## 5 Toxicological Data
### Subchronic and Chronic Toxicological Data
In the second series of tests, adult rats were given either 2.5% or 15% date seed fiber in their food. The feed was provided ad libitum and the feed intake was not restricted. The study showed that the test animals consumed more food when the diet had a higher crude fiber content. At the conclusion of the experiment, the test animals were euthanized and their livers were removed for subsequent histological examination. The inclusion of date seed fiber in animal feed impacted the lipid metabolism of growing and adult male rats, leading to fatty infiltration in the liver at all stages of development. The livers of the test animals showed increased lipid deposits. The results of the feeding study confirmed an increase in serum lipids and cholesterol even at low dosages of date seed flour. Additionally, the overall weight gain was better than that of the control group, which received cellulose [29]. In another study, alternative treatments for diabetes were tested in rats [51]. Over a ten-week period, test animals (40 adult male Sprague-Dawley rats) were divided into diabetic and non-diabetic groups and given an oral dose of 200 mg/kg b.w. date seed extract daily, or an i.p. dose of aloxan monohydrate to treat diabetes. The test group consuming only the DS extract showed no deviations compared to the control group in NO, MDA, AST, ALT, amyloid A, CRP, and glucose, and only a slight increase in TGs, but no change in cholesterol levels, LDL, and HDL. Therefore, neither positive nor negative effects can be expected from daily oral consumption of date seed beverages. Interestingly, the diabetic test groups treated with date seed coffee drinks and aloxan monohydrate showed a decrease in NO, MDA, amyloid A, and CRP, as well as in AST and ALT, total cholesterol, TGs, LDL, but an increase in HDL compared to the diabetic group treated with aloxan monohydrate alone. But, overall, no harmful effects related to the consumption of date seed coffee have been shown [51]. Similar results were obtained in a 28-day study in adult male Wistar albino rats [89]. The rats were fed a high-fat diet ad libitum. The test groups were divided and received either medication with atorvastatin (for the treatment of dyslipidemia and the prevention of cardiovascular disease) or date seed extracts from different cultivars in two doses of 300 or 600 mg/kg b.w. rat daily. Over time, the high-fat diet resulted in increased body and liver weight, increased food intake, and higher glucose and insulin levels. Compared to the high-fat diet control group, the groups fed date seed extracts (from the Barhi and Ruthana cultivars) had lower liver and total body weights. Histological findings showed a decrease in the lipid deposition and a good hepatocyte and parenchymal structure. After repeated consumption of date seed extracts, leptin, AST and ALT, TGs, and total cholesterol were reduced, with an increase in HDL and a reduction in LDL in the blood concentration being confirmed, but no change in the glucose or insulin levels. Overall, no adverse effects were observed after the daily consumption of either 300 or 600 mg/kg b.w. date seed extract. Similar metabolic effects to the test substance atorvastatin were observed in the animals treated with the date seed extract [89]. [PERSON] and [PERSON] [51] conducted parallel observations in human volunteers in addition to the diabetic animal study. The human study was conducted between 2016 and 2021 and included 55 female volunteers who were non-diabetic and with a BMI around 30, which can be considered overweight. The group was divided into two groups, and the second group received date seed coffee at a dose of 7 \(\pm\) 0.5 g/day for at least 3 months. Group 1 was the control group and did not receive any test substance. Group 2 showed a significant decrease in various serum markers, including NO (34.7%), MDA (43.9%), amyloid A (79.6%), CRP (60.9%), HbA1c (33.8%), and troponin T (16.4%) compared to group 1.