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. 2024 Mar 5;13(2):tfae025. doi: 10.1093/toxres/tfae025

Safety assessment of transgenic maize CC-2 by 90-day feeding study in Sprague-Dawley rats

Chen Chen 1, Lili Shi 2, Hongmei Mao 3, Chao Han 4, Jinpeng Zhao 5, Qin Zhuo 6, Yan Li 7,✉
PMCID: PMC10939339  PMID: 38496381

Abstract

Objective

The purpose of this study was to assess the safety of transgenic maize CC-2 through a 90-day feeding study in Sprague-Dawley rats.

Methods

Transgenic maize CC-2 and its parental counterpart maize Zhengdan 958 were respectively incorporated into diets at levels of 70%, 35% or 17.5% (w/w) and were administrated to rats (n = 10/sex/group) for 90 days. An additional control group of rats (n = 10/sex/group) were fed with the AIN93 breeding diet. All formulated diets were nutritionally balanced.

Results

There was no death and obvious toxic symptom in all rats. Food consumption, body weight, total food consumption rate, hematology, urinalysis, organ weight and organ coefficient were comparable between transgenic groups and the corresponding dose parental groups. There were significant differences of food consumption rate on some timepoint between high dose transgenic group and high dose parental group; male rats in high dose transgenic group showed significantly higher ALT/AST than high dose parental group on the middle or end of the experiment; but the differences showed no biological significance. There were no significant differences of other serum biochemistry parameters and pathological changes.

Conclusion

The results in this study demonstrated that the transgenic maize CC-2 didn't cause any related toxicity in rats.

Keywords: transgenic maize cc-2, 90-day feeding study, rats, diets, parental, toxicity

Introduction

Maize is important as food and animal feed source. In recent years, biotechnology has provided a new route for maize breeding for herbicide and pest resistance. The genetically modified (GM) maize has become one of the most widely GM plants in the world.1 CC-2 is a transgenic maize that expresses the macroACC gene which is tolerant to the herbicide glyphosate.

Glyphosate is the broad-spectrum and widely used herbicide,2 which was developed and promoted by Monsanto in 1971. Its physical and chemical properties are stable.2 Glyphosate could inhibit plant growth and make plant death, the mechanism was as follows: glyphosate could inhibit the 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS), cause a large amount of shikimic acid accumulation in plants, and inhibit the synthesis of tryptophan acid, tyrosine, phenylalanine, etc.3,4 The herbicide resistant maize CC-2 transfected with maroACC gene was obtained by China Agricultural University, which optimized CP4EPSPS gene according to the characteristics of maize codon, and obtained EPSPS gene suitable for high expression in maize. At the same time, the chloroplast signal peptide of sorghum EPSPS gene was cloned,5 and linked to the optimized EPSPS gene, named maroACC. The maroACC gene was transferred into the hybrid of HiIIA and HiIIB maize inbred lines with high transformation efficiency by gene gun co-transformation method and screened by glyphosate.6 In this study, MaroACC gene (similar to CP4 EPSPS) was transformed into maize to produce transgenic maize CC-2, then EPSPS’s active sites were changed, and the transgenic maize was resistant to glyphosate.

Since the occurence of transgenic crop commercialization, the GM product market share has rapidly increased.7 In 2017, biotech maize cultivated area was 59.7 million hectares of the global 188 million hectares maize.8 The advantage of genetical modification technology was crop yields increasing, chemical pesticide use reducing and farmer profits increasing.9 Although GM plants would be as safe as the parental species of these plants in many studies,10 the safety of the GM crops (including GM maize) used as feed/food has raised concerns. GM plant biosafety has become the object of recent research work.11

The European Food Safety Authority (EFSA) and Organization for Economic Cooperation and Development (OECD) have developed a risk assessment process for GM crops.12,13 The concept of “substantial equivalence” have been proposed for the safety assessment of GM crops/foods.14 However, only substantial equivalence concept is not sufficient to assess unintended effects, and animal feeding trials were needed for toxicity assessment of GM crops.15 The 90-day oral toxicity study of rodents has been recommended to assess the potential adverse effects of transgenic foods by FAO/WHO and EFSA.16,17 In Europe, the 90-day oral toxicity of rodents should be mandatory for adverse effect assessment of transgenic food.18 It is also needed in China to perform the 90-day feeding study using whole genetically modified food/feed.7,19

In this study, the 90-day feeding study was carried out in Sprague-Dawley (SD) rats for assessing the safety of transgenic maize CC-2 (CC-2 for short). The safety of CC-2 is compared with its non-transgenic counterpart Zhengdan958 and a common commercial AIN93 breeding feed. The dose design of the study was performed according to 90-day feeding study on transgenic crops.20 The detecting parameters were carried out according to guidance of 90-day oral toxicity study of China National Food Safety Standard (GB15193.13-2015).21

Materials and methods

Maize materials

Transgenic maize CC-2 with maroACC gene transforming and its none-transgenic counterpart maize Zhengdan 958 were received from China Agricultural University (Beijing, China). The maize was stored at 4–8 °C.

Identification verification of both kinds of maize

The endogenous reference gene zSSIIb of maize and the identification-specific gene of CC-2 maize were detected.

Compositional analysis and experimental diet formulation

Three batches of maize samples at different time and places were selected, Compositional analysis were performed in samples randomly selected from each kind of maize. Energy, protein, fat, carbonhydrate, ash, moisture, minerals and fiber were detected in maize for diet formulation. There was no significant difference in composition. Finally, the three batches of maize were mixed into feed.

In the safety evaluation of GM, it is considered to give as much transgenic crops as possible without affecting the nutrient intake. Based on this, the incorporation ratio of GM maize of feed in this study was determined to be 70% by referring to domestic and foreign studies and according to the requirements of AIN93 nutrient composition framework.

Based on the results above and AIN93G guidelines,22 flours of CC-2 and Zhengdan958 maize were formulated into rodent diets at 70%, 35% and 17.5% levels, all diets were produced in granulates, vacuum-packed and sterilized by irradiation using Co60. All diets were produced by Xiaoshuyoutai (Beijing) Biotechnology Co., Ltd (Beijing, China). After the diet was made, the nutrient composition of each group was detected and analyzed to avoid the interference of nutrient differences on the experimental results. The compositional analysis was performed by Nutrition Testing Center of Beijing Institute of Nutritional Resources.

Animals and housing

Specific pathogen-free (SPF) SD rats were purchased from Vital River Laboratory Animal Technology Co, Ltd (Beijing, China), whose body weights at receipt were 50–70 g. All animals were adaptively fed for 3 days after arrival. Two rats were housed in one cage and provided with food and water ad libitum. The suitable environmental conditions of rats were 22 ± 2 °C room temperature and 40%–70% relative humidity, 12 h light/dark photocycle and 15 times of air changes/h.

Study design

140 SD rats were randomly divided into seven groups according to weight, 10 rats/sex/group. Rats in six experimental groups were respectively fed with diets formulated with CC-2 or Zhengdan 958 maize at 70%, 35% or 17.5% (w/w) concentrations. AIN93 feeding diet was provided to rats in negative control group. The study was approved by the Animal Ethic Committee of National Institute of Nutrition and Health, Chinese Center for Disease Control and Prevention.

Clinical observations

All animals were observed once daily for activity, the color of fur, ingestion, excretion, the time of toxicity symptom occurence and mortality. Animals were also observed for growth and development. Individual body weight was measured pre-test, once a week thereafter and at sacrifice after fasting.7 Diet consumption of rats was measured weekly. The increase of weight, the diet consumption (rate) of each week and total experiment were determined. Rats of high dose groups and control group were examined for the eyes (cornea, crystalline lens, bulbar conjunctiva, iris) pre-test and at the end of experiment.

Haematology and hemagglutination

Since 16 h after fasting on the 45th Day, blood samples were collected from orbital sinus of rats, and put in ethylenediaminetetraacetic acid (EDTA) tubes to prevent coagulation for haematology examination. Since 16 h after fasting on the 90th Day, blood samples were collected from abdominal aorta under sodium pentobarbital intraperitoneally administered, put in EDTA tubes for haematology examination, and put in sodium citrate tubes (BD company, America) for hemagglutination examination. The following haematology parameters were detected by an haemacytometer analyzer (Sysmex XT-1800i, Japan): erythrocyte count (RBC), white blood cell count (WBC), haemoglobin concentration (HBG), Hematokrit (HCT), platelet count (PLT), the percentage of neutrophils (NEU), lymphocytes (LYM), monocytes (MO), eosinophils (EOS) and basophils (BAS). The following hemagglutination parameters were detected by an automatic coagulation analyzer (Sysmex CS-2000i, Japan): prothrombin time (PT) and active partial thromboplastin time (APTT).

Blood biochemistry

Since 16 h after fasting on the middle of the experiment, orbital sinus blood was obtained in serum collection tubes (BD company, America). Then serum chemistry was detected as follows: alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactic dehydrogenase (LDH), total protein (TP), albumin (ALB), glucose (GLU), blood urea nitrogen (BUN), creatinine (CREA), total cholesterol (CHO), triglyceride (TG). Since 16 h after fasting at the end of the experiment, blood samples were collected from abdominal aorta of rats under sodium pentobarbital intraperitoneally administered; not only the above parameters were detected, but also the following parameters [chlorine (Cl−), potassium (K+) and sodium (Na+)] were detected. All the blood chemistry parameters were detected using an automatic biochemical analyser (Hitachi, Japan).

Urine chemistry

At the end of the experiment, urine was collected for urinalysis. The following parameters were detected by urine analyzer (URIT-180, China): protein, gravity, glucose, pH, blood, etc.

Organ weight and histopathology

At the end of the study, the body weight of rats were measured after fasting, rats were sacrificed through anesthesia bleeding. Necropsy were performed and gross pathology were observed including an examination of the abdominal cavities, thoracic cavities, cranial cavities, main organs and tissues. The weight of brain, heart, liver, kidney, adrenal glands, spleen, thymus, testes, epididymis, ovary and uterus were recorded, and paired organs were weighed together. Organ-to-body weight ratios (relative organ weights) were calculated. Brain, heart, lung, liver, kidneys, adrenal glands, spleen, stomach, duodenum, jejunum, ileum, thymus, thyroid, testes, epididymis, prostate, ovary and uterus were sampled and fixed in 10% neutral-buffered formalin. Paraffin sections were prepared, routine HE staining was performed to observe the histological and pathological changes of the organs under the microscope, and abnormal changes were recorded. Firstly, the animal organ specimens of transgenic maize high-dose group, parent maize high-dose group and control AIN93G group were examined. If abnormalities were found, the animal specimens of other groups were made to pathological sections and observed under microscope. The histopathological examination was commissioned by Institute of Experimental Animals, Chinese Academy of Medical Sciences.

Statistical analysis

Data were presented as the form of mean ± SD, and the SPSS Software (version 25.0) was used for statistical analysis. Levene’s test was used to assess the homogeneity. If the variations were homogeneous, one way analysis of variance (ANOVA) was carried out; If the variations were not homogeneous, nonparametric test was carried out. If there were significant differences through ANOVA or nonparametric test, comparisons between multiple groups were conducted through LSD post hoc test (when equal variances assumed) or Tamhane’s T2 post hoc test (when equal variances not assumed). P-value less than 0.05 referred to statistical significance.

Results

Verification of identification

Both kinds of maize were processed for verification of identification before 90-day feeding study. As was shown in Fig. 1A, the endogenous gene zSSIIb could be detected in both kinds of maize; Fig. 1B showed that identification-specific genes of CC-2 maize were only detected in transgenic maize. The results showed that both kinds of maize were identified correct and were able to used in the following study.

Fig. 1.

Fig. 1

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The amplification results of maize endogenous gene zSSIIb (A) and CC-2 maize identification-specific genes (B).

Compositional analysis

Energy, moisture, ash, fat, protein, carbonhydrate, calcium and phosphate in the diet of each group were consistent to the theoretical values of AIN93G (shown in Table 1).

Table 1.

The main nutritional components of diets in each group.

Energy
(kJ/100 g)		 Crude protein
(g/100 g)		 Crude fat
(g/100 g)		 Carbonhydrate
(g/100 g)		 Crude ash
(g/100 g)		 Water
(g/100 g)		 Calcium
(g/100 g)		 Phosphate
(g/100 g)		 Crude fibre
(g/100 g)
Control 1934.5 17.3 7.8 65.4 2.2 7.3 0.524 0.29 30
CC-2(17.5%) 1888.7 16.6 7.9 64.6 2.5 8.4 0.554 0.31 27
CC-2(35%) 1906.5 16.8 7.6 66.1 2.3 7.2 0.551 0.31 27
CC-2(70%) 1947.9 17.3 7.9 65.5 2.5 6.8 0.531 0.29 31
Zhengdan 958 maize(17.5%) 1863.0 16.8 7.5 64.7 2.2 8.8 0.492 0.29 25
Zhengdan 958 maize(35%) 1874.3 17.9 7.7 64.3 2.2 7.9 0.551 0.28 24
Zhengdan 958 maize(70%) 1930.2 18.1 7.9 64.6 2.2 7.2 0.531 0.28 29
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Clinical observation

No treatment-related adverse clinical symptoms and no mortality were observed in the experiment. There was no obvious abnormality in ocular examination in high groups and AIN93G group.

Body weight and food consumption

The weekly body weight (Fig. 2A for female body weight and Fig. 2B for male body weight), food consumption (Table 2 for female food consumption and Table 3 for male food consumption) and food utilization (Table 4 for female and Table 5 for male) were calculated among all groups in the feeding study. Total body weight gain, total food consumption and total food utilization were calculated between rats of all groups (Table 6). There was no test-related differences in male or female weight when compared to the corresponding dose parental group.

Fig. 2.

Fig. 2

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Female mean weekly body weight (A) and male mean weekly body weight (B) of rats administrated with different diets for 90 days.

Table 2.

Food consumption of female rats fed with different diets for 90 days (Inline graphic±SD) (the unit of the number was g).

Time Control (n = 5) CC-2(17.5%) (n = 5) CC-2(35%)
(n = 5)		 CC-2(70%) (n = 5) Zhengdan 958 maize (17.5%)
(n = 5)		 Zhengdan 958 maize (35%)
(n = 5)		 Zhengdan 958 maize (70%)
(n = 5)
The 1st week 92.1 ± 6.6 97.7 ± 6.1 102.2 ± 7.4 106.2 ± 6.5 99.8 ± 4.6 97.3 ± 7.7 102.7 ± 8.3
The 2nd week 114.2 ± 6.7 116.2 ± 7.0 123.3 ± 6.8 127.7 ± 8.2 116.1 ± 7.3 122.3 ± 7.0 118.5 ± 5.1
The 3rd week 111.1 ± 9.7 117.0 ± 7.9 117.7 ± 7.1 121.0 ± 8.7 116.7 ± 9.6 119.6 ± 6.4 111.2 ± 10.0
The 4th week 128.4 ± 10.0 135.2 ± 7.8 140.3 ± 7.2 142.0 ± 9.8 136.2 ± 10.9 145.7 ± 11 132.9 ± 5.0
The 5th week 127.9 ± 8.6 137.2 ± 9.3 141.7 ± 4.1* 143.1 ± 8.4* 133.7 ± 4.7 145.4 ± 9.0** 130.3 ± 8.2
The 6th week 128.9 ± 12.5 142.4 ± 11.4 137.9 ± 5.5 145.7 ± 8.5 139.4 ± 5.0 147.1 ± 11.6 134.5 ± 6.1
The 7th week 120.2 ± 5.6 118.9 ± 9.1 127.2 ± 5.7 130.4 ± 10.9 119.6 ± 4.4 128.5 ± 3.8 126.4 ± 9.5
The 8th week 122.0 ± 7.0 127.1 ± 6.2 125.4 ± 9.9 132.4 ± 16.8 134.8 ± 5.8 136.6 ± 8.2 138.2 ± 6.3
The 9th week 113.1 ± 7.6 124.3 ± 6.5 120.8 ± 9.9 130.9 ± 15.5* 128.3 ± 7.3 135.0 ± 8.2** 125.4 ± 9.6
The 10th week 113.4 ± 5.6 120.7 ± 14.7 121.0 ± 7.8 126.5 ± 9.6 131.5 ± 9.5* 134.6 ± 12.3** 119.7 ± 4.7
The 11th week 115.9 ± 10.5 127.9 ± 11.3 122.0 ± 6.2 130.9 ± 6.3 132.0 ± 12.3 130.3 ± 9.4 127.7 ± 13.8
The 12th week 110.9 ± 3.4 122.6 ± 14.3 120.2 ± 7.1 132.5 ± 7.1** 124.1 ± 8.9 124.6 ± 4.2 125.9 ± 13.3
The 13th week 110.0 ± 10.6 115.1 ± 9.6 110.5 ± 7.7 114.4 ± 9.2 110.6 ± 8.1 113.6 ± 8.9 108.7 ± 7.1
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* P<0.05.

** P<0.01 as compared with the control group.

P > 0.05 as compared with the corresponding dose parental group.

Table 3.

Food consumption of male rats fed with different diets for 90 days (Inline graphic±SD) (the unit of the number was g).

Time Control (n = 5) CC-2(17.5%) (n = 5) CC-2(35%)
(n = 5)		 CC-2(70%) (n = 5) Zhengdan 958 maize (17.5%)
(n = 5)		 Zhengdan 958 maize (35%)
(n = 5)		 Zhengdan 958 maize (70%)
(n = 5)
The 1st week 108.0 ± 8.8 118.5 ± 6.7 116.9 ± 7.2 128.0 ± 9.3 116.6 ± 11.6 118.0 ± 8.3 117.8 ± 8.8
The 2nd week 149.7 ± 6.1 154.1 ± 7.2 158.5 ± 7.0 171.7 ± 7.1** 154.7 ± 13.7 156.8 ± 12.2 159.6 ± 10.8
The 3rd week 156.4 ± 9.2 167.5 ± 7.5 175.7 ± 11.9 183.9 ± 12.2** 172.0 ± 14.8 166.8 ± 13.2 172.6 ± 12.4*
The 4th week 179.4 ± 9.4 193.3 ± 10.0 203.4 ± 16.0 214.2 ± 12.2** 199.9 ± 14.7 195.0 ± 17.3 207.4 ± 17.6*
The 5th week 183.5 ± 11.3 194.4 ± 12.9 207.3 ± 18.5 223.0 ± 16.3** 199.8 ± 12.6 191.7 ± 15.5 211.1 ± 14.6
The 6th week 189.2 ± 11.3 197.7 ± 16.8 208.3 ± 15.2 222.9 ± 16.8** 205.2 ± 10.0 196.7 ± 12.9 207.8 ± 10.9
The 7th week 164.4 ± 7.5 177.4 ± 20.1 190.9 ± 12.3 199.2 ± 13.9** 167.6 ± 27.2 173.3 ± 20.6 191.9 ± 12.1*
The 8th week 171.1 ± 10.8 182.5 ± 17.4 188.6 ± 11.7 205.7 ± 11.4* 182.8 ± 7.1 182.1 ± 17.6 200.1 ± 16.9
The 9th week 172.3 ± 12.0 180.0 ± 13.8 186.5 ± 12.7 203.4 ± 8.4** 182.4 ± 4.4 181.4 ± 12.6 197.5 ± 20.1*
The 10th week 170.8 ± 11.2 178.7 ± 13.3 187.2 ± 11.1 200.2 ± 9.7** 182.2 ± 9.3 182.1 ± 10.6 189.1 ± 15.0
The 11th week 173.2 ± 10.1 184.3 ± 16.2 190.1 ± 11.0 203.6 ± 14.6 184.2 ± 8.1 189.3 ± 12.5 192.5 ± 17.7
The 12th week 171.6 ± 5.1 185.1 ± 15.4 193.0 ± 14.5 204.4 ± 16.3** 187.7 ± 13.7 179.5 ± 9.7 197.8 ± 18.7*
The 13th week 163.6 ± 8.9 175.2 ± 15.3 184.6 ± 10.5 197.4 ± 12.8** 177.8 ± 16.5 176.3 ± 10.7 190.4 ± 19.0*
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* P<0.05.

** P<0.01 as compared with the control group.

P > 0.05 as compared with the corresponding dose parental group.

Table 4.

Food utilization of female rats fed with different diets for 90 days (Inline graphic±SD).

Time Control (n = 5) CC-2(17.5%) (n = 5) CC-2(35%)
(n = 5)		 CC-2(70%) (n = 5) Zhengdan 958 maize (17.5%)
(n = 5)		 Zhengdan 958 maize (35%)
(n = 5)		 Zhengdan 958 maize (70%)
(n = 5)
The 1st week 51.8 ± 1.5 49.8 ± 1.6 51.9 ± 1.8 46.8 ± 3.5 50.9 ± 1.9 49.3 ± 4.5 48.9 ± 4.0
The 2nd week 39.0 ± 2.0 37.5 ± 0.7 35.7 ± 1.6 34.6 ± 1.3 36.0 ± 2.5 36.4 ± 3.9 36.0 ± 2.7
The 3rd week 30.2 ± 2.0 28.3 ± 1.6 31.6 ± 2.9 29.9 ± 1.6 32.3 ± 1.5 32.0 ± 5.9 30.3 ± 4.3
The 4th week 25.4 ± 1.4 21.8 ± 3.0 22.9 ± 1.4 23.0 ± 2.5 22.0 ± 2.1 23.7 ± 1.0 20.6 ± 3.4
The 5th week 19.2 ± 4.0 16.5 ± 2.5 17.8 ± 3.5 15.4 ± 1.5 17.0 ± 2.3 13.8 ± 3.2 16.5 ± 2.5
The 6th week 14.1 ± 2.5 15.9 ± 3.3 13.9 ± 2.4 16.7 ± 3.6 16.9 ± 2.9 15.1 ± 4.1 13.3 ± 2.2
The 7th week 8.3 ± 3.8 9.0 ± 4.4 11.0 ± 3.6 8.6 ± 2.7 6.0 ± 4.4 9.8 ± 3.5 8.8 ± 2.2
The 8th week 11.9 ± 3.5 11.1 ± 2.5 11.2 ± 4.2 16.2 ± 1.9 15.2 ± 4.8 9.8 ± 3.1 11.5 ± 3.2
The 9th week 8.8 ± 4.6 9.6 ± 5.4 7.0 ± 2.3 7.0 ± 3.9 8.7 ± 4.1 11.2 ± 2.0 8.6 ± 3.8
The 10th week 4.8 ± 1.0 5.8 ± 4.6 6.3 ± 3.2 4.7 ± 5.0 9.3 ± 3.1 6.9 ± 2.6 8.0 ± 4.3
The 11th week 9.3 ± 3.8 10.3 ± 4.1 9.6 ± 3.7 9.1 ± 4.2 12.9 ± 5.5 6.7 ± 5.2 6.6 ± 3.1
The 12th week 4.8 ± 2.6 8.1 ± 4.5 5.8 ± 5.5 8.8 ± 4.2 5.9 ± 4.4 5.0 ± 3.0 5.7 ± 4.4
The 13th week 10.6 ± 5.0 8.0 ± 3.0 6.2 ± 3.1 7.4 ± 4.5 5.8 ± 3.4 7.8 ± 4.2 5.1 ± 4.2
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P > 0.05 as compared with the control group; P > 0.05 as compared with the corresponding dose parental group.

Table 5.

Food utilization of male rats fed with different diets for 90 days (Inline graphic±SD).

Time Control (n = 5) CC-2(17.5%) (n = 5) CC-2(35%)
(n = 5)		 CC-2(70%) (n = 5) Zhengdan 958 maize (17.5%)
(n = 5)		 Zhengdan 958 maize (35%)
(n = 5)		 Zhengdan 958 maize (70%)
(n = 5)
The 1st week 65.4 ± 0.5 62.2 ± 8.8 60.7 ± 6.6 58.1 ± 8.1 58.6 ± 5.2 60.9 ± 7.3 57.5 ± 5.8
The 2nd week 47.3 ± 2.1 48.5 ± 0.8 48.9 ± 2.1 44.5 ± 0.9 47.2 ± 2.4 46.6 ± 0.8 45.9 ± 2.5
The 3rd week 46.5 ± 1.0 43.0 ± 2.1 42.2 ± 4.5* 40.7 ± 1.1** 44.1 ± 2.0 42.8 ± 1.6 43.9 ± 1.7
The 4th week 39.3 ± 4.7 37.3 ± 2.6 39.2 ± 3.2 35.7 ± 1.9 36.5 ± 1.8 37.0 ± 1.7 36.3 ± 1.6
The 5th week 30.0 ± 3.0 28.4 ± 1.9 27.4 ± 2.9 27.2 ± 2.3 27.8 ± 1.5 27.3 ± 1.1 27.9 ± 0.5
The 6th week 26.2 ± 5.0 26.70 ± 1.8 26.6 ± 2.0 28.5 ± 1.0*** 25.0 ± 0.9 25.5 ± 1.0 25.3 ± 1.4
The 7th week 15.9 ± 2.4 14.6 ± 2.8 14.8 ± 3.1 17.3 ± 1.7 18.9 ± 2.5 13.8 ± 1.6 17.6 ± 0.9
The 8th week 19.6 ± 1.3 19.3 ± 2.6 20.2 ± 2.9 19.1 ± 3.3 16.9 ± 2.9 18.9 ± 3.8 15.0 ± 4.5
The 9th week 14.9 ± 2.4 12.8 ± 2.1 10.8 ± 4.4 13.8 ± 2.4 11.9 ± 2.9 14.6 ± 3.0 17.2 ± 3.0
The 10th week 12.9 ± 3.9 12.7 ± 1.0 14.4 ± 2.9 13.8 ± 3.5 11.4 ± 3.5 16.3 ± 3.2 9.7 ± 2.3
The 11th week 13.5 ± 3.6 13.2 ± 0.8 14.3 ± 1.5 13.1 ± 2.8*** 12.9 ± 0.9 12.0 ± 1.3 17.6 ± 2.6
The 12th week 11.5 ± 2.2 12.4 ± 3.1 14.0 ± 4.4 14.1 ± 5.7 11.1 ± 2.8 10.7 ± 5.9 9.9 ± 2.2
The 13th week 10.6 ± 4.0 9.9 ± 1.7 10.6 ± 1.2 11.4 ± 4.2 11.0 ± 3.1 9.2 ± 3.6 11.0 ± 2.2
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* P<0.05

** P<0.01 as compared with the control group.

*** P<0.05 as compared with the corresponding dose parental group.

Table 6.

Total body weight gain, total food consumption and total food utilization of rats fed with different diets for 90 days (Inline graphic±SD).

Sex Groups Number of rats Total body weight gain Total food consumption Total food utilization
Female Control 10 269.4 ± 26.4 1502.2 ± 72.7 17.8 ± 0.7
CC-2(17.5%) 10 276.9 ± 31.2 1602.3 ± 89.9 17.3 ± 0.4
CC-2(35%) 10 280.2 ± 17.4 1610.3 ± 51.4 17.4 ± 0.3
CC-2(70%) 10 288.8 ± 26.8 1683.5 ± 105.0** 17.1 ± 0.4
Zhengdan 958 maize(17.5%) 10 288.2 ± 36.1 1622.6 ± 60.7 17.7 ± 0.8
Zhengdan 958 maize(35%) 10 281.4 ± 34.3 1680.6 ± 89.2** 16.7 ± 1.0
Zhengdan 958 maize(70%) 10 261.9 ± 21.0 1602.1 ± 65.7 16.3 ± 0.7**
Male Control 10 557.1 ± 62.5 2153.1 ± 107.0 25.8 ± 0.8
CC-2(17.5%) 10 573.1 ± 65.9 2288.7 ± 163.6 25.0 ± 0.7
CC-2(35%) 10 603.2 ± 71.8 2391.0 ± 139.3 25.2 ± 1.2
CC-2(70%) 10 634.5 ± 43.7 2557.6 ± 133.7** 24.8 ± 0.4
Zhengdan 958 maize(17.5%) 10 566.1 ± 44.7 2313.1 ± 120.4 24.4 ± 0.6
Zhengdan 958 maize(35%) 10 563.8 ± 52.4 2289.0 ± 164.4 24.6 ± 1.2
Zhengdan 958 maize(70%) 10 595.0 ± 74.6 2435.3 ± 176.0* 24.4 ± 1.3
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* P<0.05

** P<0.01 as compared with the control group

P > 0.05 as compared with the corresponding dose parental group.

As shown in Tables 2 and 3, there was no significant difference in food intake of female and male rats in each transgenic group compared with the corresponding dose parental group (P > 0.05). Compared with the control group, the food intake of female rats were shown below: the food intake significantly increased at the 5th week in CC-2(35%) group (P < 0.05), and also significantly increased at the 5th, 9th and 12th week in the CC-2(70%) group (P < 0.05, P < 0.05, P < 0.01); the food consumption of the Zhengdan 958 maize (17.5%) group increased at the 10th week (P < 0.05), and the food consumption of the Zhengdan 958 maize (35%) group increased at the 5th, 9th and 10th week (P < 0.01). Compared to the control group, the food consumption of male rats were shown below: the food consumption of the CC-2 (70%) group significantly increased at weeks 2–7, 9–10, 12–13 (P < 0.01) and week 8 (P < 0.05), while that of the Zhengdan 958 maize (70%) group significantly increased at weeks 3–4, 7, 9 and 12–13 (P < 0.05).

As shown in Tables 4 and 5, compared with the corresponding dose parental group, there was no significant difference in the food utilization rate of female rats in each transgenic group (P > 0.05); the food utilization rate of male rats in CC-2 (70%) group increased at the 6th week and decreased at the 11th week (P < 0.05), and there was no significant difference at other time points. Compared with the control group, there was no significant difference in food utilization rate of female rats in transgenic groups (P > 0.05); the food utilization rate of male rats significantly decreased in CC-2 (35% and 70%) groups at the 3rd week (P < 0.05 or P < 0.01).

As shown in Table 6, there were no significant differences in total food consumption, total weight gain and total food utilization rate of male and female rats in each transgenic group compared with the corresponding dose parental group (P > 0.05). Compared with the control group, the total food consumption of male rats in Zhengdan 958 (70%) and CC-2 (70%) groups significantly increased (P < 0.05 or P < 0.01); The total food consumption of female rats in the CC-2 (70%) group and Zhengdan 958 (35%) group significantly increased (P < 0.01); the total food utilization rate of the Zhengdan 958 (70%) group significantly decreased (P < 0.01).

In conclusion, the food utilization rate of male rats in the CC-2 (70%) group was significantly different from that in the corresponding dose parent group at individual time points, but there was no difference between CC-2 (70%) group and the control group, which was analyzed as normal fluctuation. No adverse effects were observed on body weight, food consumption and food utilization of rats after 90 days of feeding transgenic maize CC-2.

Haematology

In this paper, medium-term or terminal haematology (Table 7 or table 8) of male and female rats were respectively presented.

Table 7.

The effects of mid-term haematology parameters of rats fed with transgenic maize CC-2 (Inline graphic±SD).

Sex Groups Number of rats WBC
(×109/L)		 RBC
(×1012/L)		 HGB
(g/L)		 HCT
(%)		 PLT
(×109/L)
Female Control 10 11.35 ± 1.88 8.70 ± 0.25 165 ± 9 47.7 ± 1.3 964 ± 205
CC-2(17.5%) 10 9.52 ± 2.74 8.79 ± 0.22 165 ± 5 47.2 ± 1.3 1,066 ± 188
CC-2(35%) 10 11.17 ± 2.49 8.66 ± 0.39 163 ± 6 47.2 ± 1.8 998 ± 192
CC-2(70%) 10 11.35 ± 4.25 8.85 ± 0.40 162 ± 6 46.8 ± 1.6 1,010 ± 156
Zhengdan 958 maize(17.5%) 10 11.38 ± 3.58 8.63 ± 0.31 163 ± 6 47.3 ± 1.5 949 ± 228
Zhengdan 958 maize(35%) 10 13.10 ± 2.47 8.76 ± 0.35 165 ± 7 47.3 ± 1.6 986 ± 130
Zhengdan 958 maize(70%) 10 11.87 ± 4.83 8.73 ± 0.49 163 ± 9 47.2 ± 2.5 998 ± 203
Male Control 10 14.16 ± 3.24 8.79 ± 0.56 170 ± 10 49.8 ± 2.7 913 ± 218
CC-2(17.5%) 10 14.79 ± 2.26 8.25 ± 0.31* 159 ± 10 47.0 ± 3.1 975 ± 182
CC-2(35%) 10 13.28 ± 1.77 8.82 ± 0.39 161 ± 8 47.1 ± 2.2 1,104 ± 158
CC-2(70%) 10 14.59 ± 2.83 8.53 ± 0.28 156 ± 10** 45.7 ± 2.5** 845 ± 272
Zhengdan 958 maize(17.5%) 10 11.57 ± 2.69 8.61 ± 0.65 160 ± 12 47.3 ± 3.4 1,016 ± 208
Zhengdan 958 maize(35%) 10 14.11 ± 3.73 8.95 ± 0.36 167 ± 6 49.2 ± 1.6 939 ± 296
Zhengdan 958 maize(70%) 10 15.30 ± 3.31 8.75 ± 0.39 157 ± 9* 46.4 ± 2.1* 1,122 ± 187
Sex Groups Number of rats neutrophil
(%) 		LYM
(%) 		MO
(%) 		EOS
(%) 		BAS
(%)
Female Control 10 7.0 ± 2.2 88.1 ± 2.4 3.9 ± 1.0 1.1 ± 0.2 0.0 ± 0.0
CC-2(17.5%) 10 8.0 ± 3.0 85.7 ± 4.0 5.0 ± 1.9 1.4 ± 0.4 0.0 ± 0.0
CC-2(35%) 10 7.6 ± 4.1 86.4 ± 6.2 4.6 ± 2.0 1.4 ± 0.5 0.0 ± 0.0
CC-2(70%) 10 7.4 ± 4.6 87.1 ± 5.5 4.4 ± 1.1 1.2 ± 0.7 0.0 ± 0.0
Zhengdan 958 maize(17.5%) 10 7.8 ± 2.7 86.7 ± 3.8 4.1 ± 1.0 1.5 ± 1.2 0.0 ± 0.0
Zhengdan 958 maize(35%) 10 7.9 ± 4.2 86.8 ± 5.3 3.9 ± 0.8 1.5 ± 0.9 0.0 ± 0.0
Zhengdan 958 maize(70%) 10 10.2 ± 5.7 83.5 ± 6.4 4.7 ± 1.2 1.5 ± 1.3 0.0 ± 0.0
Male Control 10 12.0 ± 2.1 82.6 ± 2.0 4.3 ± 0.5 1.1 ± 0.9 0.0 ± 0.0
CC-2(17.5%) 10 11.3 ± 2.5 83.2 ± 2.9 4.3 ± 1.5 1.1 ± 0.8 0.1 ± 0.0
CC-2(35%) 10 11.5 ± 3.2 83.2 ± 3.7 4.3 ± 1.0 0.9 ± 0.3 0.1 ± 0.1
CC-2(70%) 10 11.4 ± 5.3 83.3 ± 6.2 4.2 ± 0.7 1.0 ± 0.7 0.1 ± 0.1
Zhengdan 958 maize(17.5%) 10 11.4 ± 2.2 82.9 ± 2.5 4.9 ± 0.8 0.8 ± 0.2 0.1 ± 0.1
Zhengdan 958 maize(35%) 10 8.8 ± 3.2 86.3 ± 3.4 4.1 ± 0.7 0.9 ± 0.3 0.1 ± 0.1
Zhengdan 958 maize(70%) 10 10.6 ± 2.0 82.9 ± 2.0 5.4 ± 0.9* 1.0 ± 0.2 0.1 ± 0.1
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Compared to control group, *P<0.05, **P<0.01; compared to the corresponding dose parental group, P > 0.05.

Table 8.

The effects of terminal haematology parameters of rats fed with transgenic maize CC-2 (Inline graphic±SD).

Sex Groups Number of rats WBC
(×109/L)		 RBC
(×1012/L)		 HGB
(g/L)		 HCT
(%)		 PLT
(×109/L)
Female Control 10 6.50 ± 1.39 8.36 ± 0.41 154 ± 6 43.3 ± 1.5 986 ± 87
CC-2(17.5%) 10 5.06 ± 1.26 8.28 ± 0.31 151 ± 4 42.6 ± 1.2 989 ± 81
CC-2(35%) 10 6.38 ± 0.77 8.42 ± 0.29 153 ± 5 43.5 ± 1.3 1,045 ± 130
CC-2(70%) 10 5.14 ± 2.08 8.56 ± 0.33 154 ± 5 43.8 ± 1.1 924 ± 118
Zhengdan 958 maize(17.5%) 10 5.05 ± 1.25 8.37 ± 0.26 152 ± 4 43.5 ± 1.2 1,029 ± 61
Zhengdan 958 maize(35%) 10 6.15 ± 1.04 8.57 ± 0.23 155 ± 4 43.9 ± 1.2 1,014 ± 121
Zhengdan 958 maize(70%) 10 6.81 ± 3.56 7.96 ± 1.55 145 ± 28 41.6 ± 7.2 1,010 ± 165
Male Control 10 7.23 ± 2.47 8.74 ± 0.46 154 ± 5 44.5 ± 1.7 985 ± 132
CC-2(17.5%) 10 6.73 ± 3.17 8.52 ± 0.44 151 ± 5 44.2 ± 2.0 1,000 ± 162
CC-2(35%) 10 7.64 ± 2.78 9.00 ± 0.45 157 ± 6 45.3 ± 2.0 1,034 ± 194
CC-2(70%) 10 8.27 ± 1.94 8.82 ± 0.37 152 ± 5 44.1 ± 1.4 958 ± 143
Zhengdan 958 maize(17.5%) 10 8.26 ± 3.54 8.75 ± 0.17 154 ± 4 45.1 ± 1.0 1,044 ± 176
Zhengdan 958 maize(35%) 10 8.72 ± 3.65 8.86 ± 0.34 153 ± 7 44.2 ± 2.8 969 ± 93
Zhengdan 958 maize(70%) 10 9.10 ± 3.74 9.20 ± 0.26* 155 ± 5 44.6 ± 1.2 1,072 ± 117
Sex Groups Number of rats NEU
(%) 		LYM
(%) 		MO
(%) 		EOS
(%) 		BAS
(%) 		PT
(s) 		APTT
(s)
Female Control 10 9.6 ± 4.2 84.9 ± 4.2 4.3 ± 1.0 1.2 ± 0.4 0.0 ± 0.0 9.2 ± 0.2 14.6 ± 0.9
CC-2(17.5%) 10 8.6 ± 3.0 85.1 ± 3.9 4.7 ± 1.2 1.7 ± 0.6 0.0 ± 0.0 9.1 ± 0.1 14.3 ± 1.2
CC-2(35%) 10 9.9 ± 3.6 84.1 ± 4.6 4.2 ± 1.0 1.8 ± 1.1 0.0 ± 0.0 8.9 ± 0.4 13.6 ± 1.6
CC-2(70%) 10 10.6 ± 3.6 83.4 ± 4.0 4.6 ± 1.3 1.4 ± 0.5 0.0 ± 0.0 9.0 ± 0.3 13.7 ± 1.1
Zhengdan 958 maize(17.5%) 10 12.0 ± 5.8 81.7 ± 5.4 4.6 ± 1.9 1.7 ± 0.7 0.0 ± 0.0 9.1 ± 0.1 14.4 ± 0.6
Zhengdan 958 maize(35%) 10 8.6 ± 3.3 85.3 ± 3.6 4.5 ± 1.2 1.6 ± 0.3 0.0 ± 0.0 9.1 ± 0.3 15.1 ± 2.0
Zhengdan 958 maize(70%) 10 10.9 ± 6.4 83.6 ± 6.1 4.3 ± 1.2 1.2 ± 0.5 0.0 ± 0.0 9.0 ± 0.2 14.6 ± 1.3
Male Control 10 14.4 ± 3.8 79.8 ± 3.9 4.6 ± 1.1 1.2 ± 0.3 0.0 ± 0.0 10.1 ± 1.0 19.0 ± 2.5
CC-2(17.5%) 10 15.7 ± 3.8 78.7 ± 4.8 4.0 ± 1.4 1.7 ± 0.4 0.0 ± 0.0 10.6 ± 1.8 20.0 ± 3.3
CC-2(35%) 10 13.7 ± 5.3 80.9 ± 5.6 3.9 ± 0.9 1.6 ± 1.0 0.0 ± 0.0 10.1 ± 0.8 17.9 ± 1.5
CC-2(70%) 10 11.8 ± 4.6 82.3 ± 5.5 5.0 ± 1.1 1.0 ± 0.4 0.0 ± 0.0 10.5 ± 1.2 19.9 ± 2.3
Zhengdan 958 maize(17.5%) 10 13.0 ± 4.4 81.1 ± 4.8 4.7 ± 1.1 1.1 ± 0.5 0.0 ± 0.0 10.7 ± 1.0 21.3 ± 2.5
Zhengdan 958 maize(35%) 10 12.4 ± 5.3 81.1 ± 6.3 5.3 ± 1.2 1.1 ± 0.6 0.0 ± 0.0 10.2 ± 1.0 20.5 ± 3.0
Zhengdan 958 maize(70%) 10 14.2 ± 7.1 80.1 ± 7.4 4.5 ± 1.1 1.2 ± 0.6 0.0 ± 0.0 10.2 ± 0.4 18.5 ± 1.5
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Compared to control group, *P<0.05; compared to the corresponding dose parental group, P > 0.05.

Medium-term haematology results were presented below (Table 7). There were no significant differences in haematology results of the transgenic group, compared to the corresponding dose parental group (P > 0.05). Compared to the female rats of the control group, there were no significant differences in haematology (P > 0.05); compared to the male rats of the control group, RBC number of CC-2(17.5%) group was statistically lower (P < 0.05), hemoglobin content/hematocrit of CC-2 (70%) group and Zhengdan 958 maize (70%) group was statistically lower (P < 0.01 or P < 0.05), monocyte percentage of Zhengdan 958 maize (70%) group was statistically higher (P < 0.05).

Terminal haematology results were presented below (Table 8). There were no significant differences in haematology results of the transgenic group, compared to the corresponding dose parental group (P > 0.05). Compared to the male rats of the control group, RBC number of Zhengdan 958 maize (70%) group was statistically higher (P < 0.05).

There were no adverse effects on haematology of rats fed with diets containing transgenic maize CC-2 for 90 days.

Blood biochemistry

Medium-term blood biochemistry results were presented below (Table 9). ALT and AST of male rats in CC-2 (70%) group were significantly higher (P < 0.05 for ALT; P < 0.01 for AST), compared to the corresponding dose parental group. There were no significant differences in blood chemistry results of female rats in the transgenic group, compared to the corresponding dose parental group (P > 0.05). Compared to the male control group, ALT, ALP, GLU of CC-2(70%) group were significantly higher (P < 0.01 for ALT and ALP; P < 0.05 for GLU), while AST, TG of Zhengdan 958 maize (70%) group were significantly higher (P < 0.01). Compared to the female control group, ALT and GLU of CC-2 (70%) group were significantly higher (P < 0.01), while GLU of zhengdan 958 maize (35%) group was significantly higher (P < 0.05).

Table 9.

The effects of mid-term blood chemistry parameters of rats fed with transgenic maize CC-2 (Inline graphic±SD).

Sex Groups Number of rats ALT
(U/L)		 AST
(U/L)		 ALP
(U/L)		 TP
(g/L)		 ALB
(g/L)
Female Control 10 31 ± 3 245 ± 40 114 ± 28 79.8 ± 6.4 58.6 ± 4.4
CC-2(17.5%) 10 34 ± 5 249 ± 23 109 ± 32 80.0 ± 3.3 59.8 ± 3.1
CC-2(35%) 10 31 ± 5 234 ± 49 108 ± 21 78.2 ± 5.6 58.8 ± 4.5
CC-2(70%) 10 46 ± 12** 238 ± 45 120 ± 30 75.4 ± 4.0 57.9 ± 2.7
Zhengdan 958 maize(17.5%) 10 28 ± 6 208 ± 41 104 ± 33 80.3 ± 4.8 59.9 ± 4.2
Zhengdan 958 maize(35%) 10 35 ± 6 240 ± 33 112 ± 38 78.9 ± 7.8 59.1 ± 5.7
Zhengdan 958 maize(70%) 10 36 ± 8 215 ± 31 104 ± 29 74.4 ± 6.4 56.2 ± 5.5
Male Control 10 41 ± 8 303 ± 72 154 ± 18 76.9 ± 6.6 50.8 ± 2.5
CC-2(17.5%) 10 42 ± 6 316 ± 32 159 ± 24 76.1 ± 5.4 50.9 ± 2.8
CC-2(35%) 10 41 ± 5 302 ± 40 180 ± 39 77.9 ± 6.5 51.9 ± 2.9
CC-2(70%) 10 54 ± 7**,*** 306 ± 49**** 202 ± 44** 73.0 ± 4.9 49.7 ± 1.5
Zhengdan 958 maize(17.5%) 10 35 ± 6 285 ± 47 172 ± 32 75.0 ± 2.7 50.8 ± 1.9
Zhengdan 958 maize(35%) 10 44 ± 9 283 ± 66 181 ± 26 76.1 ± 4.4 52.3 ± 2.9
Zhengdan 958 maize(70%) 10 44 ± 7 225 ± 52** 177 ± 27 70.8 ± 2.4 49.4 ± 1.5
Sex Groups Number of rats BUN
(mmol/L) 		CRE
(μmol/L) 		GLU
(mmol/L) 		CHO
(mmol/L) 		TG
(mmol/L) 		LDH
(U/L)
Female Control 10 5.12 ± 0.77 36.9 ± 2.4 2.64 ± 0.68 2.06 ± 0.54 0.31 ± 0.19 3,195 ± 391
CC-2(17.5%) 10 6.15 ± 0.79 40.5 ± 4.2 2.85 ± 0.78 2.39 ± 0.41 0.28 ± 0.14 3,320 ± 205
CC-2(35%) 10 5.70 ± 1.13 40.2 ± 3.8 2.93 ± 0.46 2.52 ± 0.46 0.42 ± 0.27 3,260 ± 545
CC-2(70%) 10 5.60 ± 1.12 41.5 ± 5.2 3.63 ± 0.49** 2.70 ± 0.53 0.35 ± 0.19 3,225 ± 482
Zhengdan 958 maize(17.5%) 10 5.49 ± 1.22 38.3 ± 3.8 3.10 ± 0.77 2.45 ± 0.59 0.39 ± 0.25 2,936 ± 489
Zhengdan 958 maize(35%) 10 5.84 ± 1.52 34.3 ± 3.8 3.44 ± 0.55* 2.72 ± 0.55 0.28 ± 0.16 3,372 ± 370
Zhengdan 958 maize(70%) 10 4.85 ± 0.93 35.6 ± 5.8 3.35 ± 0.67 2.42 ± 0.50 0.18 ± 0.09 2,991 ± 362
Male Control 10 5.71 ± 0.80 36.9 ± 2.0 2.48 ± 0.88 2.74 ± 0.31 0.76 ± 0.24 3,340 ± 514
CC-2(17.5%) 10 5.61 ± 0.74 37.1 ± 2.2 2.90 ± 0.77 2.80 ± 0.40 0.9 ± 0.28 3,568 ± 319
CC-2(35%) 10 5.40 ± 1.23 39.0 ± 2.0 3.03 ± 0.83 2.75 ± 0.42 1.18 ± 0.47 3,597 ± 365
CC-2(70%) 10 6.40 ± 1.37 37.9 ± 2.6 3.38 ± 0.64* 2.80 ± 0.46 1.16 ± 0.48 3,665 ± 483
Zhengdan 958 maize(17.5%) 10 5.82 ± 1.27 36.2 ± 2.8 2.56 ± 0.54 2.48 ± 0.43 0.87 ± 0.42 3,599 ± 398
Zhengdan 958 maize(35%) 10 5.85 ± 0.81 39.1 ± 2.2 3.28 ± 0.57 2.61 ± 0.58 1.00 ± 0.50 3,530 ± 752
Zhengdan 958 maize(70%) 10 5.39 ± 0.84 38.2 ± 1.9 3.26 ± 0.77 2.67 ± 0.48 1.74 ± 0.69** 2,976 ± 752
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Compared to control group, *P<0.05, **P<0.01; compared to the corresponding dose parental group, ***P < 0.05, ****P < 0.01.

Terminal blood biochemistry results were shown below (Table 10). ALT of female rats in CC-2 (70%) group were significantly higher (P < 0.05), compared to the corresponding dose parental group. Compared to the female control group, ALT of CC-2(70%) group was significantly higher (P < 0.01), GLU of Zhengdan 958 maize (35% or 70%) and CC-2 (35% or 70%) maize groups was significantly higher (P < 0.05, P < 0.01, P < 0.01, P < 0.01), CHO of Zhengdan 958 maize (35%) group was significantly higher (P < 0.01). Compared to the male control group, GLU of CC-2 (35%) and Zhengdan 958 (70%) maize groups was significantly higher (P < 0.05 or P < 0.01), TG of Zhengdan 958 (70%) group was significantly higher (P < 0.05), Cl− of CC-2 (35%, 70%) groups and Zhengdan 958 (17.5%, 35% and 70%) groups was significantly lower (P < 0.01).

Table 10.

The effects of terminal blood chemistry parameters of rats fed with transgenic maize CC-2 (Inline graphic±SD).

Sex Groups Number of rats ALT
(U/L)		 AST
(U/L)		 TP
(g/L)		 ALB
(g/L)		 BUN
(mmol/L)		 CRE
(μmol/L)		 GLU
(mmol/L)
Female Control 10 19 ± 2 91 ± 17 67.4 ± 4.8 48.8 ± 3.8 6.62 ± 1.44 61.2 ± 6.3 4.71 ± 0.97
CC-2(17.5%) 10 19 ± 4 100 ± 10 68.1 ± 3.3 50.2 ± 3.0 6.86 ± 1.19 68.1 ± 5.9 5.65 ± 1.29
CC-2(35%) 10 19 ± 5 97 ± 16 68.4 ± 5.5 50.7 ± 4.6 6.43 ± 1.46 62.8 ± 10.6 6.26 ± 0.53*
CC-2(70%) 10 30 ± 6**,*** 120 ± 20 67.3 ± 4.0 50.0 ± 3.2 6.62 ± 2.42 68.9 ± 16.4 7.96 ± 1.52**
Zhengdan 958 maize(17.5%) 10 20 ± 8 101 ± 24 68.8 ± 4.2 51.0 ± 3.9 6.80 ± 1.78 69.7 ± 12.1 6.00 ± 1.29
Zhengdan 958 maize(35%) 10 20 ± 3 101 ± 20 68.5 ± 4.0 51.2 ± 4.0 6.80 ± 2.10 64.9 ± 10.3 7.14 ± 1.25**
Zhengdan 958 maize(70%) 10 22 ± 5 102 ± 23 64.3 ± 6.2 47.3 ± 7.1 7.37 ± 2.05 67.0 ± 9.8 7.25 ± 1.34**
Male Control 10 30 ± 5 130 ± 29 62.8 ± 2.8 41.0 ± 1.4 6.14 ± 1.13 56.5 ± 4.2 6.88 ± 2.19
CC-2(17.5%) 10 31 ± 9 150 ± 21 63.0 ± 3.3 41.0 ± 2.8 6.59 ± 0.90 55.6 ± 6.9 7.70 ± 1.49
CC-2(35%) 10 31 ± 8 136 ± 42 62.8 ± 3.1 40.3 ± 2.0 6.15 ± 0.82 55.3 ± 7.5 9.29 ± 2.42*
CC-2(70%) 10 46 ± 21 147 ± 44 60.9 ± 2.9 39.4 ± 1.4 5.90 ± 0.81 54.1 ± 4.3 8.98 ± 1.91
Zhengdan 958 maize(17.5%) 10 28 ± 6 112 ± 23 62.1 ± 2.5 40.5 ± 1.9 6.69 ± 0.95 58.7 ± 6.6 6.72 ± 1.53
Zhengdan 958 maize(35%) 10 38 ± 13 139 ± 40 61.0 ± 4.4 39.5 ± 2.8 6.48 ± 0.68 61.3 ± 5.3 8.18 ± 2.09
Zhengdan 958 maize(70%) 10 44 ± 28 138 ± 37 62.2 ± 3.2 40.4 ± 2.2 6.15 ± 0.80 57.7 ± 5.8 10.00 ± 1.53**
Sex Groups Number of rats CHO
(mmol/L) 		TG
(mmol/L) 		ALP
(U/L) 		K +
(mmol/L) 		Na +
(mmol/L) 		Cl −
(mmol/L)
Female Control 10 1.62 ± 0.40 0.19 ± 0.13 45 ± 10 4.39 ± 0.29 143.6 ± 1.2 100.5 ± 0.7
CC-2(17.5%) 10 1.96 ± 0.39 0.23 ± 0.14 43 ± 10 4.34 ± 0.24 143.8 ± 0.8 100.3 ± 1.6
CC-2(35%) 10 2.07 ± 0.47 0.30 ± 0.16 43 ± 7 4.71 ± 0.50 143.5 ± 1.1 100.7 ± 1.0
CC-2(70%) 10 2.10 ± 0.50 0.24 ± 0.07 56 ± 21 4.48 ± 0.46 143.0 ± 1.2 100.9 ± 1.0
Zhengdan 958 maize(17.5%) 10 2.07 ± 0.42 0.18 ± 0.05 44 ± 15 4.22 ± 0.27 143.8 ± 1.4 100.7 ± 1.0
Zhengdan 958 maize(35%) 10 2.35 ± 0.47** 0.22 ± 0.09 41 ± 16 4.41 ± 0.34 143.4 ± 0.7 100.2 ± 1.1
Zhengdan 958 maize(70%) 10 2.02 ± 0.34 0.22 ± 0.08 43 ± 8 4.20 ± 0.42 144.1 ± 1.1 101.1 ± 0.9
Male Control 10 1.99 ± 0.30 0.62 ± 0.29 68 ± 12 4.97 ± 0.39 143.9 ± 1.2 102.5 ± 1.2
CC-2(17.5%) 10 2.31 ± 0.38 0.80 ± 0.37 73 ± 10 5.31 ± 0.49 143.8 ± 1.5 101.4 ± 1.9
CC-2(35%) 10 2.14 ± 0.39 0.84 ± 0.51 77 ± 16 5.36 ± 0.39 143.4 ± 1.6 100.1 ± 0.8**
CC-2(70%) 10 2.09 ± 0.36 0.74 ± 0.35 82 ± 16 5.09 ± 0.25 144.5 ± 0.9 100.0 ± 1.6**
Zhengdan 958 maize(17.5%) 10 1.87 ± 0.36 0.46 ± 0.27 78 ± 13 4.99 ± 0.37 145.1 ± 1.1 100.3 ± 1.7**
Zhengdan 958 maize(35%) 10 1.89 ± 0.25 0.60 ± 0.25 69 ± 9 5.25 ± 0.82 143.5 ± 1.1 99.7 ± 1.3**
Zhengdan 958 maize(70%) 10 2.19 ± 0.49 1.12 ± 0.68* 73 ± 13 5.27 ± 0.40 143.7 ± 1.4 99.2 ± 1.6**
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Compared to control group, *P<0.05, **P<0.01; compared to the corresponding dose parental group, ***P < 0.05.

Compared with the corresponding dose parental group and control group, ALT of male rats in the medium-term CC-2 (70%) group and female rats in the end CC-2 (70%) group increased statistically significantly, but the above data were basically within the range of historical normal values in our laboratory, and no abnormalities were found in other liver function indicators and organ histopathology, so there was no obvious biological significance. AST of male rats in the mid-term CC-2 (70%) maize group was significantly higher than that in the parental Zhengdan 958 (70%) maize group, but there was no significant difference compared with the control group, which was analyzed as natural fluctuation. There was no significant difference in other blood chemistry parameters between each CC-2 dose group and the corresponding dose parental group. No adverse effects were found on blood chemistry parameters of rats after 90 days of feeding with transgenic maize CC-2.

Urine analysis

As was shown in the Table 11, compared to the female control group, urine specific gravity (SG) of Zhengdan 958 (35%) group was significantly higher (P < 0.05), while there was no significant difference in urine analysis of rats in transgenic group compared to the corresponding dose parental group. There was no adverse effects on urine analysis of rats fed with diets containing transgenic maize CC-2 for 90 days.

Table 11.

The effects of terminal urine parameters of rats fed with transgenic maize CC-2 (positive number/total number).

Sex Groups Number of rats Leukocyte Ketone body bilirubin urobilinogen Occult blood Protein Glucose Nitrosamine PH Gravity of urine
Female Control 10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 1/10 1.015 ± 0.013 5.2 ± 0.3
CC-2(17.5%) 10 0/10 0/10 0/10 0/10 0/10 9/10 0/10 1/10 1.015 ± 0.013 5.2 ± 0.3
CC-2(35%) 10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 1.018 ± 0.013 5.4 ± 0.4
CC-2(70%) 10 0/10 0/10 0/10 0/10 0/10 8/10 0/10 2/10 1.020 ± 0.013 5.4 ± 0.4
Zhengdan 958 maize(17.5%) 10 0/10 0/10 1/10 0/10 0/10 10/10 0/10 2/10 1.023 ± 0.012 5.2 ± 0.4
Zhengdan 958 maize(35%) 10 0/10 0/10 0/10 0/10 1/10 9/10 0/10 0/10 1.025 ± 0.011 5.8 ± 0.4*
Zhengdan 958 maize(70%) 10 0/10 0/10 0/10 0/10 0/10 7/10 0/10 2/10 1.028 ± 0.008 5.5 ± 0.4
Male Control 10 0/10 0/10 0/10 0/10 0/10 5/10 0/10 0/10 1.030 ± 0.000 5.3 ± 0.4
CC-2(17.5%) 10 0/10 0/10 0/10 0/10 0/10 5/10 0/10 3/10 1.028 ± 0.008 5.5 ± 0.4
CC-2(35%) 10 0/10 0/10 0/10 0/10 1/10 7/10 0/10 0/10 1.028 ± 0.008 5.8 ± 0.4
CC-2(70%) 10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 1.030 ± 0.000 6.0 ± 0.4
Zhengdan 958 maize(17.5%) 10 0/10 0/10 0/10 0/10 2/10 3/10 0/10 0/10 1.030 ± 0.000 5.5 ± 0.5
Zhengdan 958 maize(35%) 10 0/10 0/10 0/10 3/10 3/10 1/10 0/10 2/10 1.030 ± 0.000 5.6 ± 0.6
Zhengdan 958 maize(70%) 10 0/10 0/10 0/10 0/10 0/10 8/10 0/10 0/10 1.030 ± 0.000 5.7 ± 0.4
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Compared to control group, *P<0.05; compared to the corresponding dose parental group, P > 0.05.

The organ weight and organ coefficient

As was shown in Table 12, compared to the corresponding dose parental group, there were no significant differences in the organ weight and organ coefficient of rats in the transgenic group (P > 0.05). Compared to the female control group, kidney coefficient of CC-2 (17.5%, 70%) and Zhengdan 958 (17.5%) maize groups was significantly lower (P < 0.05, P < 0.01, P < 0.05). Compared to the male control group, the anatomical weight of male rats in CC-2 (70%) maize group was significantly higher (P < 0.05), brain coefficient of the above group was significantly lower (P < 0.01), epididymis weight of rats in Zhengdan 958 (70%) group was significantly higher (P < 0.05), adrenal gland weight and coefficient of Zhengdan 958 (35%) group were significantly higher (P < 0.05).

Table 12.

The effects of organ weight and organ coefficient of rats fed with transgenic maize CC-2 (Inline graphic±SD).

Sex Group Number of rats Weight before sacrifice
(g)		 Heart Liver Kidney
Weight
(g)		 Organ
coefficient
(%)		 Weight
(g)		 Organ coefficient
(%)		 Weight
(g)		 Organ coefficient
(%)
Female control 10 343.1 ± 27.6 1.098 ± 0.089 0.322 ± 0.038 8.014 ± 0.795 2.336 ± 0.139 2.323 ± 0.246 0.679 ± 0.069
CC-2(17.5%) 10 350.7 ± 29.1 1.084 ± 0.134 0.309 ± 0.029 8.071 ± 0.795 2.300 ± 0.073 2.098 ± 0.222 0.599 ± 0.045*
CC-2(35%) 10 354.7 ± 19.3 1.090 ± 0.113 0.307 ± 0.029 8.192 ± 0.847 2.316 ± 0.267 2.306 ± 0.209 0.652 ± 0.066
CC-2(70%) 10 363.3 ± 28.6 1.161 ± 0.171 0.319 ± 0.032 8.262 ± 0.782 2.277 ± 0.172 2.132 ± 0.220 0.587 ± 0.050**
Zhengdan 958 maize(17.5%) 10 362.6 ± 40.0 1.096 ± 0.081 0.304 ± 0.024 8.240 ± 0.722 2.280 ± 0.117 2.178 ± 0.169 0.604 ± 0.055*
Zhengdan 958 maize(35%) 10 355.3 ± 33.9 1.063 ± 0.105 0.300 ± 0.025 8.343 ± 0.828 2.351 ± 0.142 2.309 ± 0.222 0.655 ± 0.087
Zhengdan 958 maize(70%) 10 334.0 ± 17.5 1.026 ± 0.092 0.307 ± 0.024 8.021 ± 0.549 2.404 ± 0.152 2.089 ± 0.191 0.625 ± 0.040
Male control 10 622.5 ± 60.2 1.872 ± 0.291 0.300 ± 0.027 15.476 ± 2.446 2.478 ± 0.207 3.613 ± 0.307 0.582 ± 0.033
CC-2(17.5%) 10 643.8 ± 62.6 1.864 ± 0.199 0.290 ± 0.024 14.436 ± 4.210 2.219 ± 0.567 3.684 ± 0.406 0.575 ± 0.071
CC-2(35%) 10 669.6 ± 70.3 2.009 ± 0.297 0.300 ± 0.030 16.731 ± 1.473 2.508 ± 0.174 3.856 ± 0.305 0.580 ± 0.062
CC-2(70%) 10 703.4 ± 40.7* 2.039 ± 0.257 0.290 ± 0.033 17.245 ± 1.887 2.449 ± 0.198 3.752 ± 0.435 0.533 ± 0.055
Zhengdan 958 maize(17.5%) 10 634.1 ± 46.6 1.950 ± 0.290 0.307 ± 0.031 15.516 ± 1.160 2.450 ± 0.134 4.054 ± 0.419 0.640 ± 0.059
Zhengdan 958 maize(35%) 10 634.9 ± 48.2 1.931 ± 0.206 0.304 ± 0.027 15.794 ± 1.768 2.483 ± 0.148 3.530 ± 0.374 0.557 ± 0.059
Zhengdan 958 maize(70%) 10 662.5 ± 70.0 1.993 ± 0.326 0.300 ± 0.034 17.992 ± 3.238 2.700 ± 0.259 3.879 ± 0.491 0.589 ± 0.078
Female control 10 0.071 ± 0.011 0.021 ± 0.004 0.551 ± 0.090 0.160 ± 0.022 0.441 ± 0.098 0.128 ± 0.025
CC-2(17.5%) 10 0.069 ± 0.020 0.020 ± 0.005 0.474 ± 0.083 0.135 ± 0.020 0.508 ± 0.165 0.143 ± 0.039
CC-2(35%) 10 0.072 ± 0.015 0.020 ± 0.005 0.539 ± 0.087 0.152 ± 0.025 0.527 ± 0.122 0.148 ± 0.031
CC-2(70%) 10 0.070 ± 0.009 0.019 ± 0.003 0.481 ± 0.112 0.132 ± 0.028 0.544 ± 0.111 0.149 ± 0.022
Zhengdan 958 maize(17.5%) 10 0.065 ± 0.006 0.016 ± 0.006 0.489 ± 0.034 0.136 ± 0.018 0.501 ± 0.079 0.140 ± 0.029
Zhengdan 958 maize(35%) 10 0.071 ± 0.009 0.020 ± 0.003 0.515 ± 0.074 0.146 ± 0.023 0.558 ± 0.193 0.157 ± 0.048
Zhengdan 958 maize(70%) 10 0.077 ± 0.012 0.023 ± 0.003 0.510 ± 0.110 0.153 ± 0.033 0.441 ± 0.074 0.132 ± 0.019
Male control 10 0.059 ± 0.019 0.010 ± 0.003 0.824 ± 0.110 0.132 ± 0.010 0.622 ± 0.221 0.099 ± 0.032
CC-2(17.5%) 10 0.072 ± 0.013 0.011 ± 0.001 0.817 ± 0.171 0.126 ± 0.019 0.715 ± 0.199 0.110 ± 0.027
CC-2(35%) 10 0.064 ± 0.009 0.010 ± 0.001 0.818 ± 0.140 0.123 ± 0.022 0.619 ± 0.092 0.093 ± 0.016
CC-2(70%) 10 0.064 ± 0.007 0.009 ± 0.001 0.940 ± 0.128 0.133 ± 0.014 0.684 ± 0.174 0.097 ± 0.021
Zhengdan 958 maize(17.5%) 10 0.071 ± 0.012 0.011 ± 0.002 0.862 ± 0.102 0.136 ± 0.015 0.634 ± 0.201 0.099 ± 0.028
Zhengdan 958 maize(35%) 10 0.077 ± 0.015* 0.012 ± 0.003* 0.824 ± 0.118 0.130 ± 0.014 0.623 ± 0.186 0.099 ± 0.032
Zhengdan 958 maize(70%) 10 0.062 ± 0.008 0.009 ± 0.001 0.901 ± 0.158 0.135 ± 0.015 0.584 ± 0.144 0.087 ± 0.016
Female control 10 1.938 ± 0.080 0.568 ± 0.052 0.612 ± 0.200 0.180 ± 0.063 0.145 ± 0.031 0.042 ± 0.008
CC-2(17.5%) 10 1.877 ± 0.100 0.539 ± 0.059 0.667 ± 0.321 0.187 ± 0.077 0.152 ± 0.027 0.044 ± 0.008
CC-2(35%) 10 1.952 ± 0.107 0.551 ± 0.034 0.591 ± 0.161 0.166 ± 0.042 0.151 ± 0.028 0.043 ± 0.008
CC-2(70%) 10 1.944 ± 0.132 0.537 ± 0.046 0.571 ± 0.130 0.158 ± 0.039 0.157 ± 0.038 0.043 ± 0.010
Zhengdan 958 maize(17.5%) 10 1.992 ± 0.109 0.555 ± 0.066 0.558 ± 0.146 0.157 ± 0.048 0.161 ± 0.050 0.046 ± 0.017
Zhengdan 958 maize(35%) 10 1.908 ± 0.081 0.542 ± 0.063 0.565 ± 0.140 0.159 ± 0.036 0.139 ± 0.030 0.040 ± 0.011
Zhengdan 958 maize(70%) 10 1.964 ± 0.087 0.589 ± 0.028 0.513 ± 0.083 0.154 ± 0.025 0.153 ± 0.031 0.046 ± 0.008
control 10 2.163 ± 0.082 0.350 ± 0.037 3.477 ± 0.286 0.565 ± 0.080 1.282 ± 0.087 0.208 ± 0.024
Male CC-2(17.5%) 10 2.110 ± 0.136 0.330 ± 0.033 3.410 ± 0.303 0.533 ± 0.062 1.405 ± 0.108 0.220 ± 0.027
CC-2(35%) 10 2.176 ± 0.101 0.327 ± 0.024 3.316 ± 0.850 0.491 ± 0.114 1.384 ± 0.254 0.206 ± 0.034
CC-2(70%) 10 2.105 ± 0.041 0.300 ± 0.018** 3.397 ± 0.272 0.485 ± 0.055 1.425 ± 0.084 0.203 ± 0.019
Zhengdan 958 maize(17.5%) 10 2.213 ± 0.097 0.350 ± 0.027 3.486 ± 0.358 0.550 ± 0.049 1.373 ± 0.134 0.217 ± 0.018
Zhengdan 958 maize(35%) 10 2.141 ± 0.117 0.339 ± 0.036 3.521 ± 0.607 0.554 ± 0.071 1.295 ± 0.104 0.205 ± 0.021
Zhengdan 958 maize(70%) 10 2.162 ± 0.094 0.329 ± 0.036 3.745 ± 0.540 0.571 ± 0.096 1.479 ± 0.152* 0.224 ± 0.025
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Compared to control group, *P<0.05, **P<0.01; compared to the corresponding dose parental group, P > 0.05.

There were no adverse effects on organ weight and coefficient of rats fed with diets containing transgenic maize CC-2 for 90 days.

Histophathology

Histopathological findings are summarized in Table 13. Histopathological findings showed that some spontaneous changes in liver, kidney, heart, pancreas, caecum and prostate. The incidences/severity of the changes showed no difference between the transgenic and control groups. No histopathological lesions were observed in other examined tissues.

Table 13.

Histopathological changes of rats fed with maize for 90 days.

Organ Lesions Control group CC-2 70% Zhengdan 958 70%
Female Male Female Male Female Male
Liver Mild hepatocyte steatosis 9 6 5 7 7 8
Moderate hepatocyte steatosis 0 3 0 0 0 0
Slight focal hepatocyte necrosis with inflammatory cell infiltration 1 0 0 0 0 0
Small focal inflammatory cell infiltration 0 0 0 0 1 1
Multifocal lytic necrosis 0 0 0 1 0 0
Spontaneous altered hepatocyte foci 0 0 0 0 1 0
Kidney Renal tubular epithelial cell swelling 1 0 0 0 0 0
Renal pelvis inflammatory cell infiltration 0 0 1 0 0 0
Heart Slight focal inflammatory cell infiltration 1 1 0 0 0 0
Focal inflammatory cell infiltration 0 0 0 2 0 0
focal myocardial cell necrosis with inflammatory cell infiltration 0 2 0 0 0 1
Focal fibrous tissue hyperplasia with inflammatory cell infiltration 0 0 0 1 0 0
Pancreas Small inflammatory cell infiltration around the pancreatic duct 1 0 0 0 0 0
Focal acinar atrophy 0 0 1 0 0 1
Focal inflammatory cell infiltration 0 0 1 0 0 0
Vacuolar degeneration of pancreatic acinar cells 0 0 0 0 0 1
Fibrous tissue hyperplasia of pancreas islet 0 0 0 0 0 1
Caecal Mucosa inflammatory cell infiltration and submucosa swelling 0 0 0 0 0 1
Prostate prostatic interstitial inflammatory cell infiltration 0 2 0 2 0 2
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Sample number was 10.

Discussion

In China, more or less 65% of maize seed produced was used as feed.7 The transgenic technology makes it possible for maize to resist the herbicide glyphosate. As was mentioned previously, transgenic maize CC-2 expressed the macroACC gene tolerant to the herbicide glyphosate. Chen D.L. reported that the genetically modified maize CC-2 (harvested in 2010) and its non-transgenic control maize (Zheng-58) were added respectively to the basal feed in a ratio of 12.5%, 25.0% and 50.0%, which were fed to SD rats during 90d, showed that the transgenic herbicide-resistant maize was as the same safety as its non-GM maize.4 In this study, transgenic maize CC-2 was planted in November of 2018 and harvested in February of 2019; transgenic maize CC-2 was added to the basal feed at the ratio of 70%, 35%, 17.5%, the highest dose was more than that of Chen D.L.’s study; the detection parameters were more than Chen D.L.’s study; there were no adverse effects observed in SD rats 90-day exposed to transgenic maize CC-2 compared with its parental maize Zhengdan 958.4

The GMO Panel concludes that maize MZIR098 is as safe as the conventional counterpart and non-GM maize reference varieties tested, maize MZIR098 was developed to confer tolerance to glufosinate-ammonium-containing herbicides and resistance to certain coleopteran pests.23 Herbicide-resistant maize MON87427 with Cp4epsps gene has no subchronic toxicity to Wistar rats24 and no adverse effects were found in neurobehavior and cognitive ability on SD rats through the three generation study of genetically modified maize with Cry1Ab and EPSPS genes.25 The dose design in this study was based on the professional standard “Safety assessment of genetically modified plant and derived products 90-day feeding test in rats (NY/T 1102-2006)”; the detecting parameters were according to the national standard for food safety “90-day oral toxicity study” (GB15193.13-2015) in China.

Transgenic plant use as food or feed is still contentious, since it’s important for consumer that food is necessary in daily life. International organizations and the world governments have introduced a series of safety evaluation and management measures to ensure the safety of genetically modified food. It was presented at the FAO/WHO Expert Advisory Meeting in 1996 that if the GM product is different in composition from the non-GM control or if there are undesired effects for the transgenic operations, 90-day feeding animal study should be conducted.4,26 According to the regulations in EU and China, a 90-day feeding study is required as part to the toxicological assessment of genetically modified plants.7,18,19

Before the 90-day feeding study, the endogenous gene zSSIIb was detected in both kinds of maize, and identification-specific genes of CC-2 maize only existed in transgenic maize. The identification results showed that both kinds of maize were identified correct and were able to used in the following study. And the nutritional composition analysis of the diet in each group was performed and then the results showed that the fundamental nutrient and energy contents were consistent between groups.

In this study, no deaths or toxic symptoms were observed in each group. The transgenic maize CC-2 showed no significant changes in body weight of rats at concentrations up to 70% compared with parental groups and the control group. The results of food utilization rate showed that the food utilization rate of male rats in the CC-2 (70%) group was significantly different from that in the Zhengdan 958 (70%) group at some time points (P < 0.05), but there was no dose–response relationship and no significant difference in total food utilization rate, so there was no biological significance.

The results of blood biochemistry showed that ALT of male rats in the mid-experiment CC-2 (70%) group and female rats in the end-experiment CC-2 (70%) group was statistically significantly higher than that of the corresponding dose parental group or the control group. The comprehensive analysis of the results was illustrated below.

The female highest ALT in CC-2 70% group in terminal experiment was within ALT historical normal value range of the female SD rat in our laboratory, so there was no obvious biological significance. Mild hepatocyte steatosis in terminal was detected in 5 female rats of CC-2 70% group, 9 female rats of control group and 7 female rats of Zhengdan 958 70% group; otherwise control and Zhengdan 958 70% group showed other pathological changes in liver. So there were no CC-2 related pathological changes in liver of female rats.

In the mid-experiment, ALT in male rats of CC-2 70% group [(54 ± 7)U/L] was significantly higher than that in control group [(41 ± 8)U/L], and significantly higher than that in parental high-dose group [(44 ± 7)U/L]. The highest ALT in the male rats of the CC-2 70% group was within the normal range of our laboratory. The pathological results showed that the rats with the highest ALT value in the CC-2 70% group had mild hepatocyte steatosis without other pathological changes in liver, and the control group and the Zhengdan 958 70% group also had mild hepatocyte steatosis. In the CC-2 70% group, 7 cases had mild hepatic steatosis and 1 case had hepatic multifocal lytic necrosis. There were 6 cases of mild hepatic steatosis and 3 cases of moderate hepatocyte steatosis in the control group. In the Zhengdan958 70% group, there were 8 cases of mild hepatic steatosis and 1 case of focal inflammatory cell infiltration. In conclusion, compared with the control group and the parental high-dose group (Zhengdan958 70% group), the male mice in the CC-2 70% group did not show obvious pathological changes related to the test substance.

AST of male rats in the mid-term CC-2 (70%) group was significantly higher than that in the corresponding dose parental group, but there was no significant difference compared with the control group, which was analyzed as natural fluctuation. There was no significant difference in other biochemical indexes between each dose group and the corresponding dose parent group. In the histopathological examination, there are some lesions in the liver, kidney, heart, pancreas, caecum and prostate. However, the incidences/severity of the lesions showed no difference between the transgenic and control groups, so the lesions were considered to be spontaneous or incidental alterations, unrelated to exposure to the transgenic maize CC-2.

The safety evaluation of GM food includes not only 90-day feeding experiment, but also long-term toxicity study, reproductive toxicity test and so on. A long-term toxicity study of transgenic rice containing cry1Ac and sck genes was reported in the literature, the feeding period was 78 weeks, and the long-term intake of high doses of the above transgenic rice did not cause unexpected adverse effects in rats.27 Hu et al. studied the reproductive toxicity of the above transgenic rice in the third generation, and found no unexpected adverse effects.28 Guo et al. studied the effects of transgenic maize with insect-resistant and herbicide-tolerant genes Cry1Ab and EPSPS on the neurobehavior and cognitive ability of the offspring of third-generation breeding rats, and found no adverse effects on the neuroethology and cognitive ability of F3 offspring.25

Similar to the reported literature, there were no adverse effects in SD rats following 90-day dietary exposure to transgenic maize CC-2 in this study.

Author contributions

Chen Chen (Performed the animal experiment, hemagglutination parameters), Lili Shi (Performed the experiment, contributed to necropsy), Hongmei Mao (Performed the animal experiment, hematology and serum biochemistry), Jinpeng Zhao (Performed the animal experiment, contributed to necropsy and histopathology), Chao Han (Performed the experiment, hematology and serum biochemistry), Qin Zhuo (Performed the animal experiments and provided fund assist), Yan Li (Designed and performed the experiments, formal analysis, provided fund assist). The manuscript was drafted by CC and proof-read by YL. All authors read and approved the final manuscript.

Funding

This study was supported by the Chinese National Major Project for the Development of Genetically Modified Organisms (2016ZX08011005).

Conflict of interest statement

The authors declare that there were no competing financial interests between themselves.

Ethics approval

The study was approved by the Animal Ethic Committee of National Institute of Nutrition and Health, Chinese Center for Disease Control and Prevention.

Contributor Information

Chen Chen, Key laboratory of Public Nutrition and Health, National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Nanwei Road of Xicheng District (100050), Beijing, P.R. China.

Lili Shi, Key laboratory of Public Nutrition and Health, National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Nanwei Road of Xicheng District (100050), Beijing, P.R. China.

Hongmei Mao, Key laboratory of Public Nutrition and Health, National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Nanwei Road of Xicheng District (100050), Beijing, P.R. China.

Chao Han, Key laboratory of Public Nutrition and Health, National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Nanwei Road of Xicheng District (100050), Beijing, P.R. China.

Jinpeng Zhao, Key laboratory of Public Nutrition and Health, National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Nanwei Road of Xicheng District (100050), Beijing, P.R. China.

Qin Zhuo, Key laboratory of Public Nutrition and Health, National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Nanwei Road of Xicheng District (100050), Beijing, P.R. China.

Yan Li, Key laboratory of Public Nutrition and Health, National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, Nanwei Road of Xicheng District (100050), Beijing, P.R. China.

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