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Subchronic toxicity study of yttrium nitrate by 90-day repeated oral exposure in rats
Regulatory Toxicology and Pharmacology 90 (2017) 116e125
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Subchronic toxicity study of yttrium nitrate by 90-day repeated oral exposure in rats Yi-Mei Wang a, *, 1, Zhou Yu b, 1, Zeng-Ming Zhao a, Li Jia a, Hai-Qin Fang b, Ting-Fen Zhang a, Xiao-Yan Yuan a, Yu-Lei Shu a, Jun He a, Hui Peng a, Li-Zhong Li a, Jun Zhao a, Xu-Dong Jia b, **, Shuang-Qing Peng a, *** a b
Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, PLA, Beijing 100071, China Key Laboratory of Food Safety Risk Assessment of Ministry of Health, China National Center for Food Safety Risk Assessment, Beijing 100021, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 June 2017 Received in revised form 28 August 2017 Accepted 29 August 2017 Available online 1 September 2017
Concerns regarding the adverse effects of long-term exposure to low levels of rare earth elements (REEs) from foods on human health have arisen in recent years. Nevertheless, no official acceptable daily intake (ADI) has yet been proposed for either total REEs or individual REE. In accordance with the Organization for Economic Co-operation and Development (OECD) testing guideline, the present study was undertaken to evaluate the subchronic toxicity of yttrium, a representative heavy REE with higher contaminated level in foods in China, to achieve a no observed adverse effect level (NOAEL) which is a critical basis for the establishment of an ADI. Yttrium nitrate was orally administered to rats at doses of 0, 10, 30 and 90 mg/kg/day for 90 days followed by a recovery period of 4 weeks. The following toxicity indices were measured: mortality, clinical signs, daily food consumption and weekly body weight; urinalysis, hematology, blood coagulation, clinical biochemistry and histopathology at the end of administration and recovery periods. No toxicologically significant changes were found in any yttrium-treated group as compared to the concurrent control group. Under the present experimental condition, the NOAEL in rats was thus set at 90 mg/kg for yttrium nitrate, i.e. 29.1 mg/kg for yttrium. © 2017 Elsevier Inc. All rights reserved.
Keywords: Rare earth elements ADI Yttrium Subchronic toxicity NOAEL
1. Introduction The rare earth elements (REEs) are a group of metals which are comprised of lanthanum, 14 lanthanides, yttrium (Y) and scandium. Due to their unique physical and chemical properties, REEs have been widely utilized in industrial and medical fields in the last decades (Du and Graedel, 2011; USEPA, 2012). The growing increases in REEs-related industrial activities imply human occupational exposure to REEs. Human environmental exposure to REEs
* Corresponding author. Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, PLA, 20 Dongdajie Street, Fengtai District, Beijing 100071, China. ** Corresponding author. *** Corresponding author. Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, PLA, 20 Dongdajie Street, Fengtai District, Beijing 100071, China. E-mail addresses: [email protected] (Y.-M. Wang), [email protected]. cn (X.-D. Jia), [email protected] (S.-Q. Peng). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.yrtph.2017.08.020 0273-2300/© 2017 Elsevier Inc. All rights reserved.
has also been found in the populations residing in REEs mining areas in China (Peng et al., 2003; Tong et al., 2004; Zhu et al., 2005; Wei et al., 2013). REEs have also been widely used in agricultural and zootechnical fields as fertilizers for crops and as feed additives for livestock, poultry and aquaculture in China (Hu et al., 2002; Pang et al., 2002; He et al., 2010). Established and growing evidence points to REEs-related marine, fresh water and soil pollution and accumulation. As a result, different levels of REEs from agricultural products of mining areas (Li et al., 2013; Jin et al., 2015; Zhuang et al., 2017) or non-mining areas (Li et al., 2012; Jiang et al., 2012; Song et al., 2016) have been detected, thereby increasing their accumulations in human body when ingested through food chain. REEs have been reported to exert miscellaneous toxicities to animals and humans via different exposure routines (Reviewed by Seishiro and Suzuki, 1996; Rim et al., 2013; Pagano et al., 2015). Concerns regarding the adverse effects of long-term exposure to low levels of REEs from foods on human health have arisen, as they can accumulate in blood, brain and bone after entering into human body (Seishiro and Suzuki, 1996). However, the existing
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toxicological studies are yet confined to few light REEs, and scanty toxicological data are available for other REEs, especially heavy REEs. More importantly, no official acceptable daily intake (ADI) value, the amount of a chemical or food contaminant that can be ingested daily over a lifetime without appreciable health risk, has yet been proposed for either total REEs or individual REE. Yttrium (Y) is a heavy REE, and has also been widely used in industrial, medical and agricultural fields. In China, relative high contaminated levels of yttrium in foods have been detected (Jiang et al., 2012; Song et al., 2016). Therefore, the potential for adverse human health effects from oral exposure to yttrium, especially through long-term consumption of yttrium-contaminated foods, warrants greater attention. Nevertheless, there are only few oral exposure toxicological studies for yttrium. Schroeder and Mitchener (1971) previously reported that the growth of mice was depressed when they were given 5 ppm of Y3þ in drinking water, and the longevity was increased in Y3þ-fed mice. Rats were fed with water dissolved different levels of Y3þ for 6 months, and it was shown that low level (0.534 mg/L) of Y3þ might improve the functions of learning and memory in rats, but high level (5340 mg/ L) of Y3þ could restrain the learning-memory functions and growth-development in rats (Wu et al., 2006). In the recent toxicological investigations performed in accordance with the guideline issued by the Organization of Economic Co-operation and Development (OECD) (OECD TG 426), the effects of yttrium on the neurobehavior and cognitive ability of rat offspring have been assessed. Dams were orally exposed to 0, 5, 15, or 45 mg/kg daily of yttrium nitrate from gestation day (GD) 6 to postnatal day (PND) 21, and their offspring were given the same doses until PND 63. The results revealed that oral exposure of rats to yttrium nitrate in doses up to 45 mg/kg daily had no adverse effects on their neurobehavioral development and cognitive ability (Li et al., 2015a,b). As well-known, for a chemical or food additive or contaminant, the no observed adverse effect level (NOAEL) which is primarily derived from the repeated dose toxicity studies in rodents, is a critical basis for the establishment of an ADI, a very critical value for food safety risk assessment. However, the general toxicity of yttrium following long-term repeated oral exposure has not been scientifically assessed in compliance with the international testing guidelines and regulatory requirements. Accordingly, no NOAEL or ADI value for yttrium has yet been developed to date. Therefore, it is extremely necessary to reveal the long-term toxicological effects of yttrium via oral exposure by using current international test guidelines. In this research, according to the OECD guidelines for the Testing of Chemicals “Repeated Dose 90-Day Oral Toxicity Study in Rodents” (OECD Test No. 408) and Good Laboratory Practice (GLP), the subchronic toxicity of yttrium to rats was evaluated and the NOAEL was estimated, which can provide scientific data for the food safety risk assessment of yttrium and REEs. Our present subchronic toxicity study reveals that repeated oral exposure of rats to yttrium nitrate in doses up to 90 mg/kg daily for 90 days exerts no adverse effects on rats, and the NOAEL is set at 90 mg/kg for yttrium nitrate, i.e. 29.1 mg/kg for yttrium. Accordingly, the ADI value for yttrium was estimated to be 145.5 mg/kg/day by using an uncertainty factor of 200. Based on the current contaminated levels of yttrium in foods and average dietary intake of yttrium for adults in China, this estimated ADI value indicates the daily intake of yttrium from foods is relative acceptable and safe for adults. 2. Materials and methods 2.1. Test substance Yttrium nitrate stock solution with the concentration of
117
218.28 mg/mL was provided by China National Center for Food Safety Risk Assessment (Beijing, China). When used, yttrium nitrate stock solution was diluted to the indicated dosing concentrations with distilled water and was adjusted to pH 6.0 by using 1 mol/L sodium hydroxide. 2.2. Animals and dosing Sprague-Dawley (SD) rats (50e70 g) were obtained from the Laboratory Animal Center of Academy of Military Medical Sciences (Beijing China). The rats were housed at Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, PLA (Beijing China). After 6 days of acclimatization and quarantine, the healthy rats were randomly assigned into control group or one of 3 yttrium dosing groups according to body weights separated by sex, with 15 rats per sex per group. All rats were housed five per sex in standard PVC cages (i.e. 3 cages per sex per group) in a ventilated room (10e20 air exchanges/hr) with controlled illumination (12 h light/dark cycle), temperature (20 Ce25 C) and humidity (40%e 70%). Feed (standard rodent diet) and tap water were provided ad libitum. Rats in 3 yttrium exposure groups were given yttrium nitrate solution by oral gavage at a daily dose of 10, 30, and 90 mg/kg body weight, respectively. Control rats received equivalent volumes of vehicle of distilled water only. The application volume for all groups was 1 mL/100 g body weight. The animals were treated with the test substance or vehicle once daily for a period of 90 days followed by a recovery period of 4 weeks. Ten rats (2 cages) per sex of each group were subjected to necropsy one day after the last administration (the end of treatment period), and the remaining 5 rats (1 cage) per sex of each group were subjected to necropsy 28 days after the last administration (the end of recovery period). All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Institute of Disease Control and Prevention, PLA (approval No.: 2013-021), and were in strict accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The protocol was generally based on the OECD guidelines for the Testing of Chemicals “Repeated Dose 90-day Oral Toxicity Study in Rodents” (OECD Test No. 408) (OECD, 1998). And the study was conducted at Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, PLA (Beijing China), a Good Laboratory Practice (GLP)-certified laboratory, and was in strict accordance with the GLP principles of China Food and Drug Administration and Ministry of Environmental Protection of China. 2.3. Observations and examinations The following parameters/indices were determined: mortality, daily clinical observation, daily food consumption, weekly body weight; urinalysis, hematology, blood coagulation, clinical biochemistry and histopathologic examination at the end of administration and recovery periods. Animals were fasted overnight before blood collection. 2.3.1. Clinical observation and mortality All animals were checked daily for any clinically abnormal signs. A check for moribund and dead animals was made once daily. If animals were in a moribund state, they were sacrificed and necropsied. 2.3.2. Body weight and food consumption Body weights were recorded on the first day of administration, weekly thereafter and prior to sacrifice. The 24 h feed intake pattern on rats was measured weekly for each cage. Briefly, at weekly interval, the supplied amount of food
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to each cage and the remnant of each cage were measured to calculate the difference, which was regarded as weekly food consumption of each cage. The average of daily feed intake per rat per cage was then used to calculate the mean food consumption in grams per rat per day for one group. 2.3.3. Urinalysis At the end of administration and recovery periods, urine samples from 5 animals per sex of each group were collected, respectively. The following parameters were analyzed using a urine analyzer (Scan 100; Combi, Germany): leukocytes, nitrite, urobilinogen, bilirubin, urinary protein, glucose, ketone body, specific gravity, pH and occult blood. Additionally, urine appearance (color and transparency) was recorded. 2.3.4. Hematology, blood coagulation and clinical biochemistry At the end of administration period (10 rats/sex/group) and recovery period (5 rats/sex/group), the overnight fasted rats were anesthetized with pentobarbital sodium (50 mg/kg), and blood was then collected from the abdominal aorta for the determinations of hematology, blood coagulation and clinical biochemistry. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. Hematology was analyzed using an automatic hematology analyzer (Nihon Kohden, Japan), and blood smears were prepared and stained to count the reticulocytes. The following parameters are measured: red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT), reticulocyte (RET), white blood cell (WBC); percentages of lymphocyte [LYM (%)], monocyte [MON (%)], neutrophil [NEUT (%)], eosinophil [EOS (%)] and basophil [BAS (%)]. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were analyzed using a semi-automatic blood coagulation analyzer (Sysmex, Japan). Clinical biochemistry was measured using an automatic biochemical analyzer (Hitachi, Japan) and an automatic electrolyte (Na/K/Cl) analyzer (Medica, USA). The measured parameters are as follows: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), total bilirubin (TBIL), alkaline phosphatase (ALP), glucose (GLU), creatinine (CREA), cholesterol (CHOl), triglyceride (TG), creatine kinase (CK); sodium ion (Naþ), potassium ion (Kþ) and chloride ion (Cl). 2.3.5. Histopathology At the end of administration and recovery periods, the overnight fasted rats were weighed and sacrificed by anesthesia and exsanguinations after blood collection, followed by a detailed gross necropsy. 2.3.5.1. Organ weights. The following organs from all animals were isolated and weighed at scheduled necropsy before fixation: brain, heart, livers, spleen, lungs, kidneys, testes, epididymides, uterus, ovaries, thymus, and adrenals. Relative organ weight was calculated as the ratio of organ weight to the terminal fasted body weight. 2.3.5.2. Histopathologic examination. Besides the organs described above for weight measurement, samples of the following tissues and organs were collected from all animals at necropsy and were fixed in 10% neutral buffered formalin: brain (cerebrum, cerebellum and pons), pancreas, oesophagus, stomach, small and large intestines (duodenum, jejunum, ileum, caecum, colon, rectum), mesenteric lymph nodes,
submandibular gland, thyroid, parathyroid glands, salivary gland, trachea, prostate, mammary glands, pituitary gland, sciatic nerve with skeletal muscle, skin, urinary bladder, spinal cord, optic nerve, thoracic aorta, and sternum with bone marrow. For the animals of control group and high dose group sacrificed at the scheduled days, the afore-listed tissues and organs were histopathologically examined after preparation of paraffin sections and haematoxylin-eosin staining. The histopathologic examinations were not extended to animals of other dosage groups when no treatment-related changes were observed in the high dose group. 2.4. Statistical analysis Statistical analyses of body weight, food consumption, parameters of hematology, blood coagulation and clinical biochemistry, and absolute and relative organ weights were performed for each gender by comparing the values of dosed animals with control animals using a one-way analysis of variance and a post-hoc Dunnet t-test. Pathological results were descriptively analyzed, and the incidences of tissue lesions were analyzed using chi-square test. These statistics were performed with SPSS software, and the significance level was set at P < 0.05. 3. Results 3.1. Clinical observation All rats survived to the scheduled necropsies. There were no abnormal clinical signs or toxic effects that could be attributed to the administration of yttrium. 3.2. Body weight and food consumption Weekly mean body weights of both male (Fig. 1A) and female rats (Fig. 1B) increased with the progress of study in all groups. Yttrium exposure didn't evoke significant effects on the body weights of rats when compared with the concurrent control group for both sexes. As shown in Fig. 2, mean daily food consumptions for both males (Fig. 2A) and females (Fig. 2B) were similar among the 4 groups during the administration and recovery periods. 3.3. Hematology and blood coagulation As presented in Table 1, at the end of treatment and recovery periods, almost all the hematology and blood coagulation parameters in any yttrium treatment group were not significantly different from the concurrent control group for both sexes, except for a slight but biologically insignificant increase in PT in 30 mg/kg yttrium-treated female rats at the end of recovery period. 3.4. Clinical biochemistry At the end of treatment and recovery periods, there were no significant differences for most clinical biochemistry parameters in any yttrium treatment group when compared with the concurrent control group of the same sex (Table 2). Although statistically significant increases or decreases in several parameters such as TP, GLU and CREA were observed in 30 or 90 mg/kg yttrium-treated groups, these changes are irregular, no dose-related dependence, and are all within the normal reference ranges of SD rats in our laboratory. Therefore, these alterations were judged to be of no toxicological significance.
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Fig. 1. Effect of yttrium nitrate on weekly mean body weight of rats. Rats were orally administered different doses of yttrium nitrate for 90 days (13 weeks) by gavage, followed by a recovery period of 4 weeks. Body weights were recorded on the first day of administration (Week 1) and weekly thereafter during the treatment period (Weeks 2e14) and recovery period (Weeks 15e18). (A) Male rats; (B) female rats. Data were expressed as means ± SD. The number of animals in each group for each sex is 15 and 5 in the treatment and recovery periods, respectively.
Fig. 2. Effect of yttrium nitrate on mean daily food consumption of rats. Rats were orally administered different doses of yttrium nitrate for 90 days (13 weeks) by gavage, followed by a recovery period of 4 weeks. Food consumption for each cage of 5 rats was determined once a week by subtracting the weight of the remaining food from the weight of the supplied food of each cage, and the average of daily feed intake per rat for each cage was used to calculate the mean daily food consumption in grams per rat per day for one group. (A) Male rats; (B) female rats. The number of cages for each sex in each group is 3 and 1 in the treatment period and recovery period, respectively. Data were expressed as means ± SD in the treatment period (Weeks 1e13 after dosing), and as a single value in recovery period (Weeks 14e17 after dosing).
3.5. Urinalysis
3.6. Organ weights and histopathology
At the end of treatment and recovery periods, urine appearance (color and transparency) and all of the 10 urinary parameters in all groups were similar, and there were no statistically or biologically significant changes in any parameter in yttrium-treated groups as compared to the concurrent control group (data not shown).
3.6.1. Organ weights At necropsies of the end of treatment and recovery periods, no significant differences in the absolute weights of the measured organs were observed between any yttrium-treated group and the concurrent control group for both sexes (Table 3). As for the relative organ weight, only increased relative lung weights of male rats were noted in 30 and 90 mg/kg of yttrium treatment groups at the end of treatment period (Table 4).
Dose (mg/kg)
Sex
RBC (1012/L)
HGB (g/L)
HCT (%)
MCH (pg)
MCHC (g/L)
PLT (109/L)
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Table 1 Effects of repeated oral exposure to yttrium nitrate for 90 days on hematology and coagulation of rats. RET ( ‰)
WBC (109/L)
LYM (%)
MON (%)
NEUT (%)
EOS (%)
BAS (%)
PT (s)
APTT (s)
154 152 155 156 152 153 155 155
± ± ± ± ± ± ± ±
6 3 8 9 10 5 9 7
42.1 40.8 42.5 41.6 41.8 40.7 42.9 42.9
± ± ± ± ± ± ± ±
1.4 1.2 2.1 3.3 3.9 2.0 2.8 2.7
19.5 20.5 19.2 20.8 19.3 21.0 19.3 20.3
± ± ± ± ± ± ± ±
0.7 0.9 0.9 0.8 1.0 1.0 0.8 0.9
366 373 363 375 364 377 362 362
± ± ± ± ± ± ± ±
7 13 7 12 12 12 11 8
829 760 803 811 827 805 846 848
± ± ± ± ± ± ± ±
108 86 106 94 103 39 83 132
23 26 23 26 25 28 22 30
± ± ± ± ± ± ± ±
5 6 6 6 9 8 7 6
7.7 6.1 6.9 5.7 9.4 5.3 6.7 6.2
± ± ± ± ± ± ± ±
1.6 1.7 4.2 1.7 10.6 1.1 1.5 2.6
92.0 92.5 91.2 91.8 91.7 92.0 91.5 91.6
± ± ± ± ± ± ± ±
1.5 1.4 1.5 1.4 1.3 1.6 1.5 1.5
0.2 0.2 0.2 0.4 0.3 0.2 0.2 0.2
± ± ± ± ± ± ± ±
0.1 0.2 0.2 0.2 0.3 0.2 0.2 0.2
7.2 6.4 7.7 6.8 7.2 6.7 7.1 6.9
± ± ± ± ± ± ± ±
1.5 1.3 1.2 1.5 1.2 1.3 1.7 1.2
0.5 0.7 0.7 0.9 0.6 0.9 0.8 1.0
± ± ± ± ± ± ± ±
0.3 0.4 0.3 0.3 0.4 0.3 0.6 0.3
0.1 0.2 0.2 0.2 0.2 0.2 0.3 0.3
± ± ± ± ± ± ± ±
0.0 0.2 0.1 0.2 0.1 0.2 0.3 0.4
14.5 13.4 14.3 13.3 14.7 13.3 14.4 13.5
± ± ± ± ± ± ± ±
0.6 0.6 0.6 0.6 0.9 0.4 0.7 0.7
27.4 20.5 27.6 21.9 28.2 22.1 25.6 22.9
± ± ± ± ± ± ± ±
2.2 2.5 3.3 2.1 6.0 2.2 3.4 2.4
End of Recovery Period 0 M 7.84 ± 0.63 F 7.22 ± 0.28 10 M 7.51 ± 0.38 F 6.98 ± 0.21 30 M 7.56 ± 0.28 F 7.15 ± 0.29 90 M 7.77 ± 0.16 F 7.30 ± 0.31
154 147 146 146 156 151 153 151
± ± ± ± ± ± ± ±
5 5 7 7 6 10 3 4
41.7 40.7 40.1 39.8 41.2 41.1 41.9 40.8
± ± ± ± ± ± ± ±
2.6 1.4 1.6 1.6 1.7 2.7 0.8 1.8
19.8 20.3 19.4 20.9 20.6 21.2 19.7 20.7
± ± ± ± ± ± ± ±
1.2 0.5 0.9 1.0 0.3 0.7 0.1 0.7
371 361 364 367 379 368 366 370
± ± ± ± ± ± ± ±
13 8 15 6 6 7 3 10
862 776 905 829 883 830 895 803
± ± ± ± ± ± ± ±
69 152 120 112 44 115 73 26
38 32 34 35 36 38 36 32
± ± ± ± ± ± ± ±
4 6 5 8 7 7 6 4
5.8 2.8 8.0 2.3 5.4 2.5 6.2 4.1
± ± ± ± ± ± ± ±
1.7 1.3 7.4 0.3 0.5 0.9 1.2 1.6
93.7 93.1 92.4 93.1 92.6 93.3 92.5 93.6
± ± ± ± ± ± ± ±
1.2 1.8 2.1 1.6 1.4 1.7 1.2 2.1
0.1 0.2 0.2 0.2 0.3 0.1 0.2 0.2
± ± ± ± ± ± ± ±
0.1 0.2 0.2 0.1 0.1 0.2 0.2 0.2
5.2 5.9 6.4 5.9 6.1 5.9 6.2 5.3
± ± ± ± ± ± ± ±
1.4 1.6 1.8 1.3 1.2 1.4 0.9 1.7
0.7 0.7 0.7 0.7 0.7 0.6 0.9 0.7
± ± ± ± ± ± ± ±
0.2 0.5 0.5 0.3 0.3 0.4 0.2 0.5
0.2 0.2 0.3 0.2 0.3 0.0 0.3 0.2
± ± ± ± ± ± ± ±
0.2 0.4 0.2 0.3 0.2 0.1 0.1 0.2
13.6 13.3 14.0 13.5 13.7 14.5 14.0 13.3
± ± ± ± ± ± ± ±
2.0 0.6 0.8 0.6 0.7 0.7* 1.4 0.5
30.8 19.9 30.0 16.7 28.2 16.6 33.4 20.4
± ± ± ± ± ± ± ±
11.1 6.4 7.8 4.7 3.7 5.3 5.5 6.2
At the end of 90 days of treatment period and 4 weeks of recovery period, blood was collected from 10 and 5 rats per group per sex for hematology and blood coagulation determinations, respectively. Data were expressed as means ± SD. *P < 0.05, significantly different from the concurrent control group of same sex. RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet; RET, reticulocyte; WBC, white blood cell; LYM (%), percentage of lymphocyte; MON (%), percentage of monocyte; NEUT (%), percentage of neutrophil; EOS (%), percentage of eosinophil; BAS (%), percentage of basophil; PT, prothrombin time; APTT, activated partial thromboplastin time.
Table 2 Effects of repeated oral exposure to yttrium nitrate for 90 days on clinical biochemistry of rats. Dose (mg/kg)
Sex
ALT (U/L)
AST (U/L)
TP (g/L)
End of Treatment Period 0 M 28 ± 4 F 22 ± 3 10 M 30 ± 4 F 24 ± 4 30 M 31 ± 7 F 23 ± 6 90 M 29 ± 4 F 25 ± 10
78 87 78 76 78 81 82 85
End of Recovery Period 0 M 34 ± 4 F 21 ± 3 10 M 69 ± 69 F 23 ± 4 30 M 34 ± 4 F 21 ± 3 90 M 36 ± 9 F 26 ± 6
86 ± 18 82 ± 10 115 ± 57 71 ± 15 85 ± 6 74 ± 15 93 ± 23 78 ± 12
± ± ± ± ± ± ± ±
22 20 22 13 22 21 19 21
ALB (g/L)
TBIL (mmol/L)
ALP (U/L)
GLU (mmol/L)
Urea (mmol/L)
CREA (mmol/L)
CHOl (mmol/L)
TG (mmol/L)
Naþ (mmol/L)
CK (U/L)
Kþ (mmol/L)
Cl (mmol/L)
50.6 62.1 48.1 57.9 46.2 59.8 47.9 60.8
± ± ± ± ± ± ± ±
2.2 5.1 4.0 5.8 7.2 4.4 1.6* 4.3
23.5 28.2 22.6 26.3 22.4 27.1 22.7 28.0
± ± ± ± ± ± ± ±
0.6 2.3 1.2 1.8 2.6 2.2 0.7 3.2
1.0 1.6 0.9 1.2 0.8 1.2 1.1 1.2
± ± ± ± ± ± ± ±
0.2 0.5 0.3 0.3 0.2 0.4 0.4 0.6
87 54 91 52 94 50 77 57
± ± ± ± ± ± ± ±
15 19 17 12 21 12 11 13
8.3 7.8 8.5 8.2 8.8 8.8 8.2 8.2
± ± ± ± ± ± ± ±
0.7 1.1 1.3 1.1 2.2 0.6* 0.8 0.5
4.5 4.7 4.6 5.5 4.8 4.8 4.5 5.6
± ± ± ± ± ± ± ±
0.6 0.7 0.7 1.5 0.9 1.0 0.9 0.7
44 51 43 47 43 50 40 53
± ± ± ± ± ± ± ±
4 5 5 5 8 7 6 4
1.43 1.80 1.44 1.74 1.37 1.97 1.48 1.72
± ± ± ± ± ± ± ±
0.17 0.28 0.25 0.24 0.22 0.43 0.26 0.24
0.61 0.47 0.59 0.44 0.51 0.56 0.52 0.36
± ± ± ± ± ± ± ±
0.19 0.17 0.22 0.12 0.21 0.44 0.23 0.11
250 272 181 199 230 255 209 237
± ± ± ± ± ± ± ±
145 147 116 125 166 143 88 99
138.1 137.1 139.0 138.4 139.7 126.3 137.7 138.8
± ± ± ± ± ± ± ±
3.0 1.5 1.5 2.7 2.0 39.6 1.8 1.2
4.10 3.87 3.99 3.84 4.14 3.84 4.04 3.86
± ± ± ± ± ± ± ±
0.23 0.24 0.27 0.32 0.18 0.27 0.18 0.22
108.4 108.0 109.1 107.4 107.8 105.5 107.6 106.1
± ± ± ± ± ± ± ±
1.1 2.7 1.4 1.3 1.5 2.8 1.6 2.4
53.6 62.2 52.0 63.3 54.7 59.9 54.4 65.0
± ± ± ± ± ± ± ±
2.1 4.1 3.6 5.6 1.8 1.5 2.1 2.1
23.4 29.0 22.3 29.8 23.5 28.2 23.5 31.2
± ± ± ± ± ± ± ±
0.8 0.8 1.4 2.6 0.9 0.9 0.9 1.3
1.5 1.5 1.1 1.3 1.3 1.1 1.2 1.6
± ± ± ± ± ± ± ±
0.5 0.4 0.3 0.4 0.3 0.4 0.4 0.8
56 35 79 40 76 39 63 41
± ± ± ± ± ± ± ±
12 8 19 18 23 15 19 13
9.4 8.3 8.5 7.6 9.4 7.9 9.4 7.7
± ± ± ± ± ± ± ±
0.5 0.5 0.8 0.5* 1.0 0.2 0.9 0.2
4.5 5.4 5.3 5.5 5.3 6.3 5.4 5.2
± ± ± ± ± ± ± ±
0.6 1.0 0.5 0.6 0.8 1.0 0.3 0.4
30 20 21 20 15 22 31 19
± ± ± ± ± ± ± ±
11 5 5 9 11* 12 5 16
1.62 1.88 1.61 2.30 1.76 1.95 1.57 1.79
± ± ± ± ± ± ± ±
0.34 0.26 0.36 0.41 0.25 0.22 0.27 0.24
0.39 0.38 0.65 0.42 0.64 0.45 0.66 0.44
± ± ± ± ± ± ± ±
0.08 0.08 0.41 0.11 0.27 0.14 0.40 0.16
243 262 232 204 254 234 257 199
± ± ± ± ± ± ± ±
138 47 69 89 47 99 161 86
139.2 137.2 137.6 137.8 137.9 138.8 137.2 138.7
± ± ± ± ± ± ± ±
3.5 1.7 1.4 2.1 2.4 2.1 2.4 1.4
4.18 3.69 4.15 3.96 4.26 3.79 3.95 3.71
± ± ± ± ± ± ± ±
0.21 0.21 0.36 0.27 0.12 0.19 0.20 0.34
107.2 112.0 107.4 107.9 107.1 108.4 107.1 107.6
± ± ± ± ± ± ± ±
2.0 2.3 1.6 1.4* 1.8 2.4* 1.7 1.7**
At the end of 90 days of treatment period and 4 weeks of recovery period, blood was collected from 10 and 5 rats per group per sex for clinical biochemistry determinations, respectively. Data were expressed as means ± SD. *P < 0.05, **P < 0.01, significantly different from the concurrent control group of same sex. ALT, alanine aminotransferase; AST, aspartate aminotransferase; TP, total protein; ALB, albumin; TBIL, total bilirubin; ALP, alkaline phosphatase; GLU, glucose; CREA, creatinine; CHOl, cholesterol; TG, triglyceride; CK, creatine kinase; Naþ, sodium ion; Kþ, potassium ion; Cl, chloride ion.
Y.-M. Wang et al. / Regulatory Toxicology and Pharmacology 90 (2017) 116e125
End of Treatment Period 0 M 7.89 ± 0.36 F 7.43 ± 0.30 10 M 8.04 ± 0.46 F 7.50 ± 0.53 30 M 7.91 ± 0.76 F 7.32 ± 0.44 90 M 8.02 ± 0.57 F 7.67 ± 0.40
Table 3 Effects of repeated oral exposure to yttrium nitrate for 90 days on the organ weights (g) of rats. Dose (mg/kg)
Sex
Body weight
End of Treatment Period 0 M 532.5 ± F 316.6 ± 10 M 521.5 ± F 323.5 ± 30 M 502.3 ± F 327.3 ± 90 M 497.5 ± F 295.3 ±
Brain
Thymus
Heart
Lungs
Liver
Spleen
Kidneys
Adrenals
Testes/Ovaries
Epididymides/Uterus
2.131 1.953 2.155 1.930 2.142 2.014 2.151 1.901
± ± ± ± ± ± ± ±
0.147 0.098 0.148 0.088 0.135 0.091 0.141 0.088
0.416 0.352 0.433 0.348 0.455 0.358 0.338 0.322
± ± ± ± ± ± ± ±
0.146 0.120 0.178 0.069 0.287 0.082 0.100 0.055
1.665 1.098 1.633 1.109 1.562 1.211 1.551 1.052
± ± ± ± ± ± ± ±
0.168 0.105 0.143 0.187 0.188 0.200 0.097 0.093
1.802 1.359 1.893 1.581 2.094 1.466 2.146 1.503
± ± ± ± ± ± ± ±
0.283 0.148 0.290 0.231 0.367 0.186 0.410 0.345
12.670 ± 0.669 7.757 ± 1.294 12.199 ± 1.794 7.819 ± 1.008 11.877 ± 1.320 8.196 ± 1.338 11.711 ± 1.224 7.280 ± 0.373
0.805 0.605 0.818 0.616 0.854 0.598 0.819 0.588
± ± ± ± ± ± ± ±
0.126 0.099 0.111 0.106 0.184 0.094 0.101 0.100
3.070 1.790 2.974 1.900 2.901 1.920 2.853 1.773
± ± ± ± ± ± ± ±
0.132 0.188 0.422 0.260 0.388 0.165 0.174 0.157
0.069 0.076 0.064 0.077 0.067 0.081 0.067 0.076
± ± ± ± ± ± ± ±
0.008 0.010 0.012 0.016 0.012 0.011 0.009 0.012
3.098 0.169 3.329 0.166 3.231 0.170 2.966 0.171
± ± ± ± ± ± ± ±
0.279 0.036 0.376 0.044 0.270 0.032 0.854 0.034
1.351 0.592 1.441 0.646 1.276 0.658 1.242 0.807
± ± ± ± ± ± ± ±
0.138 0.144 0.136 0.346 0.158 0.222 0.264 0.350
End of Recovery Period 0 M 588.5 ± 47.1 F 326.8 ± 54.9 10 M 584.1 ± 65.8 F 323.8 ± 32.3 30 M 609.6 ± 56.0 F 312.3 ± 18.1 90 M 582.6 ± 54.2 F 314.3 ± 12.1
2.289 1.970 2.232 1.977 2.213 2.064 2.187 1.914
± ± ± ± ± ± ± ±
0.112 0.150 0.087 0.059 0.122 0.069 0.130 0.086
0.265 0.293 0.268 0.280 0.326 0.273 0.340 0.290
± ± ± ± ± ± ± ±
0.098 0.046 0.051 0.070 0.062 0.040 0.043 0.060
1.690 1.122 1.745 1.054 1.765 1.083 1.762 1.072
± ± ± ± ± ± ± ±
0.198 0.108 0.245 0.131 0.188 0.070 0.232 0.135
1.879 1.335 1.859 1.486 2.002 1.403 2.004 1.551
± ± ± ± ± ± ± ±
0.158 0.238 0.242 0.187 0.337 0.187 0.127 0.324
13.069 ± 1.339 7.990 ± 1.492 14.016 ± 2.343 8.254 ± 0.743 13.903 ± 1.479 7.586 ± 0.387 13.970 ± 2.458 7.737 ± 0.749
0.905 0.559 0.925 0.549 0.896 0.536 0.802 0.588
± ± ± ± ± ± ± ±
0.163 0.107 0.109 0.053 0.179 0.109 0.160 0.011
3.026 1.874 2.939 1.933 3.074 1.832 2.942 1.833
± ± ± ± ± ± ± ±
0.241 0.301 0.253 0.150 0.277 0.085 0.400 0.153
0.063 0.078 0.054 0.075 0.059 0.067 0.061 0.074
± ± ± ± ± ± ± ±
0.008 0.015 0.014 0.011 0.017 0.008 0.010 0.006
3.084 0.163 3.466 0.179 2.685 0.164 3.355 0.169
± ± ± ± ± ± ± ±
0.184 0.028 0.329 0.032 1.045 0.029 0.443 0.021
1.319 0.552 1.549 0.743 1.220 0.872 1.394 0.604
± ± ± ± ± ± ± ±
0.159 0.169 0.126 0.168 0.481 0.420 0.093 0.070
At the end of 90 days of treatment period and 4 weeks of recovery period, the main organs listed were isolated from 10 and 5 rats per group per sex and weighed, respectively. Data were expressed as means ± SD.
Table 4 Effects of repeated oral exposure to yttrium nitrate for 90 days on the relative organ weights to body weight (%) of rats. Dose (mg/kg)
Sex
Brain
End of Treatment Period 0 M 0.402 F 0.625 10 M 0.415 F 0.603 30 M 0.428 F 0.626 90 M 0.434 F 0.644 End of Recovery Period 0 M 0.390 F 0.614 10 M 0.386 F 0.615 30 M 0.366 F 0.663 90 M 0.377 F 0.610
Thymus
Heart
Lungs
Liver
Spleen
Kidneys
Adrenals
Testes/Ovaries
Epididymides/Uterus
± ± ± ± ± ± ± ±
0.045 0.072 0.030 0.070 0.034 0.080 0.039 0.029
0.078 0.110 0.082 0.107 0.092 0.109 0.068 0.109
± ± ± ± ± ± ± ±
0.026 0.027 0.030 0.015 0.061 0.015 0.021 0.017
0.313 0.349 0.314 0.342 0.311 0.373 0.313 0.357
± ± ± ± ± ± ± ±
0.031 0.032 0.018 0.037 0.019 0.062 0.030 0.034
0.340 0.432 0.363 0.490 0.418 0.453 0.433 0.510
± ± ± ± ± ± ± ±
0.059 0.048 0.044 0.068 0.073* 0.067 0.088* 0.122
2.382 2.444 2.331 2.416 2.365 2.501 2.351 2.467
± ± ± ± ± ± ± ±
0.107 0.166 0.207 0.151 0.202 0.110 0.163 0.131
0.151 0.191 0.157 0.191 0.170 0.183 0.165 0.199
± ± ± ± ± ± ± ±
0.018 0.017 0.017 0.031 0.037 0.014 0.019 0.031
0.578 0.568 0.569 0.588 0.576 0.592 0.575 0.600
± ± ± ± ± ± ± ±
0.032 0.048 0.043 0.048 0.043 0.059 0.034 0.049
0.013 0.024 0.012 0.024 0.013 0.025 0.013 0.046
± ± ± ± ± ± ± ±
0.001 0.003 0.003 0.004 0.002 0.003 0.002 0.064
0.582 0.053 0.641 0.051 0.645 0.052 0.594 0.058
± ± ± ± ± ± ± ±
0.046 0.009 0.079 0.011 0.052 0.011 0.174 0.011
0.254 0.188 0.279 0.195 0.255 0.204 0.250 0.273
± ± ± ± ± ± ± ±
0.026 0.045 0.043 0.082 0.033 0.073 0.055 0.119
± ± ± ± ± ± ± ±
0.028 0.094 0.048 0.057 0.045 0.040 0.035 0.042
0.044 0.090 0.046 0.086 0.054 0.088 0.058 0.092
± ± ± ± ± ± ± ±
0.015 0.011 0.010 0.018 0.011 0.014 0.005 0.019
0.288 0.348 0.299 0.327 0.290 0.349 0.302 0.340
± ± ± ± ± ± ± ±
0.033 0.046 0.026 0.046 0.017 0.038 0.022 0.032
0.321 0.410 0.318 0.461 0.330 0.451 0.347 0.491
± ± ± ± ± ± ± ±
0.039 0.047 0.017 0.053 0.060 0.066 0.050 0.084
2.218 2.442 2.389 2.590 2.281 2.431 2.386 2.457
± ± ± ± ± ± ± ±
0.070 0.183 0.194 0.074 0.138 0.063 0.225 0.158
0.154 0.171 0.159 0.170 0.148 0.171 0.137 0.187
± ± ± ± ± ± ± ±
0.023 0.017 0.017 0.015 0.026 0.030 0.024 0.009
0.514 0.575 0.505 0.599 0.508 0.587 0.504 0.582
± ± ± ± ± ± ± ±
0.010 0.040 0.033 0.029 0.070 0.025 0.035 0.029
0.011 0.025 0.010 0.024 0.010 0.021 0.010 0.024
± ± ± ± ± ± ± ±
0.001 0.007 0.002 0.005 0.003 0.002 0.002 0.002
0.526 0.050 0.600 0.055 0.442 0.053 0.582 0.054
± ± ± ± ± ± ± ±
0.039 0.006 0.094 0.007 0.171 0.011 0.108 0.005
0.226 0.168 0.266 0.234 0.203 0.276 0.240 0.193
± ± ± ± ± ± ± ±
0.041 0.039 0.012 0.075 0.089 0.125 0.020 0.027
Y.-M. Wang et al. / Regulatory Toxicology and Pharmacology 90 (2017) 116e125
28.9 41.4 43.3 34.4 42.6 48.6 30.7 11.8
At the end of 90 days of treatment period and 4 weeks of recovery period, the main organs listed were isolated from 10 and 5 rats per group per sex and weighed, respectively, and the relative organ weights to body weight (%) were calculated. Data were expressed as means ± SD. *P < 0.05, significantly different from the concurrent control group of same sex. 121
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3.6.2. Histopathologic examination At necropsies, adhesion of lung and thoracic cavity in one male rat of 90 mg/kg group was observed at the end of treatment period, and local brown change in the lung of one male of 30 mg/kg group was noted at the end of recovery period. Atrophies of testis and epididymis observed in one male of 90 mg/kg group were considered to be the frequently spontaneous pathological changes in this strain of this age, and were not treatment-related. At the end of treatment and recovery periods, microscopic examinations showed slight or mild chronic focal inflammatory cell infiltration in the heart and liver of several rats, and the incidence and severity were comparable between yttrium-treated and control groups. Chronic interstitial inflammation in the lungs was also observed in several yttrium-treated and control rats, with similar incidence and severity. Based on the historical occurrence rates of chronic inflammations in the heart, liver and lung of control SD rats from our previous repeated oral exposure toxicological studies, we consider these chronic inflammations observed in the present study are frequently spontaneous background changes in SD rats, rather than treatment-related pathological changes. In addition, slight or mild prostate epithelial proliferation and uterus dilatation, which are frequently spontaneous pathological changes in rats, were noted in few yttrium-treated rats, and were not associated with yttrium treatment. At the end of treatment period, foreign body granulomatous inflammation in the lungs, as evidenced by eosinophilic granulocyte infiltration and especially distinctive macrophage accumulation were observed in the 3 yttrium-treated groups. These pulmonary lesions were occasional in 10 mg/kg group (Fig. 3B; Table 5), while with more incidence and severity in 30 and 90 mg/ kg treatment groups (Fig. 3C and D; Table 5). At the end of recovery period, moderate ameliorations of these pulmonary lesions were
noted (Fig. 3F; Table 5). The increased relative lung weights along with pulmonary foreign body granuloma observed in 30 and 90 mg/kg of yttrium-treated rats indicated the pulmonary lesions might be yttrium treatmenterelated.
4. Discussion Since the widespread uses in agriculture for decades in China, REEs have accumulated in soils and aquatic environments, which can transfer to the agricultural products. REEs have been detected in different dietary samples, such as cereals, vegetables, beans, tea, aquatic products, meats and fresh eggs (Jiang et al., 2012; Zhou et al., 2014; Song et al., 2016), thereby increasing their bioaccumulations in human body through food intakes. Nevertheless, to our knowledge, no contaminated level limits in foods for either total REEs or individual element have been established worldwidely except China. In 2005, China developed the maximal contaminated levels of total rare earth oxides (REOs) in foods: 2.0 mg/kg for cereals and tea, and 0.7 mg/kg for vegetables and fruits. However, limit for individual RRE in any dietary sample has yet not been established. In addition, the existing toxicological studies are focused on only a few REEs, rarely on heavy REEs. As a result, the health risk posed by dietary intake of total REEs or individual element cannot be accurately assessed. Yttrium is one of the heavy REEs with higher concentrations in foods and higher total dietary intake. In a survey of 16 REEs in the major foods in China during 2009e2010, it was found that the concentrations of yttrium in the investigated foods were relative high (Jiang et al., 2012). The average content of yttrium in the major foods was 16.9 mg/kg with the maximum of 3470 mg/kg; and in fresh vegetables, the average content of yttrium was 34.1 mg/kg with the maximum of 3470 mg/kg. The 4th China total diet survey in 2007
Fig. 3. Histopathologic changes in the lungs of rats orally gavaged yttrium nitrate. Rats were orally administered different doses (10, 30 and 90 mg/kg) of yttrium nitrate or distilled water for 90 days by gavage followed by a recovery period of 4 weeks. At the end of administration and recovery periods, the rats were subjected to euthanasia and histopathologic changes in the lungs were evaluated by routine procedures. The presented images were the representatives of histopathologic changes of the lungs. A (chronic interstitial inflammation), B (chronic interstitial inflammation), C (foreign body granuloma) and D (foreign body granuloma) indicated control, 10, 30 and 90 mg/kg yttrium-treated rats at the end of treatment period, respectively; E and F indicated control and 90 mg/kg yttrium-treated rats at the end of recovery period, respectively. Magnification, 200.
Y.-M. Wang et al. / Regulatory Toxicology and Pharmacology 90 (2017) 116e125
123
Table 5 The severity and incidence of histopathologic changes in the lungs of rats received repeated oral exposure to yttrium nitrate. Histopathologic changes
End of Treatment Period
End of Recovery Period
Control
10 mg/kg
30 mg/kg
90 mg/kg
Control
10 mg/kg
30 mg/kg
90 mg/kg
Chronic interstitial inflammation
1 (2/20) 2 (7/20) 3 (4/20)
1 (4/20) 2 (9/20) 3 (1/20)
1 (2/20) 2 (6/20) 3 (2/20)
1 (7/20) 2 (6/20) 3 (2/20)
1 (4/10) 3 (1/10)
1 (4/10) 2 (2/10)
1 (4/10) 2 (4/10)
1 (2/10) 2 (3/10)
Foreign body granuloma
e
2 (1/20)
1 (2/20) 2 (3/20) 3 (1/20)
1 (5/20) 2 (3/20)
e
3 (1/10)
1 (1/10) 2 (3/10)
1 (3/10) 2 (1/10)
Numbers before the brackets indicate the degree of histopathologic changes: 1-minimal; 2-mild; 3-moderate; 4-severe. Numbers in the brackets indicate the incidence of histopathologic changes. “d” means no observed changes.
showed that based on the data from 12 different provinces, the concentrations of several REEs including yttrium in different dietary samples in China were higher than other REEs (Zhou et al., 2014). And the average dietary intake of yttrium was 250 mg/day for male adults (Zhou et al., 2014), i.e. approximate to 4 mg/kg/day. Therefore additional attention should be focused on the food safety risk posed by this element. However, duo to the scarcity of longterm repeated oral exposure toxicological studies, especially studies performed in compliance with the regulatory requirements, no NOAEL for yttrium has yet been developed, accordingly, no official ADI value has yet been proposed for this element. To achieve a NOAEL and an estimated ADI for yttrium if possible, we conducted a subchronic oral exposure toxicity study in rats in accordance with the OECD guideline and GLP principles. The present subchronic toxicity study revealed that repeated oral exposure of rats to yttrium nitrate up to 90 mg/kg daily (i.e. 29.1 mg/kg for yttrium) for 90 days produced no biologically or toxicologically significant changes or abnormalities in the following parameters: mortality, clinical signs, daily food consumption and weekly body weights, urinalysis, hematology, blood coagulation, clinical biochemistry, and histopathology of all the main organs/ tissues except lung. In several yttrium-treated rats particularly in 30 and 90 mg/kg groups, macrophage accumulation and foreign body granuloma in the lungs were observed, suggesting the presence or deposition of yttrium crystals in the lung. Distribution and deposition of yttrium in the lung had earlier been found in rats instilled intratracheally (Hirano et al., 1990) or injected intravenously (Nakamura et al., 1997) yttrium chloride. Intratracheally instilled yttrium chloride was found to be accumulated in alveolar macrophages and pneumonocytes, and the pulmonary clearance of yttrium was very slow with a half-life of 168 days (Hirano et al., 1990). Granulomatous lesions in the lungs were also observed following intratracheal instillation of yttrium chloride (Hirano et al., 1990). It had also been revealed that inhaled REEs such as cerium and lanthanum could cause granulomatous lesions in the lungs (Haley, 1991; Suzuki et al., 1992; Aalapati et al., 2014; Nemmar et al., 2017). However, there is no evidence that oral exposure to yttrium leads to the pulmonary deposition and lesions. In the present study, the observed bronchus-derived distribution of pulmonary granulomatous lesion in orally yttrium-treated rats is similar to that induced by intratracheal instillation of yttrium chloride (Hirano et al., 1990). In addition, yttrium nitrate solution is easily crystallized under the condition of pH > 6.0 in our current study. Considering these facts, we thus speculate there is the possibility that during the performance of long-term oral gavage, accidental intratracheal instillations or inhalations of microscale yttrium nitrate droplets into the tracheas and lung may occur. In pulmonary environment, this accidental intratracheal instillations or inhalations of yttrium solution can lead to the crystallization of yttrium nitrate and subsequent deposition, thereby inducing
pulmonary macrophage accumulation and foreign body granuloma in the lungs. In order to validate this possibility, we conducted a short-term repeated intratracheal instillation exposure test in rats using 2 concentrations of yttrium nitrate solutions (1.0 and 9.0 mg/mL), which are same to those used for 10 and 90 mg/kg dosages in the aforementioned oral exposure test, respectively. Rats (5 per sex per group) were intratracheally instilled daily 100 mL distilled water or yttrium nitrate solutions (1.0 and 9.0 mg/mL) for 14 days. All animals were subjected to histopathologic examinations of the lungs following 14-day exposure. Significant foreign body granuloma in the lungs was observed in almost all intratracheally yttrium nitrate instilled-rats (Fig. 4), which is similar to those observed in the aforementioned 90-day repeated oral exposure test. No abnormal pulmonary changes in distilled water-instilled rats were noted. Based on these findings, we consider the pulmonary foreign body granuloma observed in orally yttrium nitrate-administered rats by gavage can be attributed to the accidental intratracheal instillations or inhalations of microscale yttrium nitrate solution into the lung and subsequent crystallization during long-term oral administration. Consequently, we exclude the lung as a direct toxic target for yttrium oral exposure. 5. Conclusion The present study suggests that repeated oral exposure to yttrium nitrate up to 90 mg/kg daily for 90 days exerts no adverse effects on rats, thus the NOAEL is set at 90 mg/kg for yttrium nitrate, i.e. 29.1 mg/kg for yttrium. Traditionally, the acceptable daily intake (ADI) or tolerable daily intake (TDI) or the provisional maximum TDI (PMTDI) for a food additive or contaminant is established based on the most critical NOAEL which is derived from toxicological studies on laboratory animals, with the uncertainties being taken into consideration. An uncertainty factor of 100 or higher is conventionally applied, considering the extrapolation differences (such as extrapolation from animals to humans, extrapolation from high to low exposure levels) and interindividual variability (Speijers, 1999). Considering the vulnerability of developing children to food and environmental contaminants and the adverse effects of yttrium on the neurobehavior, learning and memory in young animals (Wu et al., 2006; Yang et al., 2008), we adopted a relative conservative uncertainty factor of 200 to estimate the ADI value for yttrium. Accordingly, the ADI of 145.5 mg/kg/day for yttrium was derived from the NOAEL of 29.1 mg/kg. According to the average dietary intake of yttrium of 250 mg/day (i.e. 4 mg/kg/ day) for male adults from the 4th China total diet survey in 2007 (Zhou et al., 2014), this estimated ADI value indicates that the daily intake of yttrium from foods is acceptable and relative safe for adults. However, more attention should be paid to the health risk of chronic dietary intake of yttrium in children due to their
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Fig. 4. Histopathologic changes in the lungs of rats intratracheally instilled yttrium nitrate. Rats were intratracheally instilled different concentrations (1.0 and 9.0 mg/mL) of yttrium nitrate solution or distilled water for 14 days, and the histopathologic changes in the lungs were then evaluated. The presented images were the representatives of histopathologic changes of the lungs. A (normal) and B (foreign body granuloma) indicated control and 1.0 mg/mL yttrium-treated rats, respectively; C (foreign body granuloma) and D (foreign body granuloma) indicated 9.0 mg/mL yttrium-treated rats. Magnification, 200.
vulnerability and in the populations residing in REEs mining areas with higher exposure. In addition, the present subchronic oral exposure toxicity study may provide scientific data for the risk assessment of yttrium and REEs, and for the revision of the Chinese food safety standards of REEs. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.yrtph.2017.08.020. References Aalapati, S., Ganapathy, S., Manapuram, S., Anumolu, G., Prakya, B.M., 2014. Toxicity and bio-accumulation of inhaled cerium oxide nanoparticles in CD1 mice. Nanotoxicology 8, 786e798. Du, X., Graedel, T.E., 2011. Uncovering the global life cycles of the rare earth elements. Sci. Rep. 1, 145. Haley, P.J., 1991. Pulmonary toxicity of stable and radioactive lanthanides. Health Phys. 61, 809e820. He, M.L., Wehr, U., Rambeck, W.A., 2010. Effect of low doses of dietary rare earth elements on growth performance of broilers. J. Anim. Physiol. Anim. Nutr. Berl. 94, 86e92. Hirano, S., Kodama, N., Shibata, K., Suzuki, K.T., 1990. Distribution, localization, and pulmonary effects of yttrium chloride following intratracheal instillation into the rat. Toxicol. Appl. Pharmacol. 104, 301e311. Hu, X., Ding, Z., Wang, X., Chen, Y., Dai, L., 2002. Effects of lanthanum and cerium on
the vegetable growth of wheat (Triticumaestivum L.) seedlings. Bull. Environ. Contam. Toxicol. 69, 727e733. Jiang, D.G., Yang, J., Zhang, S., Yang, D.J., 2012. A survey of 16 rare Earth elements in the major foods in China. Biomed. Environ. Sci. 25, 267e271. Jin, S.L., Huang, Y.Z., Wang, F., Xu, F., Wang, X.L., Gao, Z., Hu, Y., Qiao, M., Li, J., Xiang, M., 2015. Rare earth elements content in farmland soils and crops of the surrounding copper mining and smelting plant in Jiangxi province and evaluation of its ecological risk. Environ. Sci. 36, 1060e1068 [Article in Chinese]. Li, C.X., Geng, X., Ma, C., Fang, H.Q., Zhi, Y., Yu, Z., Jia, X.D., Xu, H.B., 2015a. Study on the neurobehavior and cognitive ability of rat offspring exposed to yttrium nitrate. Chin. J. Food Hyg. 27, 104e109 [Article in Chinese]. Li, C.X., Ma, C., Fang, H.Q., Zhi, Y., Yu, Z., Xu, H.B., Jia, X.D., 2015b. Neurobehavioral assessment of rats exposed to yttrium nitrate during development. Biomed. Environ. Sci. 28, 281e290. Li, X., Chen, Z., Chen, Z., Zhang, Y., 2013. A human health risk assessment of rare earth elements in soil and vegetables from a mining area in Fujian Province, Southeast China. Chemosphere 93, 1240e1246. Li, Y., Yang, J.L., Jiang, Y., 2012. Trace rare earth element detection in food and agricultural products based on flow injection walnut shell packed microcolumn preconcentration coupled with inductively coupled plasma mass spectrometry. J. Agric. Food Chem. 60, 3033e3041. Nakamura, Y., Tsumura, Y., Tonogai, Y., Shibata, T., Ito, Y., 1997. Differences in behavior among the chlorides of seven rare earth elements administered intravenously to rats. Fundam. Appl. Toxicol. 37, 106e116. Nemmar, A., Al-Salam, S., Beegam, S., Yuvaraju, P., Ali, B.H., 2017. The acute pulmonary and thrombotic effects of cerium oxide nanoparticles after intratracheal instillation in mice. Int. J. Nanomed. 12, 2913e2922. OECD, 1998. OECD Guidelines for the Testing of Chemicals. Test No. 408: Repeated Dose 90-day Oral Toxicity Study in Rodents (21 Sep 1998). http://www.oecdilibrary.org/environment/test-no-408-repeated-dose-90-day-oral-toxicitystudy-in-rodents_9789264070707-en. Pagano, G., Guida, M., Tommasi, F., Oral, R., 2015. Health effects and toxicity mechanisms of rare earth elements-Knowledge gaps and research prospects. Ecotoxicol. Environ. Saf. 115, 40e48. Pang, X., Li, D., Peng, A., 2002. Application of rare-earth elements in the agriculture of China and its environmental behavior in soil. Environ. Sci. Pollut. Res. Int. 9,
Y.-M. Wang et al. / Regulatory Toxicology and Pharmacology 90 (2017) 116e125 143e148. Peng, R.L., Pan, X.C., Xie, Q., 2003. Relationship of the hair content of rare earth elements in young children aged 0 to 3 years to that in their mothers living in a rare earth mining area of Jiangxi. Chin. J. Prev. Med. 37, 20e22 [Article in Chinese]. Rim, K.T., Koo, K.H., Park, J.S., 2013. Toxicological evaluations of rare earths and their health impacts to workers: a literature review. Saf. Health Work 4, 12e26. Schroeder, H.A., Mitchener, M., 1971. Scandium, chromium (VI), gallium, yttrium, rhodium, palladium, indium in mice: effects on growth and life span. J. Nutr. 101, 1431e1438. Seishiro, H., Suzuki, K.T., 1996. Exposure, metabolism, and toxicity of rare earths and related compounds. Environ. Health Perspect. 104, 85e95. Song, Y., Liu, Z.P., Zhang, L., Sui, H.X., Mao, W.F., Bao, H.H., Yong, L., Li, N., 2016. Exposure assessment of rare earth elements in brick tea among Chinese adults. Chin. J. Food Hyg. 28, 249e233. [Article in Chinese]. Speijers, G.J.A., 1999. Precision of estimates of an ADI (or TDI or PTWI). Regul. Toxicol. Pharmacol. 30, S87eS93. Suzuki, K.T., Kobayashi, E., Ito, Y., Ozawa, H., Suzuki, E., 1992. Localization and health effects of lanthanum chloride instilled intratracheally into rats. Toxicology 76, 141e152. Tong, S.L., Zhu, W.Z., Gao, Z.H., Meng, Y.X., Peng, R.L., Lu, G.C., 2004. Distribution characteristics of rare earth elements in children's scalp hair from a rare earths
125
mining area in southern China. J. Environ. Sci. Health A Tox. Hazard. Subst. Environ. Eng. 39, 2517e2532. USEPA, 2012. Rare Earth Elements: a Review of Production, Processing, Recycling, and Associated Environmental Issues. EPA 600/R-12/572. www.epa.gov/ord. Wei, B., Li, Y., Li, H., Yu, J., Ye, B., Liang, T., 2013. Rare earth elements in human hair from a mining area of China. Ecotoxicol. Environ. Saf. 96, 118e123. Wu, M.Y., Zhang, P., Yang, W.D., Liu, J.S., Mo, W.X., 2006. Effect of long-term intake of Y3þ in drinking water on learning-memory functions and growth-development of rats. J. Hyg. Res. 35, 283e285 [Article in Chinese]. Yang, W.D., Zhang, H.P., Liu, J.S., Xia, W.X., 2008. Effect of long-term intake of Y3þ on learning and memory of rats. Chin. J. Public Health 24, 183e184 [Article in Chinese]. Zhou, R., Liu, L.P., Yue, B., Li, X.W., Zhao, Y.F., Wu, Y.N., 2014. Dietary intake assessment for rare earth elements from 4th China total diet study. Mod. Prev. Med. 41, 1975e1978 [Article in Chinese]. Zhu, W., Xu, S., Shao, P., Zhang, H., Wu, D., Yang, W., Feng, J., Feng, L., 2005. Investigation on liver function among population in high background of rare earth area in South China. Biol. Trace Elem. Res. 104, 1e8. Zhuang, M., Zhao, J., Li, S., Liu, D., Wang, K., Xiao, P., Yu, L., Jiang, Y., Song, J., Zhou, J., Wang, L., Chu, Z., 2017. Concentrations and health risk assessment of rare earth elements in vegetables from mining area in Shandong, China. Chemosphere 168, 578e582.
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Subchronic toxicity study of yttrium nitrate by 90-day repeated oral exposure in rats Yi-Mei Wang a, *, 1, Zhou Yu b, 1, Zeng-Ming Zhao a, Li Jia a, Hai-Qin Fang b, Ting-Fen Zhang a, Xiao-Yan Yuan a, Yu-Lei Shu a, Jun He a, Hui Peng a, Li-Zhong Li a, Jun Zhao a, Xu-Dong Jia b, **, Shuang-Qing Peng a, *** a b
Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, PLA, Beijing 100071, China Key Laboratory of Food Safety Risk Assessment of Ministry of Health, China National Center for Food Safety Risk Assessment, Beijing 100021, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 June 2017 Received in revised form 28 August 2017 Accepted 29 August 2017 Available online 1 September 2017
Concerns regarding the adverse effects of long-term exposure to low levels of rare earth elements (REEs) from foods on human health have arisen in recent years. Nevertheless, no official acceptable daily intake (ADI) has yet been proposed for either total REEs or individual REE. In accordance with the Organization for Economic Co-operation and Development (OECD) testing guideline, the present study was undertaken to evaluate the subchronic toxicity of yttrium, a representative heavy REE with higher contaminated level in foods in China, to achieve a no observed adverse effect level (NOAEL) which is a critical basis for the establishment of an ADI. Yttrium nitrate was orally administered to rats at doses of 0, 10, 30 and 90 mg/kg/day for 90 days followed by a recovery period of 4 weeks. The following toxicity indices were measured: mortality, clinical signs, daily food consumption and weekly body weight; urinalysis, hematology, blood coagulation, clinical biochemistry and histopathology at the end of administration and recovery periods. No toxicologically significant changes were found in any yttrium-treated group as compared to the concurrent control group. Under the present experimental condition, the NOAEL in rats was thus set at 90 mg/kg for yttrium nitrate, i.e. 29.1 mg/kg for yttrium. © 2017 Elsevier Inc. All rights reserved.
Keywords: Rare earth elements ADI Yttrium Subchronic toxicity NOAEL
1. Introduction The rare earth elements (REEs) are a group of metals which are comprised of lanthanum, 14 lanthanides, yttrium (Y) and scandium. Due to their unique physical and chemical properties, REEs have been widely utilized in industrial and medical fields in the last decades (Du and Graedel, 2011; USEPA, 2012). The growing increases in REEs-related industrial activities imply human occupational exposure to REEs. Human environmental exposure to REEs
* Corresponding author. Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, PLA, 20 Dongdajie Street, Fengtai District, Beijing 100071, China. ** Corresponding author. *** Corresponding author. Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, PLA, 20 Dongdajie Street, Fengtai District, Beijing 100071, China. E-mail addresses: [email protected] (Y.-M. Wang), [email protected]. cn (X.-D. Jia), [email protected] (S.-Q. Peng). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.yrtph.2017.08.020 0273-2300/© 2017 Elsevier Inc. All rights reserved.
has also been found in the populations residing in REEs mining areas in China (Peng et al., 2003; Tong et al., 2004; Zhu et al., 2005; Wei et al., 2013). REEs have also been widely used in agricultural and zootechnical fields as fertilizers for crops and as feed additives for livestock, poultry and aquaculture in China (Hu et al., 2002; Pang et al., 2002; He et al., 2010). Established and growing evidence points to REEs-related marine, fresh water and soil pollution and accumulation. As a result, different levels of REEs from agricultural products of mining areas (Li et al., 2013; Jin et al., 2015; Zhuang et al., 2017) or non-mining areas (Li et al., 2012; Jiang et al., 2012; Song et al., 2016) have been detected, thereby increasing their accumulations in human body when ingested through food chain. REEs have been reported to exert miscellaneous toxicities to animals and humans via different exposure routines (Reviewed by Seishiro and Suzuki, 1996; Rim et al., 2013; Pagano et al., 2015). Concerns regarding the adverse effects of long-term exposure to low levels of REEs from foods on human health have arisen, as they can accumulate in blood, brain and bone after entering into human body (Seishiro and Suzuki, 1996). However, the existing
Y.-M. Wang et al. / Regulatory Toxicology and Pharmacology 90 (2017) 116e125
toxicological studies are yet confined to few light REEs, and scanty toxicological data are available for other REEs, especially heavy REEs. More importantly, no official acceptable daily intake (ADI) value, the amount of a chemical or food contaminant that can be ingested daily over a lifetime without appreciable health risk, has yet been proposed for either total REEs or individual REE. Yttrium (Y) is a heavy REE, and has also been widely used in industrial, medical and agricultural fields. In China, relative high contaminated levels of yttrium in foods have been detected (Jiang et al., 2012; Song et al., 2016). Therefore, the potential for adverse human health effects from oral exposure to yttrium, especially through long-term consumption of yttrium-contaminated foods, warrants greater attention. Nevertheless, there are only few oral exposure toxicological studies for yttrium. Schroeder and Mitchener (1971) previously reported that the growth of mice was depressed when they were given 5 ppm of Y3þ in drinking water, and the longevity was increased in Y3þ-fed mice. Rats were fed with water dissolved different levels of Y3þ for 6 months, and it was shown that low level (0.534 mg/L) of Y3þ might improve the functions of learning and memory in rats, but high level (5340 mg/ L) of Y3þ could restrain the learning-memory functions and growth-development in rats (Wu et al., 2006). In the recent toxicological investigations performed in accordance with the guideline issued by the Organization of Economic Co-operation and Development (OECD) (OECD TG 426), the effects of yttrium on the neurobehavior and cognitive ability of rat offspring have been assessed. Dams were orally exposed to 0, 5, 15, or 45 mg/kg daily of yttrium nitrate from gestation day (GD) 6 to postnatal day (PND) 21, and their offspring were given the same doses until PND 63. The results revealed that oral exposure of rats to yttrium nitrate in doses up to 45 mg/kg daily had no adverse effects on their neurobehavioral development and cognitive ability (Li et al., 2015a,b). As well-known, for a chemical or food additive or contaminant, the no observed adverse effect level (NOAEL) which is primarily derived from the repeated dose toxicity studies in rodents, is a critical basis for the establishment of an ADI, a very critical value for food safety risk assessment. However, the general toxicity of yttrium following long-term repeated oral exposure has not been scientifically assessed in compliance with the international testing guidelines and regulatory requirements. Accordingly, no NOAEL or ADI value for yttrium has yet been developed to date. Therefore, it is extremely necessary to reveal the long-term toxicological effects of yttrium via oral exposure by using current international test guidelines. In this research, according to the OECD guidelines for the Testing of Chemicals “Repeated Dose 90-Day Oral Toxicity Study in Rodents” (OECD Test No. 408) and Good Laboratory Practice (GLP), the subchronic toxicity of yttrium to rats was evaluated and the NOAEL was estimated, which can provide scientific data for the food safety risk assessment of yttrium and REEs. Our present subchronic toxicity study reveals that repeated oral exposure of rats to yttrium nitrate in doses up to 90 mg/kg daily for 90 days exerts no adverse effects on rats, and the NOAEL is set at 90 mg/kg for yttrium nitrate, i.e. 29.1 mg/kg for yttrium. Accordingly, the ADI value for yttrium was estimated to be 145.5 mg/kg/day by using an uncertainty factor of 200. Based on the current contaminated levels of yttrium in foods and average dietary intake of yttrium for adults in China, this estimated ADI value indicates the daily intake of yttrium from foods is relative acceptable and safe for adults. 2. Materials and methods 2.1. Test substance Yttrium nitrate stock solution with the concentration of
117
218.28 mg/mL was provided by China National Center for Food Safety Risk Assessment (Beijing, China). When used, yttrium nitrate stock solution was diluted to the indicated dosing concentrations with distilled water and was adjusted to pH 6.0 by using 1 mol/L sodium hydroxide. 2.2. Animals and dosing Sprague-Dawley (SD) rats (50e70 g) were obtained from the Laboratory Animal Center of Academy of Military Medical Sciences (Beijing China). The rats were housed at Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, PLA (Beijing China). After 6 days of acclimatization and quarantine, the healthy rats were randomly assigned into control group or one of 3 yttrium dosing groups according to body weights separated by sex, with 15 rats per sex per group. All rats were housed five per sex in standard PVC cages (i.e. 3 cages per sex per group) in a ventilated room (10e20 air exchanges/hr) with controlled illumination (12 h light/dark cycle), temperature (20 Ce25 C) and humidity (40%e 70%). Feed (standard rodent diet) and tap water were provided ad libitum. Rats in 3 yttrium exposure groups were given yttrium nitrate solution by oral gavage at a daily dose of 10, 30, and 90 mg/kg body weight, respectively. Control rats received equivalent volumes of vehicle of distilled water only. The application volume for all groups was 1 mL/100 g body weight. The animals were treated with the test substance or vehicle once daily for a period of 90 days followed by a recovery period of 4 weeks. Ten rats (2 cages) per sex of each group were subjected to necropsy one day after the last administration (the end of treatment period), and the remaining 5 rats (1 cage) per sex of each group were subjected to necropsy 28 days after the last administration (the end of recovery period). All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Institute of Disease Control and Prevention, PLA (approval No.: 2013-021), and were in strict accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The protocol was generally based on the OECD guidelines for the Testing of Chemicals “Repeated Dose 90-day Oral Toxicity Study in Rodents” (OECD Test No. 408) (OECD, 1998). And the study was conducted at Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, PLA (Beijing China), a Good Laboratory Practice (GLP)-certified laboratory, and was in strict accordance with the GLP principles of China Food and Drug Administration and Ministry of Environmental Protection of China. 2.3. Observations and examinations The following parameters/indices were determined: mortality, daily clinical observation, daily food consumption, weekly body weight; urinalysis, hematology, blood coagulation, clinical biochemistry and histopathologic examination at the end of administration and recovery periods. Animals were fasted overnight before blood collection. 2.3.1. Clinical observation and mortality All animals were checked daily for any clinically abnormal signs. A check for moribund and dead animals was made once daily. If animals were in a moribund state, they were sacrificed and necropsied. 2.3.2. Body weight and food consumption Body weights were recorded on the first day of administration, weekly thereafter and prior to sacrifice. The 24 h feed intake pattern on rats was measured weekly for each cage. Briefly, at weekly interval, the supplied amount of food
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to each cage and the remnant of each cage were measured to calculate the difference, which was regarded as weekly food consumption of each cage. The average of daily feed intake per rat per cage was then used to calculate the mean food consumption in grams per rat per day for one group. 2.3.3. Urinalysis At the end of administration and recovery periods, urine samples from 5 animals per sex of each group were collected, respectively. The following parameters were analyzed using a urine analyzer (Scan 100; Combi, Germany): leukocytes, nitrite, urobilinogen, bilirubin, urinary protein, glucose, ketone body, specific gravity, pH and occult blood. Additionally, urine appearance (color and transparency) was recorded. 2.3.4. Hematology, blood coagulation and clinical biochemistry At the end of administration period (10 rats/sex/group) and recovery period (5 rats/sex/group), the overnight fasted rats were anesthetized with pentobarbital sodium (50 mg/kg), and blood was then collected from the abdominal aorta for the determinations of hematology, blood coagulation and clinical biochemistry. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. Hematology was analyzed using an automatic hematology analyzer (Nihon Kohden, Japan), and blood smears were prepared and stained to count the reticulocytes. The following parameters are measured: red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT), reticulocyte (RET), white blood cell (WBC); percentages of lymphocyte [LYM (%)], monocyte [MON (%)], neutrophil [NEUT (%)], eosinophil [EOS (%)] and basophil [BAS (%)]. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were analyzed using a semi-automatic blood coagulation analyzer (Sysmex, Japan). Clinical biochemistry was measured using an automatic biochemical analyzer (Hitachi, Japan) and an automatic electrolyte (Na/K/Cl) analyzer (Medica, USA). The measured parameters are as follows: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), total bilirubin (TBIL), alkaline phosphatase (ALP), glucose (GLU), creatinine (CREA), cholesterol (CHOl), triglyceride (TG), creatine kinase (CK); sodium ion (Naþ), potassium ion (Kþ) and chloride ion (Cl). 2.3.5. Histopathology At the end of administration and recovery periods, the overnight fasted rats were weighed and sacrificed by anesthesia and exsanguinations after blood collection, followed by a detailed gross necropsy. 2.3.5.1. Organ weights. The following organs from all animals were isolated and weighed at scheduled necropsy before fixation: brain, heart, livers, spleen, lungs, kidneys, testes, epididymides, uterus, ovaries, thymus, and adrenals. Relative organ weight was calculated as the ratio of organ weight to the terminal fasted body weight. 2.3.5.2. Histopathologic examination. Besides the organs described above for weight measurement, samples of the following tissues and organs were collected from all animals at necropsy and were fixed in 10% neutral buffered formalin: brain (cerebrum, cerebellum and pons), pancreas, oesophagus, stomach, small and large intestines (duodenum, jejunum, ileum, caecum, colon, rectum), mesenteric lymph nodes,
submandibular gland, thyroid, parathyroid glands, salivary gland, trachea, prostate, mammary glands, pituitary gland, sciatic nerve with skeletal muscle, skin, urinary bladder, spinal cord, optic nerve, thoracic aorta, and sternum with bone marrow. For the animals of control group and high dose group sacrificed at the scheduled days, the afore-listed tissues and organs were histopathologically examined after preparation of paraffin sections and haematoxylin-eosin staining. The histopathologic examinations were not extended to animals of other dosage groups when no treatment-related changes were observed in the high dose group. 2.4. Statistical analysis Statistical analyses of body weight, food consumption, parameters of hematology, blood coagulation and clinical biochemistry, and absolute and relative organ weights were performed for each gender by comparing the values of dosed animals with control animals using a one-way analysis of variance and a post-hoc Dunnet t-test. Pathological results were descriptively analyzed, and the incidences of tissue lesions were analyzed using chi-square test. These statistics were performed with SPSS software, and the significance level was set at P < 0.05. 3. Results 3.1. Clinical observation All rats survived to the scheduled necropsies. There were no abnormal clinical signs or toxic effects that could be attributed to the administration of yttrium. 3.2. Body weight and food consumption Weekly mean body weights of both male (Fig. 1A) and female rats (Fig. 1B) increased with the progress of study in all groups. Yttrium exposure didn't evoke significant effects on the body weights of rats when compared with the concurrent control group for both sexes. As shown in Fig. 2, mean daily food consumptions for both males (Fig. 2A) and females (Fig. 2B) were similar among the 4 groups during the administration and recovery periods. 3.3. Hematology and blood coagulation As presented in Table 1, at the end of treatment and recovery periods, almost all the hematology and blood coagulation parameters in any yttrium treatment group were not significantly different from the concurrent control group for both sexes, except for a slight but biologically insignificant increase in PT in 30 mg/kg yttrium-treated female rats at the end of recovery period. 3.4. Clinical biochemistry At the end of treatment and recovery periods, there were no significant differences for most clinical biochemistry parameters in any yttrium treatment group when compared with the concurrent control group of the same sex (Table 2). Although statistically significant increases or decreases in several parameters such as TP, GLU and CREA were observed in 30 or 90 mg/kg yttrium-treated groups, these changes are irregular, no dose-related dependence, and are all within the normal reference ranges of SD rats in our laboratory. Therefore, these alterations were judged to be of no toxicological significance.
Y.-M. Wang et al. / Regulatory Toxicology and Pharmacology 90 (2017) 116e125
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Fig. 1. Effect of yttrium nitrate on weekly mean body weight of rats. Rats were orally administered different doses of yttrium nitrate for 90 days (13 weeks) by gavage, followed by a recovery period of 4 weeks. Body weights were recorded on the first day of administration (Week 1) and weekly thereafter during the treatment period (Weeks 2e14) and recovery period (Weeks 15e18). (A) Male rats; (B) female rats. Data were expressed as means ± SD. The number of animals in each group for each sex is 15 and 5 in the treatment and recovery periods, respectively.
Fig. 2. Effect of yttrium nitrate on mean daily food consumption of rats. Rats were orally administered different doses of yttrium nitrate for 90 days (13 weeks) by gavage, followed by a recovery period of 4 weeks. Food consumption for each cage of 5 rats was determined once a week by subtracting the weight of the remaining food from the weight of the supplied food of each cage, and the average of daily feed intake per rat for each cage was used to calculate the mean daily food consumption in grams per rat per day for one group. (A) Male rats; (B) female rats. The number of cages for each sex in each group is 3 and 1 in the treatment period and recovery period, respectively. Data were expressed as means ± SD in the treatment period (Weeks 1e13 after dosing), and as a single value in recovery period (Weeks 14e17 after dosing).
3.5. Urinalysis
3.6. Organ weights and histopathology
At the end of treatment and recovery periods, urine appearance (color and transparency) and all of the 10 urinary parameters in all groups were similar, and there were no statistically or biologically significant changes in any parameter in yttrium-treated groups as compared to the concurrent control group (data not shown).
3.6.1. Organ weights At necropsies of the end of treatment and recovery periods, no significant differences in the absolute weights of the measured organs were observed between any yttrium-treated group and the concurrent control group for both sexes (Table 3). As for the relative organ weight, only increased relative lung weights of male rats were noted in 30 and 90 mg/kg of yttrium treatment groups at the end of treatment period (Table 4).
Dose (mg/kg)
Sex
RBC (1012/L)
HGB (g/L)
HCT (%)
MCH (pg)
MCHC (g/L)
PLT (109/L)
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Table 1 Effects of repeated oral exposure to yttrium nitrate for 90 days on hematology and coagulation of rats. RET ( ‰)
WBC (109/L)
LYM (%)
MON (%)
NEUT (%)
EOS (%)
BAS (%)
PT (s)
APTT (s)
154 152 155 156 152 153 155 155
± ± ± ± ± ± ± ±
6 3 8 9 10 5 9 7
42.1 40.8 42.5 41.6 41.8 40.7 42.9 42.9
± ± ± ± ± ± ± ±
1.4 1.2 2.1 3.3 3.9 2.0 2.8 2.7
19.5 20.5 19.2 20.8 19.3 21.0 19.3 20.3
± ± ± ± ± ± ± ±
0.7 0.9 0.9 0.8 1.0 1.0 0.8 0.9
366 373 363 375 364 377 362 362
± ± ± ± ± ± ± ±
7 13 7 12 12 12 11 8
829 760 803 811 827 805 846 848
± ± ± ± ± ± ± ±
108 86 106 94 103 39 83 132
23 26 23 26 25 28 22 30
± ± ± ± ± ± ± ±
5 6 6 6 9 8 7 6
7.7 6.1 6.9 5.7 9.4 5.3 6.7 6.2
± ± ± ± ± ± ± ±
1.6 1.7 4.2 1.7 10.6 1.1 1.5 2.6
92.0 92.5 91.2 91.8 91.7 92.0 91.5 91.6
± ± ± ± ± ± ± ±
1.5 1.4 1.5 1.4 1.3 1.6 1.5 1.5
0.2 0.2 0.2 0.4 0.3 0.2 0.2 0.2
± ± ± ± ± ± ± ±
0.1 0.2 0.2 0.2 0.3 0.2 0.2 0.2
7.2 6.4 7.7 6.8 7.2 6.7 7.1 6.9
± ± ± ± ± ± ± ±
1.5 1.3 1.2 1.5 1.2 1.3 1.7 1.2
0.5 0.7 0.7 0.9 0.6 0.9 0.8 1.0
± ± ± ± ± ± ± ±
0.3 0.4 0.3 0.3 0.4 0.3 0.6 0.3
0.1 0.2 0.2 0.2 0.2 0.2 0.3 0.3
± ± ± ± ± ± ± ±
0.0 0.2 0.1 0.2 0.1 0.2 0.3 0.4
14.5 13.4 14.3 13.3 14.7 13.3 14.4 13.5
± ± ± ± ± ± ± ±
0.6 0.6 0.6 0.6 0.9 0.4 0.7 0.7
27.4 20.5 27.6 21.9 28.2 22.1 25.6 22.9
± ± ± ± ± ± ± ±
2.2 2.5 3.3 2.1 6.0 2.2 3.4 2.4
End of Recovery Period 0 M 7.84 ± 0.63 F 7.22 ± 0.28 10 M 7.51 ± 0.38 F 6.98 ± 0.21 30 M 7.56 ± 0.28 F 7.15 ± 0.29 90 M 7.77 ± 0.16 F 7.30 ± 0.31
154 147 146 146 156 151 153 151
± ± ± ± ± ± ± ±
5 5 7 7 6 10 3 4
41.7 40.7 40.1 39.8 41.2 41.1 41.9 40.8
± ± ± ± ± ± ± ±
2.6 1.4 1.6 1.6 1.7 2.7 0.8 1.8
19.8 20.3 19.4 20.9 20.6 21.2 19.7 20.7
± ± ± ± ± ± ± ±
1.2 0.5 0.9 1.0 0.3 0.7 0.1 0.7
371 361 364 367 379 368 366 370
± ± ± ± ± ± ± ±
13 8 15 6 6 7 3 10
862 776 905 829 883 830 895 803
± ± ± ± ± ± ± ±
69 152 120 112 44 115 73 26
38 32 34 35 36 38 36 32
± ± ± ± ± ± ± ±
4 6 5 8 7 7 6 4
5.8 2.8 8.0 2.3 5.4 2.5 6.2 4.1
± ± ± ± ± ± ± ±
1.7 1.3 7.4 0.3 0.5 0.9 1.2 1.6
93.7 93.1 92.4 93.1 92.6 93.3 92.5 93.6
± ± ± ± ± ± ± ±
1.2 1.8 2.1 1.6 1.4 1.7 1.2 2.1
0.1 0.2 0.2 0.2 0.3 0.1 0.2 0.2
± ± ± ± ± ± ± ±
0.1 0.2 0.2 0.1 0.1 0.2 0.2 0.2
5.2 5.9 6.4 5.9 6.1 5.9 6.2 5.3
± ± ± ± ± ± ± ±
1.4 1.6 1.8 1.3 1.2 1.4 0.9 1.7
0.7 0.7 0.7 0.7 0.7 0.6 0.9 0.7
± ± ± ± ± ± ± ±
0.2 0.5 0.5 0.3 0.3 0.4 0.2 0.5
0.2 0.2 0.3 0.2 0.3 0.0 0.3 0.2
± ± ± ± ± ± ± ±
0.2 0.4 0.2 0.3 0.2 0.1 0.1 0.2
13.6 13.3 14.0 13.5 13.7 14.5 14.0 13.3
± ± ± ± ± ± ± ±
2.0 0.6 0.8 0.6 0.7 0.7* 1.4 0.5
30.8 19.9 30.0 16.7 28.2 16.6 33.4 20.4
± ± ± ± ± ± ± ±
11.1 6.4 7.8 4.7 3.7 5.3 5.5 6.2
At the end of 90 days of treatment period and 4 weeks of recovery period, blood was collected from 10 and 5 rats per group per sex for hematology and blood coagulation determinations, respectively. Data were expressed as means ± SD. *P < 0.05, significantly different from the concurrent control group of same sex. RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet; RET, reticulocyte; WBC, white blood cell; LYM (%), percentage of lymphocyte; MON (%), percentage of monocyte; NEUT (%), percentage of neutrophil; EOS (%), percentage of eosinophil; BAS (%), percentage of basophil; PT, prothrombin time; APTT, activated partial thromboplastin time.
Table 2 Effects of repeated oral exposure to yttrium nitrate for 90 days on clinical biochemistry of rats. Dose (mg/kg)
Sex
ALT (U/L)
AST (U/L)
TP (g/L)
End of Treatment Period 0 M 28 ± 4 F 22 ± 3 10 M 30 ± 4 F 24 ± 4 30 M 31 ± 7 F 23 ± 6 90 M 29 ± 4 F 25 ± 10
78 87 78 76 78 81 82 85
End of Recovery Period 0 M 34 ± 4 F 21 ± 3 10 M 69 ± 69 F 23 ± 4 30 M 34 ± 4 F 21 ± 3 90 M 36 ± 9 F 26 ± 6
86 ± 18 82 ± 10 115 ± 57 71 ± 15 85 ± 6 74 ± 15 93 ± 23 78 ± 12
± ± ± ± ± ± ± ±
22 20 22 13 22 21 19 21
ALB (g/L)
TBIL (mmol/L)
ALP (U/L)
GLU (mmol/L)
Urea (mmol/L)
CREA (mmol/L)
CHOl (mmol/L)
TG (mmol/L)
Naþ (mmol/L)
CK (U/L)
Kþ (mmol/L)
Cl (mmol/L)
50.6 62.1 48.1 57.9 46.2 59.8 47.9 60.8
± ± ± ± ± ± ± ±
2.2 5.1 4.0 5.8 7.2 4.4 1.6* 4.3
23.5 28.2 22.6 26.3 22.4 27.1 22.7 28.0
± ± ± ± ± ± ± ±
0.6 2.3 1.2 1.8 2.6 2.2 0.7 3.2
1.0 1.6 0.9 1.2 0.8 1.2 1.1 1.2
± ± ± ± ± ± ± ±
0.2 0.5 0.3 0.3 0.2 0.4 0.4 0.6
87 54 91 52 94 50 77 57
± ± ± ± ± ± ± ±
15 19 17 12 21 12 11 13
8.3 7.8 8.5 8.2 8.8 8.8 8.2 8.2
± ± ± ± ± ± ± ±
0.7 1.1 1.3 1.1 2.2 0.6* 0.8 0.5
4.5 4.7 4.6 5.5 4.8 4.8 4.5 5.6
± ± ± ± ± ± ± ±
0.6 0.7 0.7 1.5 0.9 1.0 0.9 0.7
44 51 43 47 43 50 40 53
± ± ± ± ± ± ± ±
4 5 5 5 8 7 6 4
1.43 1.80 1.44 1.74 1.37 1.97 1.48 1.72
± ± ± ± ± ± ± ±
0.17 0.28 0.25 0.24 0.22 0.43 0.26 0.24
0.61 0.47 0.59 0.44 0.51 0.56 0.52 0.36
± ± ± ± ± ± ± ±
0.19 0.17 0.22 0.12 0.21 0.44 0.23 0.11
250 272 181 199 230 255 209 237
± ± ± ± ± ± ± ±
145 147 116 125 166 143 88 99
138.1 137.1 139.0 138.4 139.7 126.3 137.7 138.8
± ± ± ± ± ± ± ±
3.0 1.5 1.5 2.7 2.0 39.6 1.8 1.2
4.10 3.87 3.99 3.84 4.14 3.84 4.04 3.86
± ± ± ± ± ± ± ±
0.23 0.24 0.27 0.32 0.18 0.27 0.18 0.22
108.4 108.0 109.1 107.4 107.8 105.5 107.6 106.1
± ± ± ± ± ± ± ±
1.1 2.7 1.4 1.3 1.5 2.8 1.6 2.4
53.6 62.2 52.0 63.3 54.7 59.9 54.4 65.0
± ± ± ± ± ± ± ±
2.1 4.1 3.6 5.6 1.8 1.5 2.1 2.1
23.4 29.0 22.3 29.8 23.5 28.2 23.5 31.2
± ± ± ± ± ± ± ±
0.8 0.8 1.4 2.6 0.9 0.9 0.9 1.3
1.5 1.5 1.1 1.3 1.3 1.1 1.2 1.6
± ± ± ± ± ± ± ±
0.5 0.4 0.3 0.4 0.3 0.4 0.4 0.8
56 35 79 40 76 39 63 41
± ± ± ± ± ± ± ±
12 8 19 18 23 15 19 13
9.4 8.3 8.5 7.6 9.4 7.9 9.4 7.7
± ± ± ± ± ± ± ±
0.5 0.5 0.8 0.5* 1.0 0.2 0.9 0.2
4.5 5.4 5.3 5.5 5.3 6.3 5.4 5.2
± ± ± ± ± ± ± ±
0.6 1.0 0.5 0.6 0.8 1.0 0.3 0.4
30 20 21 20 15 22 31 19
± ± ± ± ± ± ± ±
11 5 5 9 11* 12 5 16
1.62 1.88 1.61 2.30 1.76 1.95 1.57 1.79
± ± ± ± ± ± ± ±
0.34 0.26 0.36 0.41 0.25 0.22 0.27 0.24
0.39 0.38 0.65 0.42 0.64 0.45 0.66 0.44
± ± ± ± ± ± ± ±
0.08 0.08 0.41 0.11 0.27 0.14 0.40 0.16
243 262 232 204 254 234 257 199
± ± ± ± ± ± ± ±
138 47 69 89 47 99 161 86
139.2 137.2 137.6 137.8 137.9 138.8 137.2 138.7
± ± ± ± ± ± ± ±
3.5 1.7 1.4 2.1 2.4 2.1 2.4 1.4
4.18 3.69 4.15 3.96 4.26 3.79 3.95 3.71
± ± ± ± ± ± ± ±
0.21 0.21 0.36 0.27 0.12 0.19 0.20 0.34
107.2 112.0 107.4 107.9 107.1 108.4 107.1 107.6
± ± ± ± ± ± ± ±
2.0 2.3 1.6 1.4* 1.8 2.4* 1.7 1.7**
At the end of 90 days of treatment period and 4 weeks of recovery period, blood was collected from 10 and 5 rats per group per sex for clinical biochemistry determinations, respectively. Data were expressed as means ± SD. *P < 0.05, **P < 0.01, significantly different from the concurrent control group of same sex. ALT, alanine aminotransferase; AST, aspartate aminotransferase; TP, total protein; ALB, albumin; TBIL, total bilirubin; ALP, alkaline phosphatase; GLU, glucose; CREA, creatinine; CHOl, cholesterol; TG, triglyceride; CK, creatine kinase; Naþ, sodium ion; Kþ, potassium ion; Cl, chloride ion.
Y.-M. Wang et al. / Regulatory Toxicology and Pharmacology 90 (2017) 116e125
End of Treatment Period 0 M 7.89 ± 0.36 F 7.43 ± 0.30 10 M 8.04 ± 0.46 F 7.50 ± 0.53 30 M 7.91 ± 0.76 F 7.32 ± 0.44 90 M 8.02 ± 0.57 F 7.67 ± 0.40
Table 3 Effects of repeated oral exposure to yttrium nitrate for 90 days on the organ weights (g) of rats. Dose (mg/kg)
Sex
Body weight
End of Treatment Period 0 M 532.5 ± F 316.6 ± 10 M 521.5 ± F 323.5 ± 30 M 502.3 ± F 327.3 ± 90 M 497.5 ± F 295.3 ±
Brain
Thymus
Heart
Lungs
Liver
Spleen
Kidneys
Adrenals
Testes/Ovaries
Epididymides/Uterus
2.131 1.953 2.155 1.930 2.142 2.014 2.151 1.901
± ± ± ± ± ± ± ±
0.147 0.098 0.148 0.088 0.135 0.091 0.141 0.088
0.416 0.352 0.433 0.348 0.455 0.358 0.338 0.322
± ± ± ± ± ± ± ±
0.146 0.120 0.178 0.069 0.287 0.082 0.100 0.055
1.665 1.098 1.633 1.109 1.562 1.211 1.551 1.052
± ± ± ± ± ± ± ±
0.168 0.105 0.143 0.187 0.188 0.200 0.097 0.093
1.802 1.359 1.893 1.581 2.094 1.466 2.146 1.503
± ± ± ± ± ± ± ±
0.283 0.148 0.290 0.231 0.367 0.186 0.410 0.345
12.670 ± 0.669 7.757 ± 1.294 12.199 ± 1.794 7.819 ± 1.008 11.877 ± 1.320 8.196 ± 1.338 11.711 ± 1.224 7.280 ± 0.373
0.805 0.605 0.818 0.616 0.854 0.598 0.819 0.588
± ± ± ± ± ± ± ±
0.126 0.099 0.111 0.106 0.184 0.094 0.101 0.100
3.070 1.790 2.974 1.900 2.901 1.920 2.853 1.773
± ± ± ± ± ± ± ±
0.132 0.188 0.422 0.260 0.388 0.165 0.174 0.157
0.069 0.076 0.064 0.077 0.067 0.081 0.067 0.076
± ± ± ± ± ± ± ±
0.008 0.010 0.012 0.016 0.012 0.011 0.009 0.012
3.098 0.169 3.329 0.166 3.231 0.170 2.966 0.171
± ± ± ± ± ± ± ±
0.279 0.036 0.376 0.044 0.270 0.032 0.854 0.034
1.351 0.592 1.441 0.646 1.276 0.658 1.242 0.807
± ± ± ± ± ± ± ±
0.138 0.144 0.136 0.346 0.158 0.222 0.264 0.350
End of Recovery Period 0 M 588.5 ± 47.1 F 326.8 ± 54.9 10 M 584.1 ± 65.8 F 323.8 ± 32.3 30 M 609.6 ± 56.0 F 312.3 ± 18.1 90 M 582.6 ± 54.2 F 314.3 ± 12.1
2.289 1.970 2.232 1.977 2.213 2.064 2.187 1.914
± ± ± ± ± ± ± ±
0.112 0.150 0.087 0.059 0.122 0.069 0.130 0.086
0.265 0.293 0.268 0.280 0.326 0.273 0.340 0.290
± ± ± ± ± ± ± ±
0.098 0.046 0.051 0.070 0.062 0.040 0.043 0.060
1.690 1.122 1.745 1.054 1.765 1.083 1.762 1.072
± ± ± ± ± ± ± ±
0.198 0.108 0.245 0.131 0.188 0.070 0.232 0.135
1.879 1.335 1.859 1.486 2.002 1.403 2.004 1.551
± ± ± ± ± ± ± ±
0.158 0.238 0.242 0.187 0.337 0.187 0.127 0.324
13.069 ± 1.339 7.990 ± 1.492 14.016 ± 2.343 8.254 ± 0.743 13.903 ± 1.479 7.586 ± 0.387 13.970 ± 2.458 7.737 ± 0.749
0.905 0.559 0.925 0.549 0.896 0.536 0.802 0.588
± ± ± ± ± ± ± ±
0.163 0.107 0.109 0.053 0.179 0.109 0.160 0.011
3.026 1.874 2.939 1.933 3.074 1.832 2.942 1.833
± ± ± ± ± ± ± ±
0.241 0.301 0.253 0.150 0.277 0.085 0.400 0.153
0.063 0.078 0.054 0.075 0.059 0.067 0.061 0.074
± ± ± ± ± ± ± ±
0.008 0.015 0.014 0.011 0.017 0.008 0.010 0.006
3.084 0.163 3.466 0.179 2.685 0.164 3.355 0.169
± ± ± ± ± ± ± ±
0.184 0.028 0.329 0.032 1.045 0.029 0.443 0.021
1.319 0.552 1.549 0.743 1.220 0.872 1.394 0.604
± ± ± ± ± ± ± ±
0.159 0.169 0.126 0.168 0.481 0.420 0.093 0.070
At the end of 90 days of treatment period and 4 weeks of recovery period, the main organs listed were isolated from 10 and 5 rats per group per sex and weighed, respectively. Data were expressed as means ± SD.
Table 4 Effects of repeated oral exposure to yttrium nitrate for 90 days on the relative organ weights to body weight (%) of rats. Dose (mg/kg)
Sex
Brain
End of Treatment Period 0 M 0.402 F 0.625 10 M 0.415 F 0.603 30 M 0.428 F 0.626 90 M 0.434 F 0.644 End of Recovery Period 0 M 0.390 F 0.614 10 M 0.386 F 0.615 30 M 0.366 F 0.663 90 M 0.377 F 0.610
Thymus
Heart
Lungs
Liver
Spleen
Kidneys
Adrenals
Testes/Ovaries
Epididymides/Uterus
± ± ± ± ± ± ± ±
0.045 0.072 0.030 0.070 0.034 0.080 0.039 0.029
0.078 0.110 0.082 0.107 0.092 0.109 0.068 0.109
± ± ± ± ± ± ± ±
0.026 0.027 0.030 0.015 0.061 0.015 0.021 0.017
0.313 0.349 0.314 0.342 0.311 0.373 0.313 0.357
± ± ± ± ± ± ± ±
0.031 0.032 0.018 0.037 0.019 0.062 0.030 0.034
0.340 0.432 0.363 0.490 0.418 0.453 0.433 0.510
± ± ± ± ± ± ± ±
0.059 0.048 0.044 0.068 0.073* 0.067 0.088* 0.122
2.382 2.444 2.331 2.416 2.365 2.501 2.351 2.467
± ± ± ± ± ± ± ±
0.107 0.166 0.207 0.151 0.202 0.110 0.163 0.131
0.151 0.191 0.157 0.191 0.170 0.183 0.165 0.199
± ± ± ± ± ± ± ±
0.018 0.017 0.017 0.031 0.037 0.014 0.019 0.031
0.578 0.568 0.569 0.588 0.576 0.592 0.575 0.600
± ± ± ± ± ± ± ±
0.032 0.048 0.043 0.048 0.043 0.059 0.034 0.049
0.013 0.024 0.012 0.024 0.013 0.025 0.013 0.046
± ± ± ± ± ± ± ±
0.001 0.003 0.003 0.004 0.002 0.003 0.002 0.064
0.582 0.053 0.641 0.051 0.645 0.052 0.594 0.058
± ± ± ± ± ± ± ±
0.046 0.009 0.079 0.011 0.052 0.011 0.174 0.011
0.254 0.188 0.279 0.195 0.255 0.204 0.250 0.273
± ± ± ± ± ± ± ±
0.026 0.045 0.043 0.082 0.033 0.073 0.055 0.119
± ± ± ± ± ± ± ±
0.028 0.094 0.048 0.057 0.045 0.040 0.035 0.042
0.044 0.090 0.046 0.086 0.054 0.088 0.058 0.092
± ± ± ± ± ± ± ±
0.015 0.011 0.010 0.018 0.011 0.014 0.005 0.019
0.288 0.348 0.299 0.327 0.290 0.349 0.302 0.340
± ± ± ± ± ± ± ±
0.033 0.046 0.026 0.046 0.017 0.038 0.022 0.032
0.321 0.410 0.318 0.461 0.330 0.451 0.347 0.491
± ± ± ± ± ± ± ±
0.039 0.047 0.017 0.053 0.060 0.066 0.050 0.084
2.218 2.442 2.389 2.590 2.281 2.431 2.386 2.457
± ± ± ± ± ± ± ±
0.070 0.183 0.194 0.074 0.138 0.063 0.225 0.158
0.154 0.171 0.159 0.170 0.148 0.171 0.137 0.187
± ± ± ± ± ± ± ±
0.023 0.017 0.017 0.015 0.026 0.030 0.024 0.009
0.514 0.575 0.505 0.599 0.508 0.587 0.504 0.582
± ± ± ± ± ± ± ±
0.010 0.040 0.033 0.029 0.070 0.025 0.035 0.029
0.011 0.025 0.010 0.024 0.010 0.021 0.010 0.024
± ± ± ± ± ± ± ±
0.001 0.007 0.002 0.005 0.003 0.002 0.002 0.002
0.526 0.050 0.600 0.055 0.442 0.053 0.582 0.054
± ± ± ± ± ± ± ±
0.039 0.006 0.094 0.007 0.171 0.011 0.108 0.005
0.226 0.168 0.266 0.234 0.203 0.276 0.240 0.193
± ± ± ± ± ± ± ±
0.041 0.039 0.012 0.075 0.089 0.125 0.020 0.027
Y.-M. Wang et al. / Regulatory Toxicology and Pharmacology 90 (2017) 116e125
28.9 41.4 43.3 34.4 42.6 48.6 30.7 11.8
At the end of 90 days of treatment period and 4 weeks of recovery period, the main organs listed were isolated from 10 and 5 rats per group per sex and weighed, respectively, and the relative organ weights to body weight (%) were calculated. Data were expressed as means ± SD. *P < 0.05, significantly different from the concurrent control group of same sex. 121
122
Y.-M. Wang et al. / Regulatory Toxicology and Pharmacology 90 (2017) 116e125
3.6.2. Histopathologic examination At necropsies, adhesion of lung and thoracic cavity in one male rat of 90 mg/kg group was observed at the end of treatment period, and local brown change in the lung of one male of 30 mg/kg group was noted at the end of recovery period. Atrophies of testis and epididymis observed in one male of 90 mg/kg group were considered to be the frequently spontaneous pathological changes in this strain of this age, and were not treatment-related. At the end of treatment and recovery periods, microscopic examinations showed slight or mild chronic focal inflammatory cell infiltration in the heart and liver of several rats, and the incidence and severity were comparable between yttrium-treated and control groups. Chronic interstitial inflammation in the lungs was also observed in several yttrium-treated and control rats, with similar incidence and severity. Based on the historical occurrence rates of chronic inflammations in the heart, liver and lung of control SD rats from our previous repeated oral exposure toxicological studies, we consider these chronic inflammations observed in the present study are frequently spontaneous background changes in SD rats, rather than treatment-related pathological changes. In addition, slight or mild prostate epithelial proliferation and uterus dilatation, which are frequently spontaneous pathological changes in rats, were noted in few yttrium-treated rats, and were not associated with yttrium treatment. At the end of treatment period, foreign body granulomatous inflammation in the lungs, as evidenced by eosinophilic granulocyte infiltration and especially distinctive macrophage accumulation were observed in the 3 yttrium-treated groups. These pulmonary lesions were occasional in 10 mg/kg group (Fig. 3B; Table 5), while with more incidence and severity in 30 and 90 mg/ kg treatment groups (Fig. 3C and D; Table 5). At the end of recovery period, moderate ameliorations of these pulmonary lesions were
noted (Fig. 3F; Table 5). The increased relative lung weights along with pulmonary foreign body granuloma observed in 30 and 90 mg/kg of yttrium-treated rats indicated the pulmonary lesions might be yttrium treatmenterelated.
4. Discussion Since the widespread uses in agriculture for decades in China, REEs have accumulated in soils and aquatic environments, which can transfer to the agricultural products. REEs have been detected in different dietary samples, such as cereals, vegetables, beans, tea, aquatic products, meats and fresh eggs (Jiang et al., 2012; Zhou et al., 2014; Song et al., 2016), thereby increasing their bioaccumulations in human body through food intakes. Nevertheless, to our knowledge, no contaminated level limits in foods for either total REEs or individual element have been established worldwidely except China. In 2005, China developed the maximal contaminated levels of total rare earth oxides (REOs) in foods: 2.0 mg/kg for cereals and tea, and 0.7 mg/kg for vegetables and fruits. However, limit for individual RRE in any dietary sample has yet not been established. In addition, the existing toxicological studies are focused on only a few REEs, rarely on heavy REEs. As a result, the health risk posed by dietary intake of total REEs or individual element cannot be accurately assessed. Yttrium is one of the heavy REEs with higher concentrations in foods and higher total dietary intake. In a survey of 16 REEs in the major foods in China during 2009e2010, it was found that the concentrations of yttrium in the investigated foods were relative high (Jiang et al., 2012). The average content of yttrium in the major foods was 16.9 mg/kg with the maximum of 3470 mg/kg; and in fresh vegetables, the average content of yttrium was 34.1 mg/kg with the maximum of 3470 mg/kg. The 4th China total diet survey in 2007
Fig. 3. Histopathologic changes in the lungs of rats orally gavaged yttrium nitrate. Rats were orally administered different doses (10, 30 and 90 mg/kg) of yttrium nitrate or distilled water for 90 days by gavage followed by a recovery period of 4 weeks. At the end of administration and recovery periods, the rats were subjected to euthanasia and histopathologic changes in the lungs were evaluated by routine procedures. The presented images were the representatives of histopathologic changes of the lungs. A (chronic interstitial inflammation), B (chronic interstitial inflammation), C (foreign body granuloma) and D (foreign body granuloma) indicated control, 10, 30 and 90 mg/kg yttrium-treated rats at the end of treatment period, respectively; E and F indicated control and 90 mg/kg yttrium-treated rats at the end of recovery period, respectively. Magnification, 200.
Y.-M. Wang et al. / Regulatory Toxicology and Pharmacology 90 (2017) 116e125
123
Table 5 The severity and incidence of histopathologic changes in the lungs of rats received repeated oral exposure to yttrium nitrate. Histopathologic changes
End of Treatment Period
End of Recovery Period
Control
10 mg/kg
30 mg/kg
90 mg/kg
Control
10 mg/kg
30 mg/kg
90 mg/kg
Chronic interstitial inflammation
1 (2/20) 2 (7/20) 3 (4/20)
1 (4/20) 2 (9/20) 3 (1/20)
1 (2/20) 2 (6/20) 3 (2/20)
1 (7/20) 2 (6/20) 3 (2/20)
1 (4/10) 3 (1/10)
1 (4/10) 2 (2/10)
1 (4/10) 2 (4/10)
1 (2/10) 2 (3/10)
Foreign body granuloma
e
2 (1/20)
1 (2/20) 2 (3/20) 3 (1/20)
1 (5/20) 2 (3/20)
e
3 (1/10)
1 (1/10) 2 (3/10)
1 (3/10) 2 (1/10)
Numbers before the brackets indicate the degree of histopathologic changes: 1-minimal; 2-mild; 3-moderate; 4-severe. Numbers in the brackets indicate the incidence of histopathologic changes. “d” means no observed changes.
showed that based on the data from 12 different provinces, the concentrations of several REEs including yttrium in different dietary samples in China were higher than other REEs (Zhou et al., 2014). And the average dietary intake of yttrium was 250 mg/day for male adults (Zhou et al., 2014), i.e. approximate to 4 mg/kg/day. Therefore additional attention should be focused on the food safety risk posed by this element. However, duo to the scarcity of longterm repeated oral exposure toxicological studies, especially studies performed in compliance with the regulatory requirements, no NOAEL for yttrium has yet been developed, accordingly, no official ADI value has yet been proposed for this element. To achieve a NOAEL and an estimated ADI for yttrium if possible, we conducted a subchronic oral exposure toxicity study in rats in accordance with the OECD guideline and GLP principles. The present subchronic toxicity study revealed that repeated oral exposure of rats to yttrium nitrate up to 90 mg/kg daily (i.e. 29.1 mg/kg for yttrium) for 90 days produced no biologically or toxicologically significant changes or abnormalities in the following parameters: mortality, clinical signs, daily food consumption and weekly body weights, urinalysis, hematology, blood coagulation, clinical biochemistry, and histopathology of all the main organs/ tissues except lung. In several yttrium-treated rats particularly in 30 and 90 mg/kg groups, macrophage accumulation and foreign body granuloma in the lungs were observed, suggesting the presence or deposition of yttrium crystals in the lung. Distribution and deposition of yttrium in the lung had earlier been found in rats instilled intratracheally (Hirano et al., 1990) or injected intravenously (Nakamura et al., 1997) yttrium chloride. Intratracheally instilled yttrium chloride was found to be accumulated in alveolar macrophages and pneumonocytes, and the pulmonary clearance of yttrium was very slow with a half-life of 168 days (Hirano et al., 1990). Granulomatous lesions in the lungs were also observed following intratracheal instillation of yttrium chloride (Hirano et al., 1990). It had also been revealed that inhaled REEs such as cerium and lanthanum could cause granulomatous lesions in the lungs (Haley, 1991; Suzuki et al., 1992; Aalapati et al., 2014; Nemmar et al., 2017). However, there is no evidence that oral exposure to yttrium leads to the pulmonary deposition and lesions. In the present study, the observed bronchus-derived distribution of pulmonary granulomatous lesion in orally yttrium-treated rats is similar to that induced by intratracheal instillation of yttrium chloride (Hirano et al., 1990). In addition, yttrium nitrate solution is easily crystallized under the condition of pH > 6.0 in our current study. Considering these facts, we thus speculate there is the possibility that during the performance of long-term oral gavage, accidental intratracheal instillations or inhalations of microscale yttrium nitrate droplets into the tracheas and lung may occur. In pulmonary environment, this accidental intratracheal instillations or inhalations of yttrium solution can lead to the crystallization of yttrium nitrate and subsequent deposition, thereby inducing
pulmonary macrophage accumulation and foreign body granuloma in the lungs. In order to validate this possibility, we conducted a short-term repeated intratracheal instillation exposure test in rats using 2 concentrations of yttrium nitrate solutions (1.0 and 9.0 mg/mL), which are same to those used for 10 and 90 mg/kg dosages in the aforementioned oral exposure test, respectively. Rats (5 per sex per group) were intratracheally instilled daily 100 mL distilled water or yttrium nitrate solutions (1.0 and 9.0 mg/mL) for 14 days. All animals were subjected to histopathologic examinations of the lungs following 14-day exposure. Significant foreign body granuloma in the lungs was observed in almost all intratracheally yttrium nitrate instilled-rats (Fig. 4), which is similar to those observed in the aforementioned 90-day repeated oral exposure test. No abnormal pulmonary changes in distilled water-instilled rats were noted. Based on these findings, we consider the pulmonary foreign body granuloma observed in orally yttrium nitrate-administered rats by gavage can be attributed to the accidental intratracheal instillations or inhalations of microscale yttrium nitrate solution into the lung and subsequent crystallization during long-term oral administration. Consequently, we exclude the lung as a direct toxic target for yttrium oral exposure. 5. Conclusion The present study suggests that repeated oral exposure to yttrium nitrate up to 90 mg/kg daily for 90 days exerts no adverse effects on rats, thus the NOAEL is set at 90 mg/kg for yttrium nitrate, i.e. 29.1 mg/kg for yttrium. Traditionally, the acceptable daily intake (ADI) or tolerable daily intake (TDI) or the provisional maximum TDI (PMTDI) for a food additive or contaminant is established based on the most critical NOAEL which is derived from toxicological studies on laboratory animals, with the uncertainties being taken into consideration. An uncertainty factor of 100 or higher is conventionally applied, considering the extrapolation differences (such as extrapolation from animals to humans, extrapolation from high to low exposure levels) and interindividual variability (Speijers, 1999). Considering the vulnerability of developing children to food and environmental contaminants and the adverse effects of yttrium on the neurobehavior, learning and memory in young animals (Wu et al., 2006; Yang et al., 2008), we adopted a relative conservative uncertainty factor of 200 to estimate the ADI value for yttrium. Accordingly, the ADI of 145.5 mg/kg/day for yttrium was derived from the NOAEL of 29.1 mg/kg. According to the average dietary intake of yttrium of 250 mg/day (i.e. 4 mg/kg/ day) for male adults from the 4th China total diet survey in 2007 (Zhou et al., 2014), this estimated ADI value indicates that the daily intake of yttrium from foods is acceptable and relative safe for adults. However, more attention should be paid to the health risk of chronic dietary intake of yttrium in children due to their
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Fig. 4. Histopathologic changes in the lungs of rats intratracheally instilled yttrium nitrate. Rats were intratracheally instilled different concentrations (1.0 and 9.0 mg/mL) of yttrium nitrate solution or distilled water for 14 days, and the histopathologic changes in the lungs were then evaluated. The presented images were the representatives of histopathologic changes of the lungs. A (normal) and B (foreign body granuloma) indicated control and 1.0 mg/mL yttrium-treated rats, respectively; C (foreign body granuloma) and D (foreign body granuloma) indicated 9.0 mg/mL yttrium-treated rats. Magnification, 200.
vulnerability and in the populations residing in REEs mining areas with higher exposure. In addition, the present subchronic oral exposure toxicity study may provide scientific data for the risk assessment of yttrium and REEs, and for the revision of the Chinese food safety standards of REEs. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.yrtph.2017.08.020. References Aalapati, S., Ganapathy, S., Manapuram, S., Anumolu, G., Prakya, B.M., 2014. Toxicity and bio-accumulation of inhaled cerium oxide nanoparticles in CD1 mice. Nanotoxicology 8, 786e798. Du, X., Graedel, T.E., 2011. Uncovering the global life cycles of the rare earth elements. Sci. Rep. 1, 145. Haley, P.J., 1991. Pulmonary toxicity of stable and radioactive lanthanides. Health Phys. 61, 809e820. He, M.L., Wehr, U., Rambeck, W.A., 2010. Effect of low doses of dietary rare earth elements on growth performance of broilers. J. Anim. Physiol. Anim. Nutr. Berl. 94, 86e92. Hirano, S., Kodama, N., Shibata, K., Suzuki, K.T., 1990. Distribution, localization, and pulmonary effects of yttrium chloride following intratracheal instillation into the rat. Toxicol. Appl. Pharmacol. 104, 301e311. Hu, X., Ding, Z., Wang, X., Chen, Y., Dai, L., 2002. Effects of lanthanum and cerium on
the vegetable growth of wheat (Triticumaestivum L.) seedlings. Bull. Environ. Contam. Toxicol. 69, 727e733. Jiang, D.G., Yang, J., Zhang, S., Yang, D.J., 2012. A survey of 16 rare Earth elements in the major foods in China. Biomed. Environ. Sci. 25, 267e271. Jin, S.L., Huang, Y.Z., Wang, F., Xu, F., Wang, X.L., Gao, Z., Hu, Y., Qiao, M., Li, J., Xiang, M., 2015. Rare earth elements content in farmland soils and crops of the surrounding copper mining and smelting plant in Jiangxi province and evaluation of its ecological risk. Environ. Sci. 36, 1060e1068 [Article in Chinese]. Li, C.X., Geng, X., Ma, C., Fang, H.Q., Zhi, Y., Yu, Z., Jia, X.D., Xu, H.B., 2015a. Study on the neurobehavior and cognitive ability of rat offspring exposed to yttrium nitrate. Chin. J. Food Hyg. 27, 104e109 [Article in Chinese]. Li, C.X., Ma, C., Fang, H.Q., Zhi, Y., Yu, Z., Xu, H.B., Jia, X.D., 2015b. Neurobehavioral assessment of rats exposed to yttrium nitrate during development. Biomed. Environ. Sci. 28, 281e290. Li, X., Chen, Z., Chen, Z., Zhang, Y., 2013. A human health risk assessment of rare earth elements in soil and vegetables from a mining area in Fujian Province, Southeast China. Chemosphere 93, 1240e1246. Li, Y., Yang, J.L., Jiang, Y., 2012. Trace rare earth element detection in food and agricultural products based on flow injection walnut shell packed microcolumn preconcentration coupled with inductively coupled plasma mass spectrometry. J. Agric. Food Chem. 60, 3033e3041. Nakamura, Y., Tsumura, Y., Tonogai, Y., Shibata, T., Ito, Y., 1997. Differences in behavior among the chlorides of seven rare earth elements administered intravenously to rats. Fundam. Appl. Toxicol. 37, 106e116. Nemmar, A., Al-Salam, S., Beegam, S., Yuvaraju, P., Ali, B.H., 2017. The acute pulmonary and thrombotic effects of cerium oxide nanoparticles after intratracheal instillation in mice. Int. J. Nanomed. 12, 2913e2922. OECD, 1998. OECD Guidelines for the Testing of Chemicals. Test No. 408: Repeated Dose 90-day Oral Toxicity Study in Rodents (21 Sep 1998). http://www.oecdilibrary.org/environment/test-no-408-repeated-dose-90-day-oral-toxicitystudy-in-rodents_9789264070707-en. Pagano, G., Guida, M., Tommasi, F., Oral, R., 2015. Health effects and toxicity mechanisms of rare earth elements-Knowledge gaps and research prospects. Ecotoxicol. Environ. Saf. 115, 40e48. Pang, X., Li, D., Peng, A., 2002. Application of rare-earth elements in the agriculture of China and its environmental behavior in soil. Environ. Sci. Pollut. Res. Int. 9,
Y.-M. Wang et al. / Regulatory Toxicology and Pharmacology 90 (2017) 116e125 143e148. Peng, R.L., Pan, X.C., Xie, Q., 2003. Relationship of the hair content of rare earth elements in young children aged 0 to 3 years to that in their mothers living in a rare earth mining area of Jiangxi. Chin. J. Prev. Med. 37, 20e22 [Article in Chinese]. Rim, K.T., Koo, K.H., Park, J.S., 2013. Toxicological evaluations of rare earths and their health impacts to workers: a literature review. Saf. Health Work 4, 12e26. Schroeder, H.A., Mitchener, M., 1971. Scandium, chromium (VI), gallium, yttrium, rhodium, palladium, indium in mice: effects on growth and life span. J. Nutr. 101, 1431e1438. Seishiro, H., Suzuki, K.T., 1996. Exposure, metabolism, and toxicity of rare earths and related compounds. Environ. Health Perspect. 104, 85e95. Song, Y., Liu, Z.P., Zhang, L., Sui, H.X., Mao, W.F., Bao, H.H., Yong, L., Li, N., 2016. Exposure assessment of rare earth elements in brick tea among Chinese adults. Chin. J. Food Hyg. 28, 249e233. [Article in Chinese]. Speijers, G.J.A., 1999. Precision of estimates of an ADI (or TDI or PTWI). Regul. Toxicol. Pharmacol. 30, S87eS93. Suzuki, K.T., Kobayashi, E., Ito, Y., Ozawa, H., Suzuki, E., 1992. Localization and health effects of lanthanum chloride instilled intratracheally into rats. Toxicology 76, 141e152. Tong, S.L., Zhu, W.Z., Gao, Z.H., Meng, Y.X., Peng, R.L., Lu, G.C., 2004. Distribution characteristics of rare earth elements in children's scalp hair from a rare earths
125
mining area in southern China. J. Environ. Sci. Health A Tox. Hazard. Subst. Environ. Eng. 39, 2517e2532. USEPA, 2012. Rare Earth Elements: a Review of Production, Processing, Recycling, and Associated Environmental Issues. EPA 600/R-12/572. www.epa.gov/ord. Wei, B., Li, Y., Li, H., Yu, J., Ye, B., Liang, T., 2013. Rare earth elements in human hair from a mining area of China. Ecotoxicol. Environ. Saf. 96, 118e123. Wu, M.Y., Zhang, P., Yang, W.D., Liu, J.S., Mo, W.X., 2006. Effect of long-term intake of Y3þ in drinking water on learning-memory functions and growth-development of rats. J. Hyg. Res. 35, 283e285 [Article in Chinese]. Yang, W.D., Zhang, H.P., Liu, J.S., Xia, W.X., 2008. Effect of long-term intake of Y3þ on learning and memory of rats. Chin. J. Public Health 24, 183e184 [Article in Chinese]. Zhou, R., Liu, L.P., Yue, B., Li, X.W., Zhao, Y.F., Wu, Y.N., 2014. Dietary intake assessment for rare earth elements from 4th China total diet study. Mod. Prev. Med. 41, 1975e1978 [Article in Chinese]. Zhu, W., Xu, S., Shao, P., Zhang, H., Wu, D., Yang, W., Feng, J., Feng, L., 2005. Investigation on liver function among population in high background of rare earth area in South China. Biol. Trace Elem. Res. 104, 1e8. Zhuang, M., Zhao, J., Li, S., Liu, D., Wang, K., Xiao, P., Yu, L., Jiang, Y., Song, J., Zhou, J., Wang, L., Chu, Z., 2017. Concentrations and health risk assessment of rare earth elements in vegetables from mining area in Shandong, China. Chemosphere 168, 578e582.