Study ID
RefID (DistillerSR)	307
Reference (authors, year, title, other info)	T.N.O. – Division for Nutrition and Food Research, 1986. Sub‐chronic (90‐day) oral toxicity study with neohesperidine dihydrochalcone (NHDC) in rats.
Source (published/unpublished)	Unpublished
Type of study and guideline
Good laboratory practice (yes/no/No, but before establishment of GLP)	Yes
Guideline studies (if yes, specify)	No
Type of study	Subchronic toxicity
Animal model
Species and strain	Rats, Wistar (breeding not reported)
Disease models (e.g. diabetes, allergy, obesity)	Not applicable
Housing conditions
Housing condition	Housed under conventional conditions, 5 per sex per cage, in suspended stainless steel cages fitted with wire mesh floors and fronts. The cages were randomised over the cage‐racks. The temperature was kept at 22 ± 2°C, the relative humidity was at least 40%. Artificial light was provided for 12 h/day continuously, from 7.30 a.m. till 7.30 p.m. The number or air changes was about 10/h. Fed a powdered stock diet provided ad libitum from weighed feeders. Tap water was supplied ad libitum in glass bottles, cleaned weekly and filled with fresh water when necessary.
Diet name and source (if reported)	Powdered stock diet (van Eck, Cothen)
Treatment
Test material	Neo‐DHC (Neohesperidine dihydrochalcone)
Provider	Sponsor
Compound purity	More than 99.5%
Vehicle used	None (direct admixture with powder rodent diet)
Dose regimen (dose level or concentration per group, and frequency) and achieved doses if available	0%, 0.2%, 1.0%, 5.0% neohesperidine dihydrochalcone in the diet Equal to: 0, 150, 757, 4,011 mg/kg bw per day in males 0, 166, 848, 4,334 mg/kg bw per day in females
Route of administration (diet, drinking water, gavage)	Diet
Period of exposure (pre‐mating, mating, gestation, lactation, adult)	Adult
Duration of the exposure	13 weeks (90 days)
Study design
Sex and age at the start of the treatment	Males and females (3,5 weeks)
Number animals/sex per group	20/males/group; 20/females/group For ophthalmoscopic examination: 20/males and 20/females of the control and highest dose group For haematology: 10/males/group and 10/females/group For clinical chemistry (aorta samples) and urinalysis: 10/males/group and 10/females/group.
Measured endpoints	Determination of neohesperidine dihydrochalcone in the diets, clinical signs, ophthalmoscopic examination, body weights, food consumption, water consumption, haematology, clinical chemistry, urinalysis, macroscopic and microscopic examination of organs and examination of intercurrent deaths.
Time of measurement/observation period	The general condition and behaviour of all rats was checked daily. The eyes of all rats of the control and top‐dose group were examined prior to the start of the study and in week 13. Body weights were recorded initially then weekly. In addition, rats were weighed on the day of autopsy. Food intake was measured per cage weekly for 12 weeks. Water intake was measured per cage daily in week 7 and in week 12. For haematology, samples of blood were collected from males on day 84 and from females on day 85. For chemical chemistry, on day 87 blood was collected from the tail after deprivation of water for 24 h and of food during the last 16 h to determine glucose levels. Blood samples were collected from the aorta of males on day 91 and of females on day 92. For urinalysis, on day 87 rats were deprived of water 24 h and food 16 h and urine was collected during the last 16 h of deprivation period. On day 86, 3‐h urine samples were collected from unfasted rats and the pH was determined. Animals were killed on 4 successive days (91 and 93 males, 92 and 94 females) and examined macro/microscopically for pathological changes.
Methods to measure the endpoints	In line with standard sub‐chronic toxicity study.
Statistical analysis
Statistical methods	Analysis of variance followed by Dunnett's test, analysis of variance followed by the LSD test, Mann–Whitney U‐test and Fisher's Test
Results
Findings reported by the study author/s	The intake of neohesperidine dihydrochalcone in the various weeks calculated, showed the normal decrease with increasing study duration for each of the three dose groups. Focal alopecia and transient diarrhea were observed in the top‐dose group. No other treatment‐related changes in general condition or behaviour. One female rat of the mid‐dose group died in day 30, but not considered treatment related. Ophthalmoscopic examination did not reveal any treatment‐related changes. Body weights were decreased in the top‐dose group in males throughout the test period and in females in the first 2 weeks. Food intake was decreased in males of the top dose group in the first 2 weeks, whereas water intake was slightly increased in week 7 but not statistically significant. There were no treatment‐related changes in haematology. Alkaline phosphatase activity and bilirubin concentration were increased in females of the top‐dose group whereas in males of this group, total protein concentration was decreased. The changes in clinical chemistry variates observed in the top dose were not accompanied by other signs. It is not clear whether these changes are of toxicological significance. Urinary pH was statistically decreased in the top‐dose of both sexes. No treatment‐related changes in volume, density, composition or sediment of urine. The weights of the filled and empty caecum were increased in the top‐dose group in both sexes. This effect is not typical for NDHC and may be due to microbiological degradation of unabsorbed NDHC., but not considered a toxic effect. The tendency towards higher water intake, the occasional diarrhoea and the decrease in urinary pH may also be attributed to an increase amount of microbial degradation products. The relative weights of the brain and testicles were increased in males of the top‐dose group, but not considered a toxic effect. At autopsy, no treatment‐related gross abnormalities were observed. At microscopic examination, no histopathological changes were observed that could be considered to be treatment related.
No observed adverse effect level, lowest observed adverse effect level, benchmark dose/benchmark dose lower bound	NOAEL: 1% neohesperidine dihydrochalcone in the diet. This level was equivalent to a nominal intake of approximately 750 and 850 mg/kg body weight per day for males and females respectively.
Further information
Deviations from the protocol: analysis for the amount of test substance in the diets were not carried out by the sponsor, but at the TNO‐CIVO Institute. On day 15 and 86, the temperature and/or humidity in the animal room slightly exceeded the ranges mentioned, but this is not considered to have influenced the outcome of the study. Intake of the test substance and red blood cell indices were calculated, although this was not specifically mentioned in the protocol. Plasma cholesterol was determined in all groups. This determination was omitted from the protocol by mistake. Cholesterol was not determined in one female of the mid‐group because not enough sample could be obtained. PH was determined individually, in freshly voided, 3‐h urine samples, instead of in pooled, 16‐h, concentrated urine. This was done because in this way more representative pH values are obtained. A few organs of different rats were not weighed at autopsy for various reasons, viz: testicles – atrophy; kidneys – hydronephrosis; adrenal – enlargement; adrenal, ovary and thyroid – lost at autopsy. The organs of the rat that died during the study were not weighed. In addition to the protocol, the weight of the full and empty caecum was determined in all rats at study termination. A few organs or tissues of different rats could not be examined microscopically since, by oversight, they were not collected for fixation or they were lost during fixation and/or processing. The number of the various organs and tissues examined was given. The organs and tissues of one female rat of the mi‐dose group were examined microscopically, because this animal died during the study. To note that this unpublished study report was subsequently published as Lina BAR, Dreef‐van der Meulen HC, Leegwater DC, 1990. Subchronic (13‐week) oral toxicity of neohesperidin dihydrochalcone in rats. Food Chemistry and Toxicology, 26(7), 507–513. https://doi.org/10.1016/0278-6915(90)90121-3.