Study ID
RefID (DistillerSR)	360
Reference (authors, year, title, other info)	Shi ZJ, Lei HH, Chen G, Yuan PH, Cao Z, Ser HL, Zhu XH, Wu F, Liu CX, Dong MY, Song YC, Guo YY, Chen C, Hu KX, Zhu YF, Zeng XA, Zhou JL, Lu YJ, Patterson AD and Zhang LM, 2021 Impaired intestinal Akkermansia muciniphila and aryl hydrocarbon Receptor ligands contribute to nonalcoholic fatty liver disease in mice. mSystems 6(1): e00985‐20. https://doi.org/10.1128/mSystems.00985‐20
Source (published/unpublished)	Published
Type of study and guideline
Good laboratory practice (yes/no/no, but before establishment of GLP)	Yes
Guideline studies (if yes, specify)	Animal experimental procedures performed according to the Chinese National Guidelines
Type of study	Subchronic toxicity
Animal model
Species and strain	Mice, C57BL/6 strain
Disease models (e.g. diabetes, allergy, obesity)	Not applicable
Housing conditions
Housing condition	Housed in a specific pathogen‐free room with a 12 h light/dark cycle and a constant temperature (22°C ± 1°C) and humidity (40% to 60%).
Diet name and source (if reported)	Standard normal‐chow diet
Test material	Neohesperidine dihydrochalcone
Provider	Sigma‐Aldrich Chemical Co.
Compound purity	96%
Vehicle used	Sterilised water or fresh sterilised solution
Dose regimen (dose level or concentration per group, and frequency) and achieved doses if available	Solution of 0.1 mg/mL were used to meet the FDA‐approved acceptable daily intake (ADI) in humans (5 mg/kg per day) based on NAS exposure doses previously calculated. Equivalent to 10 mg/kg bw per day.
Route of administration (diet, drinking water, gavage)	Drinking water or solution
Period of exposure (pre‐mating, mating, gestation, lactation, adult)	Adult
Duration of the exposure	11 weeks
Study design
Sex and age at the start of the treatment	Females (6 weeks)
Number animals/sex/group	10 females/group. 4 groups.
Measured endpoints	Food intake, water intake, body weight, liver histopathology and clinical biochemistry analyses: alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate transaminase (AST), total bile acid (TBA), total bilirubin (TBIL), direct bilirubin (d‐BIL), glucose (GLC), triglycerides (TG), total cholesterol (TC), high‐density lipoprotein (HDL), low‐density lipoprotein (LDL), urea nitrogen (BUN), and creatinine (CREA) in serum.
Time of measurement/observation period	Food intake, water intake and the body weights of mice were monitored and recorded every week. Urine and faecal samples were collected every week over the experimental period. At the end of the experiments, the mice were sacrificed under isoflurane anaesthesia after 8 h of fasting. All the samples, including serum, liver, colon, ileum and caecal content samples, were immediately collected and stored at 280°C for the following experiments.
Methods to measure the endpoints	In line with standard sub‐chronic toxicity study
Statistical analysis
Statistical methods	All experimental values are presented as means 6 standard deviations (SD). OriginLab software (Origin 2017) and GraphPad Prism software (Graph‐Pad 7.0) were used for data analysis and graphical illustrations. All data between different groups were statistically analysed by the Mann–Whitney U test or the Kruskal–Wallis analysis of variance (ANOVA) test. p Values of 0.05 were considered significant.
Results
Findings reported by the study author/s	No significant histopathological changes were observed in the livers of mice after NHDC consumption. Compared with controls, mice with NHDC consumption exhibited no marked changes in their volumes of drinking water, food intake, or body weights. It is noteworthy that NHDC consumption had minimal effects, including no change in LPS level, inflammatory cytokines, and lipid metabolism in the livers or sera of mice. NHDC had no significant impact on the bacterial community. NHDC consumption significantly increased levels of propionic acid and isobutyric acid (SCFAs).
No observed adverse effect level, lowest observed adverse effect level, benchmark dose/benchmark dose lower bound	Not reported
Further information
Other measured endpoints: urine and faecal samples collection; quantification of tryptophan metabolites in faecal, serum, liver, and colon tissues; quantification of short‐chain fatty acids (SCFAs) in the caecal contents and of long‐chain fatty acids (LCFAs) in the livers and sera; gut microbiota analysis.