (Group 3).

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Footnotes

1

For definition of the italicized terms, see Preamble, pp. 26–29.

©International Agency for Research on Cancer, 1993.
Bookshelf ID: NBK513608
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