* National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; and
Battelle Laboratories, Columbus, Ohio 43201
Received September 30, 2002; accepted October 31, 2002
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ABSTRACT |
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Key Words: food additives; fragrance additives; GRAS list; microencapsulation; toxicity; malignant lymphoma; vinyl aldehyde; rats; mice; nephropathy.
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INTRODUCTION |
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Citral was selected for carcinogenicity studies because of its widespread use as a flavoring and fragrance ingredient. Human exposure can be anticipated to occur primarily through the oral route, but also through contact with skin. Citral administered dermally has previously been shown in multiple species to be a sensitizing agent. Citral was found to be severely irritating to albino angora rabbits, male Hartley guinea pigs, and humans (Basketter and Scholes, 1992; Cardullo et al., 1989
; Motoyoshi et al., 1979
). It was positive in the local lymph node assay (Basketter and Scholes, 1992
). Citral also has been shown to induce benign and atypical prostatic hyperplasia in rats when applied dermally for one or more months (Engelstein et al., 1996
, Kessler et al., 1998
, Scolnik et al., 1994
; Servadio et al., 1986
).
Because the most widespread exposure to citral likely occurs from consumption of foods, administration through the diet was preferable. However, because citral volatilizes rapidly and binds to reactive moieties in the diet, traditional feeding studies were not possible (Kuhn et al., 1991). A microencapsulation technique was developed to allow for administration of citral in the diet with minimal loss (Kuhn et al., 1991
). A comparative study in F344/N rats and B6C3F1 mice exposed to citral for 14 days by oral gavage or through microencapsulated citral in the diet showed that microencapsulation was an acceptable alternative to gavage administration (Dieter et al., 1993
). Previous work also has shown that the bioavailability and toxicity of a similar compound, cinnamaldehyde, administered in microcapsules was not altered compared to corn oil gavage (Hébert et al., 1994
; Yuan et al., 1993
).
The present studies were performed to characterize the toxicity of citral when administered in the diet to F344/N rats and B6C3F1 mice. The details of these studies have been reported in a Technical Report (NTP, 2001). Major findings from these studies are presented here.
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MATERIALS AND METHODS |
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Microcapsule formulation and analyses.
Citral was microencapsulated by Midwest Research Institute (MRI, Kansas City, MO). Microcapsules loaded with neat citral and placebos (empty microcapsules) were prepared in several batches by a proprietary process using food-grade sugar and starch to produce dry microspheres. The batches were homogenized and passed through 40- over 140-mesh sieves and were stored in amber glass bottles at room temperature before shipping to the testing laboratory, Battelle (Columbus, OH). The citral load of the microcapsules, determined by HPLC, was 32.9%. The microcapsules were stored in amber glass bottles, protected from light, at approximately 5°C during the studies. The stability of the microcapsules was monitored during the 14-week and two-year studies; no loss of citral from the microcapsules was detected.
Preparation and analysis of dose formulations.
The dose formulations were prepared with NTP-2000 feed (Zeigler Brothers, Inc., Gardners, PA) every two to four weeks during the 14-week studies and approximately every four weeks during the two-year studies. Dose formulations were analyzed at three timepoints (14-week studies) and every 9 to 12 weeks (two-year studies) and were within 10% of the target concentrations.
Fourteen-week studies.
Four-week-old male and female F344/N rats and B6C3F1 mice were obtained from Taconic Laboratory Animals and Services (Germantown, NY) and were quarantined for 11 to 15 days. Animals were approximately six weeks old on the first day of the studies. Before the studies began, five male and five female rats and mice were randomly selected for parasite evaluation and gross observation for evidence of disease. All tests for viral titers from rats and mice were negative.
Groups of 20 male and female rats and 10 male and female mice were fed the following diets: control (without microcapsules), placebo control (with empty microcapsules), 3900, 7800, 15,600, or 31,300 mg microencapsulated citral/kg diet (ppm) for 14 weeks. Placebo and/or loaded microcapsules were combined with feed to a concentration of 10% microcapsules. Feed and water were available ad libitum. Rats and female mice were housed five per cage, and male mice were housed individually. Clinical findings were recorded weekly for rats and mice. Feed consumption was recorded twice weekly or once weekly (male mice). The animals were weighed initially, weekly thereafter, and at the end of the studies.
Blood was collected from the retroorbital sinus of 10 designated rats from each group under carbon dioxide anesthesia on days 4 and 22 for hematology and clinical pathology and then euthanized with CO2. Using the same method, blood was collected from all core study rats and mice surviving to the end of the studies for hematology (both species) and clinical chemistry (rats) analyses. Blood samples for hematology analyses were placed in microcollection tubes containing potassium EDTA. Erythrocyte, platelet, leukocyte counts, hematocrit values, hemoglobin concentration, mean cell volume, mean cell hemoglobin, and mean cell hemoglobin concentration were determined using a Serono-Baker System 9000 hematology analyzer (Serono-Baker Diagnostics, Allentown, PA) with reagents supplied by the manufacturer. Differential leukocyte counts and erythrocyte and platelet morphologies were determined microscopically from blood smears stained with a modified Wright-Giemsa stain on a Hema-Tek® slide stainer (Miles Laboratory, Ames Division, Elkhart, IN). A Miller disc was used to determine reticulocyte counts from smears prepared with blood stained with new methylene blue. For clinical chemistry analyses, blood samples from rats were placed into microcollection serum separator tubes and centrifuged. The serum samples were analyzed using a Hitachi 704® chemistry analyzer (Boehringer Mannheim, Indianapolis, IN) using commercially available reagents. Clinical chemistry endpoints included: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids.
Necropsies were performed on all core study animals. The heart, right kidney, liver, lung, right testis, and thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 µm, and stained with hematoxylin and eosin. A complete histopathologic examination was performed on all core study untreated control and vehicle control rats and mice, 15,600 ppm rats, and 31,300 ppm rats and mice.
Two-year studies.
Groups of 50 male and 50 female rats and mice were fed the following diets: control (without microcapsules), placebo control (with microcapsules), 500 (mice only), 1000, 2000, or 4000 (rats only) ppm microencapsulated citral for up to two years. Placebo and/or loaded microcapsules were combined with feed to a concentration of 1.25% microcapsules. Male rats were housed three per cage, female rats and mice were housed five per cage, and male mice were housed individually. Feed and water were available ad libitum. Feed consumption was measured over a one-week period approximately every four weeks by cage. Cages were changed once (male mice) or twice weekly; cages and racks were rotated every two weeks.
All animals were observed twice daily for moribundity and mortality. Clinical findings and body weights were recorded initially (body weights only), on week 2, week 6, every four weeks thereafter, and at the end of the studies. Complete necropsies and microscopic examinations were performed on all rats and mice. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 µm, and stained with hematoxylin and eosin for microscopic examination.
All rodent studies were conducted at Battelle Columbus Laboratories, Columbus, Ohio, accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC, Rockville, MD); Institutional Animal Use and Care Committees approved the experimental protocols. Animal use was in accordance with the United States Public Health Service policy on humane care and use of laboratory animals and the Guide for the Care and Use of Laboratory Animals. Developmental chemistry efforts were conducted at Midwest Research Institute, Kansas City, MO.
Statistical methods.
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related effects on survival used Coxs (1972)
method for testing two groups for equality and Tarones (1975)
life table test to identify dose-related trends. Organ and body weight data were analyzed with the parametric multiple comparison procedures of Dunnett (1955)
and Williams (1971
, 1972)
. Hematology and clinical chemistry data were analyzed using the nonparametric multiple comparison methods of Shirley (1977)
and Dunn (1964)
. Extreme values were identified by the outlier test of Dixon and Massey (1951)
. Average severity values were analyzed for significance with the Mann-Whitney U-test (Hollander and Wolfe, 1973
). The Poly-k test (Bailer and Portier, 1988
; Portier and Bailer, 1989
) was used to assess neoplasm and nonneoplastic lesion prevalence.
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RESULTS |
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No gross lesions were observed that could be attributed to citral exposure. Microscopically, exposure of rats to citral was associated with forestomach epithelial hyperplasia and hyperkeratosis (Table 2). Characterized by minimal to mild thickening of the stratified squamous epithelium and of the cornified superficial layer of the mucosa, forestomach epithelial hyperplasia and hyperkeratosis were observed in several 31,300 ppm males and females, with a greater incidence in females.
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The incidences of bone marrow atrophy were significantly increased in 15,600 and 31,300 ppm males and females (Table 2). In the groups receiving 31,300 ppm, atrophy was of mild severity and was characterized by decreased myelopoietic cells with a relative increase in the adipose cells in the marrow spaces. Hemorrhage was also present in all males and nine females exposed to 31,300 ppm and was attributed to loss of vascular sinus integrity and extravasation of erythrocytes throughout the marrow spaces. Minimal atrophy, without accompanying hemorrhage, was considered a borderline lesion in the 15,600 ppm groups. It was not clear if the bone marrow lesions were a direct effect of citral toxicity or were due to inanition, but it was probable that inanition contributed to the lesions.
Also in the 31,300 ppm group, thymic atrophy was observed in males and females (data not shown). Aspermia was observed in the testes of all 31,300 males (data not shown). These lesions only occurred in the highest exposure group and were likely related to inanition and the moribund condition of the animals.
Because of an apparent early palatability problem that resulted in a reduction in body weight gain in males exposed to 7800 ppm and the fact that they never recovered from this initial effect, 4000 ppm was selected as the high dose for the two-year study. For females, the reduction in body weight gain at the end of the study was approximately the same for the 3900 and 7800 ppm groups, however, females in the 7800 ppm group were slightly more adversely affected by treatment in the first two weeks. Therefore, 4000 ppm was selected as the high dose for the two-year studies. Lower doses of 1000 and 2000 ppm also were used in the two-year study to evaluate dose-response relationships.
Two-year study.
In the two-year rat study, survival of all exposed groups of males (control 22/50, low dose 32/50, mid dose 35/50, and high dose 34/50) was significantly greater than that of the vehicle control group, and survival of females was similar to that of the vehicle control group (control 40/50, low dose 36/50, mid dose 36/50, and high dose 36/50). Mean body weights of rats exposed to 4000 ppm were generally less than those of the vehicle controls from week 49 (males) or 25 (females) to the end of the study (Fig. 1). Feed consumption by exposed groups was similar to that by vehicle controls (data not shown). Dietary concentrations of 1000, 2000, and 4000 ppm delivered average daily doses of approximately 50, 100, and 210 mg citral/kg body weight to males and females. There were no clinical findings attributed to citral exposure.
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Mice
Fourteen-week study.
In the second week of the study, four males in the 31,300 ppm group were killed moribund, all other animals survived to the end of the study (Table 3). Surviving animals exposed to 31,300 ppm lost weight during the study. Final mean body weights and body weight gains were significantly decreased in all exposed groups of males and females. Food consumption by females exposed to citral at 7800 ppm or greater was less than the vehicle controls during the first week of the study but by the end of the study measured consumption was greater than that of the vehicle controls. The increased feed consumption was due to the mice scattering feed, an indication of poor palatability. Thus, intake calculations for the 3900, 7800, 15,600, and 31,300 ppm groups were possibly slightly inflated. Based on estimated consumption, mice received average daily doses of approximately 745, 1840, 3915, and 8110 mg citral/kg body weight to males and 790, 1820, 3870, and 7550 mg/kg to females.
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The wall of the forestomach of many male and female mice exposed to 15,600 and 31,300 ppm of citral was variably thickened (25 times normal) and the mucosa (squamous epithelium) and submucosa often rugose (data not shown). Although thickened, all three main components (mucosa, submucosa, and muscle) appeared proportional to each other and to those of control animals. Therefore, this alteration was considered the result of a contracted stomach rather than a pathological alteration. There did, however, appear to be an excessive amount of keratin (hyperkeratosis) on the surface of the epithelium of these animals, but it was minimal. Keratin is normally produced by the forestomach epithelium and is naturally removed by physical contact with feed. Reduced feed intake by these animals may have contributed to this condition.
The incidences of ovarian atrophy were significantly increased in females exposed to 15,600 or 31,300 ppm; the atrophy was moderate in the 15,600 ppm females, and marked in the 31,300 ppm females (data not shown). This lesion was likely a secondary effect due to the poor condition of mice exposed to 15,600 or 31,300 ppm.
Reduced body weights were observed in all exposed mice. Typically, in the absence of other information, high exposure concentrations for two-year studies are chosen based on the concentration that causes less than a 10% reduction in body weight. Because male and female mice exposed to the lowest dose tested (3900 ppm) in the 14-week study exceeded this percentage, the high exposure concentration chosen for the two-year studies was lowered to 2000 ppm. Lower doses of 1000 and 500 ppm also were used in the two-year study to evaluate dose-response relationships.
Two-year study.
In the two-year mouse study, survival of exposed groups of males and females was similar to that of the vehicle control groups (controls, 43/50, low dose 40/50, mid dose 42/50, high dose, 40/50 for males; and 41/50, 45/50, 43/50, and 40/50, respectively, for females). Mean body weights of mice exposed to 1000 or 2000 ppm were generally less than vehicle controls and mean body weights of 500 ppm females were less from week 30 until the end of the study (Fig. 2). Feed consumption by the exposed groups was similar to that by the vehicle controls (data not shown). Dietary concentrations of 500, 1000, and 2000 ppm delivered estimated average daily doses of approximately 60, 120, and 260 mg citral/kg body weight to males and females, respectively. There were no clinical findings attributed to citral exposure.
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DISCUSSION |
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In this study, only data from vehicle controls were presented. However, statistical analyses were performed to determine differences in the incidences of nonneoplastic or neoplastic lesions between placebo and untreated controls (NTP, 2001). For all nonneoplastic lesions that were significantly different, an informal review of their incidences in other NTP studies was performed to verify that they were within a normal range. In all cases, the lesions occurred at the frequencies expected by chance, suggesting that the differences were due to biological variation and not to ingestion of microcapsules. The only neoplastic lesion that was different between placebo and untreated controls was the incidence of uterine stromal polyps in female rats, which was significantly lower in vehicle controls (10%) when compared to untreated controls (28%) and outside the lower end of the historical control range for the NTP-2000 diet (female: range, 1231%). While this response is statistically significant, it is not believed to be biologically relevant as the incidences are on the low and high end of the control range and probably reflect normal biological variation.
In the 14-week rat study, nephropathy with renal tubule granular casts was observed in some male rats exposed to 3900 ppm and most male rats exposed to 7800 or 15,600 ppm. The presence of granular casts and exacerbation of spontaneous nephropathy is suggestive of 2u-globulin nephropathy.
2u-Globulin is a protein produced by male rats under the influence of testosterone, therefore production begins with sexual maturity and starts declining later in life (Charbonneau et al., 1987
). Some is filtered through the glomerulus with a portion being lost in the urine and a portion reabsorbed via the cytoplasm of the proximal renal tubular epithelium. With chemicals that induce
2u-globulin, the amount of hyaline droplets within the proximal renal tubule epithelium is increased and can be detected microscopically (Charbonneau et al., 1987
). There was no apparent increase in the amount of hyaline droplets in this study as determined by H&E and Mallory Heidenhain stains. Additionally, in the two-year study, no compound-related increases in kidney neoplasms were observed in male rats exposed to citral. Therefore, it was considered unlikely that renal lesions were mediated by
2u-globulin.
Citral has been extensively studied for its effect on the induction of benign and atypical hyperplasia in the ventral prostate of male rats (Engelstein et al., 1996; Kessler et al., 1998
; Servadio et al., 1986
). In the present study, careful examination did not reveal any effect on male accessory glands, including all lobes of the prostate. A comparative study of citral-induced benign and atypical hyperplasia in Wistar, Sprague-Dawley, Fischer 344, and ACI/Ztm rats demonstrated that strain genotype and endocrine background play a role in the development of this disease (Scolnik et al., 1994
). The animal model chosen for the current study, the Fischer 344/N rat, was shown to be refractory to citral-induced prostatic hyperplasia (Scolnik et al., 1994
).
In the two-year mouse study, the incidences of malignant lymphoma in females occurred with a positive trend. Malignant lymphoma is a common spontaneous systemic neoplasm that most often arises in the spleen and lymph nodes in the B6C3F1 mouse. They may also arise in the thymus, particularly when induced by chemicals. When detected in lymph nodes and thymus, lymphomas were easily diagnosed. However, malignant lymphoma in the spleen was often difficult to distinguish from lymphoid hyperplasia.
Several arguments support an association of malignant lymphoma in female mice with citral administration. In addition to the positive trend in the incidences of malignant lymphoma, the incidence in the 2000 ppm group was significantly greater than that in the vehicle controls and exceeded the incidences of lymphoma in control female mice in all but one study using the NTP-2000 diet. The incidences of malignant lymphoma in 1000 and 2000 ppm females were significantly greater than that in untreated and vehicle control groups combined (Table 4).
Conversely, while the incidence of malignant lymphoma in the 2000 ppm group was significantly increased, it was within the historical ranges for control female mice given the NTP-2000 and NIH-07 diets for two years. In addition, this is a common neoplasm and there was a low incidence in the vehicle controls compared to historical control ranges. Based on these arguments the malignant lymphoma response was considered an equivocal or uncertain finding.
In conclusion, in the 14-week studies, the kidney appeared to be a target organ for citral toxicity in male rats. Other lesions observed in rats and mice were primarily noted in exposure groups that were higher than those selected for the two-year study. In the two-year study, citral appeared to exacerbate kidney mineralization in rats and ulceration of the oral mucosa in mice. Citral was not carcinogenic in F344/N rats or male B6C3F1 mice. However, there was a marginal increase in malignant lymphoma in female mice that may have been related to citral. The daily citral exposures (mg/kg/day) achieved in rats and mice at the lowest dose tested in the two-year study represents approximately 10 times the average daily intake of 5 mg/kg/day in humans (Council of Europe, 1974).
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NOTES |
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REFERENCES |
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