Toxicological Evaluation of Chicken-Breast Meat with High-Dose Irradiation

Journal of Integrative Agriculture© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.T oxicological Evaluation of Chicken-Breast Meat with High-Dose IrradiationZHU Jia-ting 1, FENG Min 1, Y AN Jian-min 1, LIU Chun-quan 2, HA Y i-ming 3, GAO Mei-xu 3, Y ANG Ping 1, W ANG Zhi-dong 3, W ANG De-ning 1, LI Shu-rong 3 and GU Gui-qiang 11Institute of Atomic Energy in Application for Agriculture, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China 2 Institute of Farm Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, P.R.China3 Institute of Agro-Products Processing Science&Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.ChinaAbstractIn this paper, toxicity and safety of high-dose irradiated chicken-breast meat were evaluated. For assays of acute toxicity,genetic toxicity, and sub-chronic toxicity, ames test, mice bone marrow erythrocyte micronucleus, and mice sperm abnormality were performed. The results showed that, in the acute oral toxicity tests, median lethal dose (more than 10 000 mg kg -1) in male and female ICR mice showed no toxicological signs. For subacute 30-d oral toxicology of irradiated chicken-breast meat with dose of 10, 15 and 25 kGy in both male and female SD rats, no noticeable toxicological effects were observed. It is concluded that chicken-breast meat with high-dose irradiation has no acute toxicity and no genotoxicity, nor harmful effects on the animal body at the tested dosage range. Therefore, high-dose irradiated chicken-breast meat is safe for pet consumption.Key words: toxicological assessment, chicken-breast meat, irradiated, high doseINTRODUCTIONGamma irradiation has been commonly employed for decontamination and sterilization of pet foods to pro-long the storage period (Lin et al . 2002, 2009; Parke et al . 2005; Liu et al . 2006; Zhu et al . 2009). There is ample published evidence showing the enormous po-tential of this method for extending shelf life of chicken meat by eliminating certain spoilage and pathogenic organisms. Many countries in the world are engaged in the commercialization of irradiation food according to the Bulletin of the Meeting of Combined Expert Com-mittee of FAO (the Food and Agriculture Organization of the United Nation)/IAEA (the International Atomic Energy Agency)/WHO (the World Health Organization)(Diehl 2002; Child et al . 2009; Tamikazu et al . 2009).At present, irradiated foods including 6 food categories and 18 food varieties have been permitted by the Minis-try of Health of the P.R.China to enter Chinese markets (Wang and Chao 2003).In recent years, with rapid development of domestic and international pet industry, huge economic benefits are brought out by pet food enterprises. China is the world’s food irradiation superpower, with a total of about 2×105 t irradiated food in 2009 (Gao et al. 2010).However, the safety of irradiated food was still drawn great attention by many consumers, a variety of stud-ies to assure the safety of irradiation technique for pet food have been carried out, but not comprehensive enough. Especially, the effects of high-dose irradiation on main nutrients, physical and chemical indicators,toxicological evaluation of pet food were still rarely re-ported (Lin et al . 2002, 2009).Received 10 November, 2011 Accepted 26 March, 2012Correspondence ZHU Jia-ting, Tel/Fax: +86-25-84391942, E-mail: zjtlw@Toxicological Evaluation of Chicken-Breast Meat with High-Dose Irradiation2089For some special demands of pet food, low-dose irradiation (below 10 kGy) is impossible to achieve com-pletely sterilization. Many researchers recognized and reported that gamma irradiation in low doses, below 10 kGy could kill most organisms without deterioration of food quality (Thayer et al. 1995; Lacroix and Quattara 2000; Mahrour et al. 2003; Javanmard et al. 2006; Waje and Kwon 2007). However, few studies on the high-dose irradiation effects on food toxicity have been re-ported (Wang et al. 1987); the safety of irradiation ster-ilization for high doses hitherto needs to be confirmed. According to the Irradiated Food Safety Management Measures (1996, China), to develop new varieties of irradiated food (over 10 kGy) should provide sensory traits, nutritional, toxicological, microorganisms, and other indicators. In this paper, a complete toxicologi-cal assessment of irradiated chicken-breast meat with high doses, including test of genotoxicity, acute oral toxicity, and a 30-d sub-chronic toxicity was conducted in order to evaluate the safety of 60Co-irradiated chicken-breast meat as a pet food.RESULTSAcute toxicity testAfter oral administration of irradiated chicken-breast meat, none of the mice had any significant symptom of poisoning or death in all dosage groups (data not shown). The LD50of irradiated chicken-breast meat in both male and female mice was more than 10000 mg kg-1 body weight (BW). No toxicological effects were observed in 14-d period, in terms of body weight, food consumption, organ weight, and gross pathological alterations. Therefore, the acute toxicity of irradiated chicken-breast meat belongs to low toxicity grade or practically non-toxic (Ministry of Health of the P.R. China 2003).Ames testNo evidence of cytotoxicity (reduced rate of spontane-ously occurring clones and visible thinning of the bac-terial lawn) was observed at all testing dosage levels of irradiated chicken-breast meat. The mean number of revertants per plate of irradiated chicken-breast meat treatment groups at all dose levels for four strains of TA97, TA98, TA100, and TA102with or without S-9, were negative. None of the treated groups has two folds or more revertant counts than the concurrent control and no dose-relationship was observed. The mean number of revertant clones of the negative control was within the historical range of the laboratory. The positive con-trol mutagens induced the increases in revertant clones, confirming the validity of the assay. The results of Ames test indicate that irradiated chicken-breast meat has no genetic toxicity (Tables 1-3); similar results were obtained from the duplicate test (data not shown). Therefore, the Ames test result for irradiated chicken-breast was negative.Micronucleus test on bone marrow cellThe micronucleus of the irradiated chicken-breast meat treatment groups at all dose levels as well as the nega-tive control group were significantly lower than thoseTable 1 The influence of irradiated chicken-breast meat (sample B) used in Salmonella typhimurium reversionDose (μg/dish)TA97TA98TA100TA102-S9+S9-S9+S9-S9+S9-S9+S910123±8132±1136±444±6173±12184±14270±18291±13 40123±9133±1037±646±4173±10181±14270±15288±18 200125±9134±1135±547±5174±8185±13272±16293±18 1000123±7132±1036±546±7171±8183±13274±19289±16 5000124±8133±1036±545±5172±13185±14273±14291±16 Concurrent125±9135±935±547±7175±10182±12270±12290±19 Solvent122±8132±938±546±6173±14182±11269±15292±18 Positive853±53**Positive control: TA97-S9500.0 used atabrine at μg/dish; TA98-S9used daunorubicin at 6.0 μg/dish; TA100-S9, TA102-S9used methyl methanesulfonate at 1.0 μL/dish;TA97+S9, TA98+S9, TA100+S9used 2-AF at 10.0 μg/dish; TA102+S9used 1.8-dihydroxyanthraquinone at 50.0 μg/dish. **, P<0.01 compared to the negative control. The resultwas mean±SD of 3 plates. Solvent control: ddH2O. The same as below.1611±114**1803±133**875±87**1740±105**1809±105**2066±134**1885±91**2090ZHU Jia-ting et al.in positive control group treated with cyclophospha-mide (CP) (P<0.01), confirming the validity of the trial. There were no significant differences of micronucleus between the negative control group and the irradiated chicken-breast meat treatment groups (P>0.05). The polychromatic erythrocytes (PCE)/normochromatic erythrocytes (NCE) ratio of each group was within nor-mal range. The result indicating that irradiated chicken-breast meat is not mutagenic in vivo at the tested dos-age 5000 mg kg-1 BW (Table 4).Mice sperm abnormality trialThe sperm abnormality of the irradiated chicken-breast meat treatment groups at three different dose levels were 2.46, 2.52 and 2.64%, respectively, which had no sig-nificant differences compared with the negative con-trol group (P>0.05). The sperm abnormality ratios in all treatment groups as well as the negative control group (2.64% sperm abnormalities) was significantly lower than those in positive control group (7.44% sperm abnormalities) treated with CP at 40 mg kg-1 (P<0.01) (Table 5). The sperm abnormality test was a method of evaluating genetic injury to male reproductive cells of mammals (including humans) in vivo test. It was indicated that no genotoxicity was found on mice sperm for irradiated chicken-breast (samples B, C and D).Subacute 30-d oral toxicology study in ratsNo decease, abnormal behaviors- or treatment-related toxic signs were observed for the mice throughout the experimental period. Table 6 shows the body weight, body weight gain and food consumption data from the 30-d study.Male and female rats’ body weight as well as body weight gain in the experiment period had no significant differences (P>0.05) except the body weight of a male rat group at d-30 (P<0.05). Further analysis on the d-30 male rat body weight using Dunnett t-test that com-pares each dose level group with the control group shows no significant difference with or without irradi-ated treatment (P>0.05).Food consumption and food utilization of the male rats had no significant differences compared with the female rats at all other dose levels at other testing weeks (P>0.05). No significant differences in total food con-sumption and total food utilization between the treat-ment and control groups.All hematological parameters had no statistically sig-nificant differences between the irradiated treatment and control groups in the end periods of the 30-d study (Table 7). All clinical chemistry measurements find-ings between the treatment and control groups in the end periods of the 30-d study were presented in TableTable 2 The influence of irradiated chicken-breast meat (sample C) used in S. typhimurium reversionDose (μg/dish)TA97aTA98TA100TA102-S9+S9-S9+S9-S9+S9-S9+S910120±7132±1235±648±4175±9184±13270±18288±15 40123±10134±736±447±7172±12181±14269±12284±14 200124±8133±937±345±4174±9185±14272±12287±16 1000122±9132±834±548±5176±9185±12272±16288±19 5000123±11134±1236±446±5173±14186±10269±12282±19 Concurrent122±9130±934±645±6175±10180±12271±13285±17 Solvent120±9133±1036±546±4176±11184±13273±16284±17 Positive853±53** 1611±114**1803±133**875±87**1740±105**1809±105**2066±134**1885±91**Table 3 The influence of irradiated chicken-breast meat (sample D) used in S. typhimurium reversionDose (μg/dish)TA97aTA98TA100TA102-S9+S9-S9+S9-S9+S9-S9+S910122±7133±935±443±6173±12180±14258±18286±15 40125±10136±737±644±7176±12185±10259±12291±18 200120±8132±1236±446±4170±8189±15258±12294±14 1000124±10137±1134±247±4174±6184±10263±16288±17 5000123±7135±1234±544±6175±10182±11260±10295±17 Concurrent125±12133±1138±545±5172±10184±15264±19287±13 Solvent122±9136±834±445±6174±11182±16262±14290±11 Positive853±53** 1611±114**1803±133**875±87**1740±105**1809±105**2066±134**1885±91**Toxicological Evaluation of Chicken-Breast Meat with High-Dose Irradiation 20918. Through the simple factor analysis of variance, the total protein (TP) levels of sample D group of male rats were significantly higher than those in the control group (P <0.05), the albumin (ALB) levels of sample B and D groups of male rats were significantly higher than those in the control group (P <0.05), the aspartate transami-nase (AST) levels of sample D group of male rats were significantly lower than those in the control group (P <0.05), and other hematological parameters had no statistically significant differences (P >0.05) between the irradiated treatment and control groups in the end periods of the 30-d study.No statistically significant differences in absolute organ weight and organ-to-body weight ratios (Table 9) were observed.In the tests of gross anatomy, the size, color, and shape of heart, lung, liver, spleen, kidney, stomach,intestine, and testis of all the tested rats were normal,as evaluated by eye observation. The pathological ex-amination was conducted by high dose group vs . the control group only. No pathological changes of liver,kidney, spleen, stomach/duodenum, testis, and ovary tissues were observed for the high-dose group vs . the control group (Table 10). In summary, no toxic side effect was observed in the 30-d study.DISCUSSIONFood irradiation is recognized by the world health com-munity as a good method for preserving food and en-suring its wholesomeness. In the last 50 years, re-search activities have concluded that when used under good manufacturing practices, irradiation will: (i) in-crease food safety and reduce foodborne illness; (ii)lengthen the shelf-life of many perishable products; and (iii) reduce the risks of post-processing contamination of intact, packaged products (Smith and Pillai 2004;Quested et al . 2010; Farkas and Mohácsi-Farkas 2011).The most important element in food irradiation is the dose used on the product. The exposure must be ad-equate to produce desired results, yet low enough tomaintain the quality of foods. In most countries, in-cluding the USA, guidelines exist and define the mini-mum and maximum exposures for various commodities.Most of the claims of adverse effects of irradiation do not apply to the typical doses approved for pasteuriza-tion and they generally only apply in theory to the much higher doses required for sterilization. High doses of irradiation sterilize food kill all microorganisms except for viruses. However, radiation-sterilized meat and poul-try products produced by current methods have beenTable 4 Effect of irradiated chicken-breast meat on bone marrow micronucleus and PCE/NCE ratioGroups Dose SexMicronucleusPCE/NCE(mg kg -1BW)Number of PCEs countedNumber of micronucleiMicronuclei frequency (‰)PCE countedPCE/NCE Sample B 50001 000×58 1.40±0.89200×50.94±0.051 000×57 1.20±1.10200×50.93±0.05Sample C 50001 000×59 1.60±1.14200×50.95±0.051 000×56 1.00±1.00200×50.94±0.05Sample D 50001 000×57 1.40±0.89200×50.92±0.041 000×59 1.60±1.14200×50.94±0.04Negative control01 000×58 1.80±0.84200×50.95±0.041 000×57 1.40±0.89200×50.92±0.04Positive control CP401 000×517536.20±4.21**200×50.73±0.051 000×518735.80±3.96**200×50.72±0.04Cyclophosphamide (CP) was treated to mice as the positive control at a dose of 40 mg kg -1. The same as in Table 5.Table 5 Effect of irradiated chicken-breast meat on sperm counts and sperm morphology in micegroupsDoseTotal spermsAbnormal typeTotal abnormalAbnormal (mg kg -1 BW)counted Ghost-likeAmorphous Kinks tail Two heads Two tails sperms Negative control 01 000×516121087322132 2.64Sample B 5 0001 000×51911781212123 2.46Sample C 5 0001 000×51510884234126 2.52Sample D 5 0001 000×52013882423132 2.64CP401 000×54934232151814193727.44**Large roundheag ratios (%)Banana shape2092ZHU Jia-ting et al .Table 6 Body weight and food consumption of rats in 30-d studyGroups Body weight (g)Body weight gain (g)Week 1Week 2Week 3Week 4 Control 43.8±9.438.0±7.528.7±5.522.6±7.0133.1±19.1 Sample B 42.2±7.939.2±5.126.2±6.224.2±7.4131.8±13.8 Sample C 42.2±8.337.6±4.825.5±4.023.8±7.5129.1±17.2 Sample D 44.5±5.536.0±5.628.2±2.824.27±8.2132.9±16.1 Control 56.2±5.364.7±8.0236.3±21.5 Sample B 62.7±3.2*65.0±6.458.2±7.154.2±4.1240.1±14.2 Sample C 59.9±4.869.0±7.757.8±7.5238.1±20.6 Sample D 59.9±3.970.3±6.159.7±4.850.5±7.6240.3±11.6Groups Weekly feed intake (g)Total feed intake (g)Week 1Week 2Week 3Week 4 Control 108.0±16.5144.8±17.3131.4±14.4166.8±20.8550.9±37.1 Sample B 107.3±13.2128.2±12.4160.7±14.2545.2±36.3 Sample C 110.5±14.9144.2±14.1129.4±12.1155.1±22.9539.1±43.5 Sample D 109.6±14.1144.7±11.5131.8±10.2161.9±15.9547.9±41.7 Control 128.3±10.0190.9±10.1197.5±22.0226.0±13.7742.6±35.9 Sample B 135.4±11.6*208.0±16.2197.4±23.5765.7±49.2 Sample C 129.4±14.2196.2±16.4186.7±22.2215.9±15.4728.2±47.4 Sample D 131.7±14.4189.3±12.7195.1±14.2218.8±14.9734.9±35.7Groups Food utilization (%)Total food utilization (%)Week 1Week 2Week 3Week 4 Control 40.5±6.026.3±4.721.8±3.113.7±4.224.2±3.0 Sample B 39.6±7.926.3±2.420.2±3.114.9±3.824.1±1.4 Sample C 38.9±9.826.2±2.919.6±2.015.1±3.323.9±2.2 Sample D 40.8±4.424.8±2.921.4±0.914.8±4.624.2±2.1 Control 44.0±5.234.0±4.132.1±3.822.6±4.931.8±2.0 Sample B 46.5±3.531.2±1.929.6±2.924.1±1.831.4±1.3 Sample C 46.8±5.935.2±2.731.1±3.923.6±5.632.7±2.3 Sample D45.9±4.637.2±3.330.7±2.523.1±3.132.7±1.4*, P <0.05.63.9±13.051.4±12.351.4±13.5224.9±6.5148.9±9.8Table 7 Termination (d-30) hematological results of 30-d feeding testItemUnit Irradiated chicken-breast meat of female rats Irradiated chicken-breast meat of male rats Control B C D Control B C D Hemoglobin concentration g L -1Erythrocyte count 1012 L -18.17±0.277.99±0.197.94±0.168.01±0.348.19±0.378.39±0.308.36±0.268.34±0.23Leukocyte count 109 L -1 6.52±1.53 6.46±1.10 6.65±1.82 6.24±1.147.38±1.237.17±1.237.03±1.657.33±1.58Platelet count 109 L -1Neutrophils %20.6±5.218.0±4.520.4±5.419.3±4.318.3±3.918.2±4.620.2±4.319.5±3.7Basophils %0.50±0.530.50±0.710.70±0.670.60±0.520.70±0.670.50±0.710.50±0.530.50±0.53Eosinophils %0.60±0.840.60±0.700.50±0.530.70±0.670.60±0.700.70±0.670.60±0.700.50±0.71Lymphocytes %74.4±4.676.6±4.974.1±4.775.3±4.377.0±3.776.1±4.574.4±4.575.2±2.8Moncocytes%133±6132±5135±4129±6131±8135±5134±6137±4715±33711±38717±38738±32722±38730±36747±46722±433.9±1.74.3±0.84.3±1.64.1±1.43.4±1.64.5±1.64.3±1.64.3±1.5Table 8 Serum biochemistry findings in SD rats after 30 d of Ac administrationItem Unit Irradiated chicken-breast meat of female ratsIrradiated chicken-breast meat of male ratsControl B C D ControlB C D ALT U L -138.8±5.734.2±4.635.5±5.935.2±4.345.2±7.942.2±10.443.6±8.644.8±8.6AST U L -1TP g L -168.7±2.871.4±3.571.3±3.173.2±2.0*71.5±3.169.3±3.969.7±3.473.2±2.4ALB g L -142.9±2.045.3±1.5*44.7±2.245.3±1.8*43.8±0.842.8±0.843.3±1.044.5±1.3CHL mmol L -1 2.10±0.19 2.15±0.26 2.09±0.24 2.11±0.31 2.04±0.27 1.98±0.25 1.99±0.25 1.97±0.23TG mmol L -10.83±0.24 1.02±0.170.91±0.260.83±0.23 1.21±0.37 1.18±0.34 1.13±0.36 1.22±0.29BUN mmol L -18.50±0.838.30±0.809.05±0.779.18±0.779.15±1.068.40±0.978.95±1.379.06±1.13UA μmol L -179.5±12.179.3±12.183.7±13.580.0±12.7CR μmol L -148.0±3.549.8±3.755.4±3.1*56.0±2.6*52.3±3.050.1±3.953.2±5.253.1±2.7GLUmmol L -15.43±0.865.38±0.405.60±0.705.38±0.715.48±0.865.43±0.685.43±0.765.73±0.58*, P <0.05 vs. the same sex control group.135.8±15.1121.8±27.8101.8±29.2116.8±19.8*121.2±13.3119.5±27.5104.7±28.5121.4±15.6117.8±10.9114.3±13.4117.2±16.5108.8±12.9Toxicological Evaluation of Chicken-Breast Meat with High-Dose Irradiation2093rated by experts as superior to their canned counter-parts in texture, appearance, and, in some instances, vitamin retention and taste (Steele and Engel 1992). The ground breaking research into irradiation of food at high doses was carried out in the USA. In September 1997, a study group appointed by WHO concluded that foods treated with doses greater than 10 kGy can be consid-ered safe and nutritionally adequate when produced under established Good Manufacturing Practice. This conclusion was based on their evaluation of all avail-able data regarding the wholesomeness of food irradi-ated at high dose (Anonymous 1997).It is more than fifty years since irradiation sterilization was used for food and currently more than thirty coun-tries have adopted this technique. In China, seven kinds of food have been approved for irradiation by the Minis-try of Health of the P.R.China. This showed clearly the technique of irradiation used for food sterilization has been accepted by market and laws. However, so far, there have no reports about whether the technique with high doses is safe to chicken breast. It is our hope to supply some toxicity and genotoxicity data for the appli-cation of high-dose irradiated chicken-breast meat.In our study we observe no acute toxic effects of the high-dose irradiated chicken-breast taken via the oral route and we conclude that the LD50is higher than 10000 mg kg-1. For sub-chronic toxicity of irradiated chicken-breast meat with dose of 10, 15 and 25 kGy in both male and female ICR mice, no noticeable toxico-logical effects were observed. And the administration of irradiated chicken-breast meat with high dose for 30 d at dietary levels was well tolerated and did not affect health, behavior and neurological parameters of SD rats. It is concluded that high-dose irradiated chicken-breast meat has no acute toxicity and no genotoxicity, nor harmful effects on the human body at the tested dos-age range, which is some concordant with the litera-ture review. Just as commented in 1983 and 1989, food irradiated with the dosage of 10-30 kGy is safe (Yang et al. 1994). The point of view was brought up by the Joint FAO/IAEA/WHO Expert Committee and it has been proved world wide by a lot of experiments (Thayer et al. 1987; Lester et al. 1996).CONCLUSIONThe lack of acute toxicity, no mutagenic effects andTable 9 Mean (±SD) organ weights (g) and organ weight to final body weight ratios (%) in the ratsOrgan MeasureIrradiated chicken-breast meat of female rats Irradiated chicken-breast meat of male ratsControl Sample B Sample C Sample D Control Sample B Sample C Sample DLiver Weight (g)10.276±1.42810.086±0.7159.861±1.13410.119±1.19215.686±1.64916.049±1.00614.962±2.28415.214±1.777 Ratio (%) 4.469±0.399 4.410±0.232 4.381±0.396 4.424±0.351 4.623±0.376 4.688±0.253 4.402±0.458 4.446±0.386 Kidney Weight(g) 2.055±0.204 2.075±0.237 1.880±0.188 1.946±0.177 2.998±0.233 2.976±0.270 2.916±0.333 2.810±0.210 Ratio (%)0.901±0.1130.905±0.0720.836±0.0670.852±0.0480.884±0.0370.870±0.0820.859±0.0500.823±0.054 Spleen Weight (g)0.602±0.0890.602±0.0780.582±0.0840.624±0.0920.864±0.1140.887±0.0930.860±0.1830.826±0.173 Ratio (%)0.263±0.0390.264±0.0350.259±0.0360.273±0.0330.255±0.0270.259±0.0280.255±0.0570.242±0.050 Testicular Weight (g) 2.923±0.203 2.948±0.209 2.837±0.115 2.823±0.089 Ratio (%)0.862±0.0420.862±0.0640.842±0.0690.828±0.050Table 10 liver, kidneys, stomach, duodenum, spleen, testicular, and ovaries pathological examination of 30-d feeding studyVisceral organs Pathological changes Negative control Sample B Sample CSample DLiver Swelling of liver cellular1/100/100/100/100/100/101/100/10 Hepatic steatosis0/101/101/101/100/100/100/100/10Sinusoidal expansion and congestion0/100/100/100/100/100/100/100/10Mesenchymal inflammatory cell infiltration and hyperplasia of fibrous tissue0/100/100/100/100/100/100/100/10 Kidney Glomerular cellularity increase0/100/100/100/100/100/100/100/10 Renal tubular epithelial cell degeneration, necrosis0/100/100/100/100/100/100/100/10Pathological casts0/100/100/100/100/100/100/100/10Interstitial congestion, inflammatory cell infiltrating0/100/100/100/100/100/100/100/10 Stomach/duodenum Mucosa slough, degeneration0/100/100/100/100/100/100/100/10 Submucosa, tunica serosa hyperemia, inflammatory cell infiltrating0/100/100/100/100/100/100/100/10 Spleen Splenic sinuses dilatation, splenic nodules atrophy0/100/100/100/100/100/100/100/10 Testicular Seminiferous tubules and spermatogenic cells dysplasia0/100/100/100/10 Ovaries Follicular, corpus luteum abnormalities0/100/100/100/102094ZHU Jia-ting et al. lack of harmful effects found in the hematology, clini-cal chemistry and histopatology in this safety assess-ment indicate that high-dose (10-25 kGy, the dose rec-ommended for quarantine and hygienic purposes) irra-diated chicken-breast meat may have a large safetymargin.MATERIALS AND METHODSMaterialsDried chicken-breast meat was purchased from JinanUniwell Pet Food Co., Ltd., China. The sample wascrushed into powder, then packaged as 100 g samples inPVC (poly-vinyl chloride) containers used for gammairradiation. Gamma irradiation was performed at the Insti-tute of Atomic Energy in Application for Agriculture,Jiangsu Academy of Agriculture Science (JAAS), China.The power of irradiation equipment of gamma rays of 60Cois 29.6 PBq, the radiation doses on the chicken-breastmeat were 10, 15 and 25 kGy, respectively. In reference tothe food safety toxicology evaluation procedures andmethods (Ministry of Health of the P.R.China 2003), thesafety toxicology for irradiated-chicken breast wasevaluated, irradiated chicken-breast meat powder (sampleB, C and D) was mixed with bidistilled water to a certaindose for assays of acute toxicity, genetic toxicity, andsub-chronic toxicity, ames test, mice bone marrow eryth-rocyte micronucleus, and mice sperm abnormality. Irradi-ated chicken-breast meat powder (sample B, C and D)was added to a basal feed at concentrations of 100 g kg-1d-1, respectively and the rats were administrated for 30-dfeeding study.Instruments and chemical agentsHematology analyzer (Sysmex XE-2100, Kobe, Japan),Auamatic biochemical analyzer (Beckman LX 20, USA),and pathologic microtome sector (A0820, AO ScientificInstruments, Buffalo, NY, USA) are the main instrumentsused. The strains of TA97, TA98, TA100, and TA102weresupplied by Shanghai Institutes of Preventive Medicine,the mixture of liver microsomal enzymes (S9) were culturedby a toxicological department of Jiangsu Center for Dis-ease Control and Prevention, China; cyclophosphamide (CP) was bought from Jiangsu Hengrui Medicine Co., Ltd., China.Animals and housingMale and female ICR mice and SD rats were purchased from Zhejiang Laboratory Animal Center in Zhejiang, China (laboratory animal reproduction license # SCXK [SH] 2008-0033). The studies were conducted in compliance with Good Laboratory Practices at Jiangsu Disease Control and Preven-tion (JDC) (laboratory animal use permit # SYXK [JS] 2008-0003. All animals were housed as groups of five animals per cage in standard plastic cages. Temperature inside the experi-mental room was controlled at 22-24°C, humidity was con-trolled at 52-58%, and light control was maintained as a 12-h cycle of dark-light throughout the test period. All animals were examined for their general physical conditions upon adop-tion and acclimatized for 1 wk before any test.Ames testMutagenicity was determined by the Ames salmonella plate test. A strain of bacterium Salmonella typhimurium is an auxotrophic mutant and requires histidine for its growth. The strains of TA97, TA98, TA100, and TA102were qualified for use in the test. The tests were performed twice by the plate incorporation test on 5 groups of different doses of irradiated-chicken breast in the test were 5000, 1000, 200, 40, and 8 μg/dish, respectively. The known carcinogens, such as daunorubicin, dexon, sodium azide, and 2-aminofluorene, were used as positive controls. Dimethyl sulfoxide was used as the solvent for the 2-aminofluorene, distilled water for other groups. The test substance will be considered to be mutagenic if the revertant clones are two or more folds the number of spontaneous revertant clones on negative sol-vent control plates. A dose response relationship was ob-served in at least 2 concentrations. The test was repeated under the same conditions to confirm the results.Mice bone marrow erythrocyte micronucleus assayFifty of 7-8 wk-old ICR mice of 25-30 g, 25 male and 25 female, were divided into five groups randomly. Cyclo-phosphamide (40 mg kg-1) was used as positive control; ddH2O was used as negative control. The irradiated-chicken breast dose levels of testing groups (sample B, C and D) were all of 5000 mg kg-1. The testing samples were freshly prepared by mixing irradiated-chicken breast with ddH2O to a total volume of 20 mL. Testing materials were ig administered once at 0.20 mL 10 g-1 at 24 h intervals. Execu-tion via cervical vertebra dislocation was conducted 6 h after the last dose administration. Sternum bone was re-moved and the bone marrow cells were pulled out and mixed with fetal bovine serum immediately following the sacrifice. One drop of the mixture was smeared onto a clean slide and air-dried. The slides were briefly flamed, then fixed with immersion in 95% methanol for 10 min, and stained in ordinary staining jars with Giemsa working solu-tion for 15 min. Stained slides were washed gently with dd。