Toxicity Test and Embryo Teratogenicity Test of Nervonic Acid in Rats

Objective: To observe the 90-day continuous feeding toxicity test and embryo teratogenicity test of nervonic acid in SD rats, so as to provide reference for clinical medication safety. According to the standard GB15193.13-2003, the toxic and side effects of nervonic acid on the body were observed after 90-day feeding at doses of 0.1, 0.3 and 0.6 g/kg. According to the standard GB15193.14-2003, nervonic acid at doses of 0.1, 0.3 and 0.6 g/kg was administered to pregnant rats from day 7 to 16 of gestation to observe its teratogenic effect on rats.

Results: After 90-day feeding of nervonic acid, hematological results showed that at 45 and 90 days, WBC, RBC and HGB decreased in the 0.6 g/kg group of female animals, and RBC and HGB decreased in the 0.3 g/kg group; WBC decreased in all dose groups of male animals at 45 days, and HGB decreased in the 0.6 g/kg group; at 90 days, WBC decreased in the 0.6 g/kg group of male rats. Blood biochemical results showed that in female rats, ALT and AST decreased, TC and GLU increased in the 0.6 g/kg group; AST decreased, TC and GLU increased in the 0.3 g/kg group; AST decreased in the 0.1 g/kg group. In male rats, ALT and AST decreased, TP, ALB and GLU increased in the 0.6 g/kg group; ALT and AST decreased in the 0.3 g/kg group. Teratogenicity test results showed that nervonic acid at 0.6 g/kg reduced the embryo survival rate of pregnant rats to a certain extent.

Conclusion: When nervonic acid is used as a health food, its service life should be limited, and the daily dosage should not exceed 0.6 g/kg.

Nervonic acid is a natural substance with high content in nerve tissue and brain tissue, which can help the brain operate efficiently, promote brain development and repair damaged nerve endings. This study mainly conducted a short-term repeated oral administration toxicity test of nervonic acid to observe the toxic reactions, toxic target organs or tissues and reversibility of toxic damage caused by continuous administration of the drug to the body, so as to provide reference for the optimal design of animal toxicity test schemes with longer experimental periods and clinical research.

Nervonic acid (CAS NO. 506-37-6; Xi'an Sost Biotech Co., Ltd., Batch No.: qckm20240606) is a white crystalline powder. Before use, it is prepared into a suspension of corresponding concentration with 0.5% sodium carboxymethylcellulose (CMC-Na; Anhui Shanhe Pharmaceutical Excipients Co., Ltd., Batch No.: 241006) as the solvent. Hematology test kit (Abbott Laboratories, USA); Urine routine test strip (Siemens Medical Diagnostics (Shanghai) Co., Ltd.); Serum electrolyte test kit (Shenzhen Yuehua Technology Development Co., Ltd.); Blood biochemistry test kit (Shenzhen Mindray Biomedical Electronics Co., Ltd.); Coagulation index test kit (Instrumentation Laboratory, Germany).

Cell-Dyn3700 Hematology Analyzer (Abbott Laboratories, USA); Urine Analyzer (Bayer AG, Germany); BS-200 Automatic Biochemical Analyzer (Shenzhen Mindray Biomedical Electronics Co., Ltd.); MI-921D Automatic Electrolyte Analyzer (Shenzhen Yuehua Technology Development Co., Ltd.); MC-4000 Coagulometer (Instrumentation Laboratory, Germany).

Ninety healthy SPF-grade SD rats (half male and half female), aged 6-8 weeks, were provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Production License No.: SCXK (Jing) 2024-0001; Certificate No.: 11400700076799). The animal experiment was carried out in an SPF-grade animal room with a temperature of 20-26℃, a daily temperature difference ≤4℃, a humidity of 40%-70%, and an air exchange rate of more than 15 times per hour. The feeding environment was monitored and managed by the SIMATIC S7-300 automatic control system, which meets the requirements of the national standard GB14925-2010 "Laboratory Animal Environment and Facilities".

The test was carried out in accordance with GB15193.13-2003 "30-Day and 90-Day Feeding Tests". After the animals were acclimated to the feeding environment for 5 days, 80 animals with a body weight difference not exceeding ±20% of the average weight were selected and randomly divided into 4 groups: control group and three test groups (nervonic acid at low, medium and high doses of 0.1, 0.3 and 0.6 g/kg respectively). The test substance was mixed into the basal feed for feeding, with contents of 2.0%, 6.0% and 12.0% respectively (the dose of the test substance was converted into the amount of feed according to the intake per 100g body weight, and the daily feed intake of rats in the 90-day feeding test was converted according to 8% of the body weight). The control group was fed with basal feed. Each group had 20 animals, half male and half female. Continuous observation was carried out for 90 days.

The animals were divided into 5 groups with 12 animals in each group: solvent control group, positive control group (vitamin A1 3000 μg/kg•bw, gavage volume 5 ml/kg) and test substance groups (nervonic acid at low, medium and high doses of 0.1, 0.3 and 0.6 g/kg•bw respectively; 0.6 g/kg•bw was the maximum concentration used, and the gavage volume was 10 ml/kg).

The test was carried out in accordance with the requirements of GB15193.14-2003 "Teratogenicity Test". After the purchased male and female rats were acclimated to the environment for 3 days, healthy rats were selected and caged at a male-to-female ratio of 1:1. Each morning, the female rats were taken out for vaginal smear. If a large number of sperm were observed under a microscope, the female rat was considered mated, separated from the male rat and regarded as a pregnant rat, and the day was recorded as day 0 of pregnancy. If no mating occurred within 5 days of caging, the male rat should be replaced. The detected pregnant rats were randomly assigned to each group, weighed and numbered, with no less than 12 animals in each group. Each group of pregnant rats was given the test substance once a day according to their body weight from day 7 to day 16 of pregnancy. The rats were sacrificed on day 20 of pregnancy, and the pregnancy status of the parent and the development of the fetus were examined by laparotomy. The uterus was quickly pulled out, the weight of the uterus with fetuses (litter weight) was weighed, the fetuses were taken out, and the number of resorbed fetuses, early dead fetuses, late dead fetuses and live fetuses was recorded and examined. The body weight and length of each litter of fetuses were recorded one by one, and the appearance of the fetuses was checked for abnormalities. Half of the live fetuses in each litter were marked for bone examination; the other half of the live fetuses were placed in Bouin's fixative for visceral examination.

SPSS17.0 software was used for statistical analysis. The counting data were tested by t-test, and the measurement data were tested by chi-square test. A P value <0.05 was considered statistically significant.

During the test, the hair of rats in the high, medium and low dose groups was white and shiny, which was no different from that in the control group, and no abnormal behaviors or manifestations were found. After 90 days of continuous feeding, there was no significant difference in the food intake of rats in the high, medium and low dose groups compared with the control group (see Table 1); there was no significant difference in the total average weight gain of rats in the high, medium and low dose groups compared with the control group (see Table 2, P < 0.05).

At 45 and 90 days of feeding, blood was collected to determine hematological indicators: white blood cell count (WBC) and its classification, red blood cell count (RBC), hemoglobin (HB), platelet (PLT), and reticulocyte. The results showed that after 45 days of feeding with nervonic acid powder, WBC, RBC and HB in the high-dose group of female animals were significantly decreased, and RBC and HB in the medium-dose group were significantly decreased. All three dose groups of nervonic acid decreased WBC in male rats, and HB in the high-dose group was decreased. There were no significant differences in other indicators compared with the control group (see Tables 3 and 4). After 90 days of feeding with nervonic acid, WBC, RBC and HB in the high-dose group of female rats were decreased, and WBC and HB in the medium-dose group were decreased. WBC in the high-dose group of male rats was decreased. There were no significant differences in other indicators compared with the control group (see Tables 5 and 6).

After 90 days of feeding, blood was collected to determine various blood biochemical indicators: serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRE), glucose (GLU), triglyceride (TG), cholesterol (TC) activity or content. The results showed that in female animals, ALT and AST decreased, TC and GLU increased in the high-dose group; AST decreased, TC and GLU increased in the medium-dose group; AST decreased in the low-dose group (see Table 7). In male rats, ALT and AST decreased, TP, ALB and GLU increased in the high-dose group; ALT and AST decreased in the medium-dose group (see Table 8). There were no significant differences in other indicators compared with the control group.

After 90 days of feeding with nervonic acid, the absolute weight and relative weight of liver, kidney, spleen, heart, brain, adrenal gland, thymus and testis were measured. The results showed that the relative weights of liver and kidney in rats of both genders in each dose group were significantly increased (P < 0.05), and there were no obvious regular changes in the weights of other organs (see Table 9).

After continuous administration of nervonic acid to rats for 90 days, the submitted heart, liver, spleen, kidney, adrenal gland, thymus, stomach, brain, duodenum, colon and gonads (testis and ovary) showed no obvious and regular pathological changes related to the test substance.

The three dose groups of the test substance (nervonic acid) inhibited the embryo survival rate of pregnant rats (see Table 10).

There were no significant differences in fetal weight, body length, bone malformation rate and visceral malformation rate between each dose group of nervonic acid and the solvent control group.

Test conclusion: When nervonic acid is used as the test substance at an added dose of 0.6 g/kg, no obvious harmful effects are observed after feeding rats for 90 days.

As a new food raw material, nervonic acid has been applied in a series of domestic and foreign foods and health products. In accordance with the declaration requirements of national new food raw materials, Xi'an Sost Biotech Co., Ltd. has studied the safety of nervonic acid in accordance with national food safety standards.

In this study, nervonic acid raw material was fed to rats for 90 days. It was found that in the 0.6 g/kg dose group, there was no significant difference in animal body weight and total food intake compared with the control group; RBC count, HB content and WBC count in hematology were decreased to a certain extent at 45 and 90 days; AST and ALT were decreased, and TC and blood glucose were increased in the 0.36 g/kg and 0.6 g/kg dose groups at 90 days. The changes of these indicators showed a dose-dependent relationship, indicating that nervonic acid has an impact on WBC, RBC, HB, TC and blood glucose. Teratogenicity test results showed that the 0.6 g/kg dose group of nervonic acid could significantly increase the number of late dead fetuses, resorbed fetuses and early dead fetuses in pregnant rats, and reduce the embryo survival rate of animals; it is speculated that it may have a certain impact on the early stage of pregnancy.

In summary, through research and comprehensive relevant literature reports, long-term and high-dose administration of nervonic acid may affect leukocytes, red blood cells, hemoglobin, blood lipids and blood glucose. Since nervonic acid has been widely used in health products and daily life, it is worth noting that the recommended daily dosage of nervonic acid in health products should not exceed 0.6 g, and its use time should be limited.