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Toxicological information

Repeated dose toxicity: oral

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Administrative data

short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Well-performed and reported study following OECD guideline 407 with GLP standards. This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Betaine >95% purity (product name BF 20, batch number 50000255)

Test animals

Details on test animals or test system and environmental conditions:
- Source: Harlan UK Ltd., Bicester, Oxon, UK
- Age at study initiation: not reported
- Weight at study initiation: not reported
- Fasting period before study: not reported
- Housing: 5 per cage in polypropylene cages with stainless-steel grid tops and floor, suspended in racks over paper for removal of excreta
- Diet (e.g. ad libitum): Rat and mouse No. 1 maintenance diet, ad libitum in stainless steel pots designed to limit spillage
- Water (e.g. ad libitum): domestic tap water ad libitum
- Acclimation period: 7 days

- Temperature (°C): 19.0-24.0°C
- Humidity (%): 34-64%
- Air changes (per hr): Minimum 15/hr with no recirculation using high efficiency filters
- Photoperiod (hrs dark / hrs light): 12 hr light/12 hr dark

Administration / exposure

Route of administration:
oral: feed
other: R/M1 (Rat and Mouse maintenance diet)
Details on oral exposure:
Within 24 hours of scheduled diet preparation, sufficient test substance was prepared by grinding the supplied material to a powder with a blender and sieving through a 0.425 mm mesh into a clean, stainless steel container for storage until required for diet mixing. Any particles too large to pass through the sieve were re-blended.
For each diet prepared, the required quantity of powered test substance was made up to 6 kg using control diet and mixed in a Hobart mixer for 20 minutes. This was subsequently blended with further control diet, to make a total of 20 kg, using a Turbula® mixer, for a minimum of 30 minutes.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Diets were extracted with 100 mL ultrapure water and shaken, first manually to ensure even wetting, and then on a horizontal flat-bed shaker for 30 minutes. After standing until the bulk of the solids had settled, the supernatant was passed through fluted filter paper and a portion of the filtrate further filtered through a 0.2 µm 25 mm diameter syringe filter to obtain a clear extract for analysis. In some cases centrifugation at 14000 rpm for 20 minutes was used for further clarification prior to the syringe filtration step. Extracts were collected into glass autosampler vials for injection and storage. Test substance content was determined by cation exchange high performance liquid chromatography of 50 µL aliquots on a 250 x 4.6 mm i.d. RCU-USP Sugar Alcohol, protected by a 4 x 3 mm i.d. Carbo-CA-2+ guard cartridge, maintained at 60°C and eluted with 5 mM calcium sulphate in water at a flow rate of 0.5 mL/min. The refractive index of the eluate was monitored and the test substance peak quantified by peak area against triplicate standard solutions of the test item at seven concentration levels.

After mixing, two samples of each diet were analysed for test substance concentration together with two samples of control diet. Stability and homogeneity of mixing was determined and the results indicated that diets prepared contained the test substance as detailed above were homogenous and stable for 35 days. The results also indicated that the diets were stable in open food pots for a 7-day period and the contents of the containers used to feed the rats were therefore replaced on a weekly basis.
Duration of treatment / exposure:
Continuous in feed
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:
0, 1, 2, 5%
nominal in diet
No. of animals per sex per dose:
20 females/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels selected were identical to those used in a previous study to investigate the sub-chronic toxicity of the test substance (TNO BIBRA Project No. 4013/1).


Observations and examinations performed and frequency:
- Time schedule: Daily

- Time schedule: Prior to the start of the study and once a week thereafter, at time of weighing.

- Time schedule for examinations: 3 days before treatment, on the first day of treament, and then twice weekly until sacrificed

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food intake for each cage of rats was measured twice weekly until sacrifice, between the intervals of each body weight measurement
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


- Time schedule for collection of blood: At scheduled necropsy (days 28 and 56)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: total erythrocyte count, total leucocyte count, haemoglobin concentration, meal cell volume, platelet count, differential leucocyte count, haematocrit, mean cell haemoglobin, mean cell haemoglobin concentration, reticulocytes, prothrombin time, activated partial thromboplastin time, fibronigen

- Time schedule for collection of blood: At scheduled necropsy
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: glucose concentration, urea concentration, total protein concentration, albumin concentration, alkaline phosphatase activity (ALKP), alanine aminotransferase activity (ALAT), lactate dehydrogenase activity (LDH), aspartate aminotransferase activity (ASAT), albumin/globulin ratio (ALB/GLOB ratio), creatinine, calcium, phosphorus (as phosphate), chloride, total bilirubin, sodium, potassium, gamma glutamyltransferase (Gamma-GT), cholesterol, triglyceride



Sacrifice and pathology:
GROSS PATHOLOGY: Yes; Animals were fasted overnight prior to scheduled necropsy (days 27 and 56). Kidney, liver, and ovaries from all animals were weighed.
HISTOPATHOLOGY: Yes; Livers were processed and embedded in wax. Sections were prepared and stained with haematoxylin and eosin and subjected to a microscopic morphological examination. Additionally, sections were prepared from frozen liver samples of each animal and stained with Oil Red O for the microscopic determination of fat deposition.
The continuous variable data from the control and test substance fed groups were tested for normality using the Kolmogorov-Smirnov (K.S.) test and homogeneity of variance using Bartlett’s test. Statistical significance was determined to be a p<0.05 in a K.S. test and at p<0.01 in a Bartlett’s test. If both tests were non-significant, the control and test groups were compared using analysis of variance followed by the least significant difference (L.S.D.) test.
If either test produced a significant result, a suitable transformation was attempted. If no suitable transformation could be made, one of the following tests was selected as the most appropriate based upon the nature and distribution of the data. In the event that the data resulted in a non-significant Bartlett’s test but a significant K.S. test, the Wilcoxon Mann-Whitney test was used. In the event that the data resulted in a non-significant K.S. test, but a significant Bartlett’s test, an appropriate t-test was used, based on whether a pooled variance was suitable or not.
In the event that the data resulted in both significant Bartlett’s and K.S. tests, the results of the Mann-Whitney and t-test were compared and the most appropriate test reported.
Incidence data from the microscopic histopathological examination was tested for differences between test substance treated and control animals using Fisher’s exact or Mann-Whitney test.
In all test comparisons, a probability level of p<0.05 in a two-sided test was taken to indicate statistical significance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
hepatocellular vacuolation
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: No test-substance related mortality was observed. Two animals were killed in extremis due to their poor general conditions (one control and one rat at 1%). One control rat was seen to have pale eyes on day 9, progressing to red staining in the corner of its left eye and a swollen abdomen (animal necropsied on day 28). One rat at 1% was noted to have pale eyes and laboured breathing on day 7 and was sacrificed on day 8.

BODY WEIGHT AND WEIGHT GAIN: The mean body weight of animals at 1% was slightly, but statistically significantly lowers than controls on day 7 only, which was largely due to the sick animal sacrificed on day 8. There were no marked differences from control weight in the body weights of female rats fed test substance at levels of 2%. The body weights of the group given 5% were consistently and statistically significant lower than controls (approximately 5% lower than control weights) throughout the 28-day treatment period. These marked differences were not observed during the reversal period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): On one occasion, between days 3 and 7, the food intake of females at 5% was statistically significantly lower than controls. Average intake of over the course of the 28-day treatment period was 0, 1146.6, 2297.6, and 5770.6 mg/kg/day for the control, 1, 2, and 5% groups, respectively.

FOOD EFFICIENCY: A formal statistical analysis was not performed as data on food intake were obtained on a cage basis while the body weight gain was based upon individual animals. Although there were no occasions where the ratios either decreased or increased in relation to increasing dose, there were a few occasions where the trend decreased with the dose given, e.g., days 0-3, 3-7, 17-21, or increased between days 34-37 during the reversal period. An index of this was taken by the ratio of control and high dose, where the conversion efficiencies of the control rats were higher than the rats fed 5% on 5/8 occasions during the treatment period and on 3/8 occasions during the reversal period.

HAEMATOLOGY: On day 28, all rats given the test substance had statistically significantly lower mean cell volume (95-97%) and mean cell haemoglobin values (94-97%) compared to control. Rats at 5% also had statistically significantly lower haemoglobin (94%) and haematocrit (95%), and higher platelet counts (117%) when compared to controls. Rats fed 1% also had slightly, but statistically significantly lower reticulocyte numbers. The prothrombin time of rats of all treated groups was shorter than controls (96-98%), although this difference was only statistically significant at ≥2%. Additionally, all groups had slightly higher fibrinogen values compared to controls, but the difference was statistically significant only at 5%. Statistically significant increases in neutrophils were observed in rats at 2%. On day 58 (after the reversal period), rats from all groups had statistically significantly lower mean cell volume values (97-98%), together with a lower, mean cell haemoglobin although this difference was statistically significant only at 1%. Female rats also had slightly, but statistically significantly lower reticulocyte numbers at 1%.

CLINICAL CHEMISTRY: On day 28, all test groups had statistically significantly higher γ-glutamyltransferase levels (185-242%) than controls. At ≥2% slightly but statistically significantly increased levels of alkaline phosphatase were observed (114-122%). Although all groups had lower aspartate aminotransferase (83-94%) and alanine aminotransferase (82-92%) levels, these differences were only statistically significant for the rats treated at 5%. Rats from this group also had slightly lower, but statistically significant sodium (99%), chloride (99%), and cholesterol (80%) levels when compared to controls, together with slightly higher bilirubin (116%) and potassium (105%) values. Rats at 1% also had statistically significantly lower chloride (99%) values and higher total protein (104%) values compared to controls. Calcium levels were slightly lower in all treated rats, but the difference was statistically significant at ≥2% (94-95%). Female rats from all treated groups had slightly higher than control urea levels, but this difference was statistically significant at 1 and 5% only (113 and 121%, respectively). On day 56, rats at 2% had slightly, but statistically significantly lower alkaline phosphatase and aspartate aminotransferase values when compared to controls. Rats at 5% had statistically significantly lower bilirubin (83%) and sodium (99%) levels and the chloride levels of all treated rat groups were very slightly (<1%) lower, but statistically significantly lower than controls. Additionally, there were higher levels of urea in all treated rats compared to controls, although the differences were statistically significant for the 1 and 5% treated groups only.

ORGAN WEIGHTS: On day 28 the mean absolute liver weight of rats at 5% was statistically significantly higher than controls. The absolute mean ovary weight of rats fed 2% was statistically significantly lower than controls. When expressed relative to body weight, the liver weights of all test substance groups were higher than controls, the difference being statistically significant at 2 and 5%. The increases observed in liver weight were directly related to the dose given. On day 56, there were no marked differences in the absolute weights of the kidneys, liver, or ovaries when compared to controls. When expressed relative to body weight, the kidney weights of rats fed 2 or 5% were statistically significantly higher than controls.

GROSS PATHOLOGY: Gross examination of rats after the 28-day feeding period revealed a high incidence (5 and 9/10, respectively) of livers exhibiting a pale appearance in the 2 and 5% treated groups. After the 28-day control diet reversal period, this gross hepatic change was present in one control and one animal at 5% only. The rat sacrificed on day 8 from the 1% treated group was found to have a fluid filled thorax (hydrothorax) and one control animal in the reversal phase sacrificed on day 28 was found to have hydrocephalus. These findings were not related to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC: Rats examined after the 28-day feeding period revealed a statistically significant increase in hepatocellular vacuolation in all treatment groups. Hepatocellular vacuolation was observed in 2, 8, 8, and 10/10 rats in 0, 1, 2, and 5% treatment groups, respectively. Moreover, the severity of the vacuolation showed a clear dose-response relationship. The Oil Red O staining demonstrated that the vacuolation was due to increased fat accumulation. Although the control animals showed a minimal to slight positive staining with the Oil Red O, the treated groups generally exhibited a clear dose-related increase in both the severity of staining as well as the area of liver which was positive. The hepatocellular vacuolation was still present in a few animals at 2 and 5% after the 28-day control diet reversal period, but the incidence and severity of this condition was similar to that of controls and this was confirmed by the Oil Red O stain. The other histopathological changes observed were common for rats of this strain and age, and occurred in a single of a few animals only or their incidences were comparable among the groups.

Effect levels

Dose descriptor:
Effect level:
> 5 771 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No other histopathological effects other than slight to moderate hepatocellular vacuolation in study groups (1, 2 and 5% groups) which were totally reversible.

Applicant's summary and conclusion

NOAEL: 5771 mg/kg/bw/day (nominal) (5% test substance in diet), based on high tolerance of test substance and reversibility of the slight hepatocellular vacuolation effects. Therefore it can be concluded that betaine is tolerated at very high levels and its effects are reversible.
Executive summary:

In a previous study to investigate the sub-chronic toxicity of the test substance to rats over a 90-day period, treatment related effects were described which included hepatic microvacuolation and changes in the clinical chemistry and haematology indicative of a treatment induced decrease in mean corpuscular volume and suspected effects on intermediary metabolism within the liver. The findings of the original study were generally seen in both sexes, but appeared to be more severe in females. The purpose of this study was to reproduce the original findings in female rats over a 28-day period ant to investigate the recovery from such effects over a 28-day period after cessation of treatment.

In this study, groups of 20 female rats of the Sprague Dawley strain were fed the test substance in the diet a nominal levels of 0 (basal diet control), 1, 2, or 5% for 28 consecutive days. Ten animals for each group were taken for necropsy on day 28 and the remainder reverted to control diet for a further 28 days, up to necropsy on day 56. During the conduct of the study all animals were monitored daily for change in clinical condition, and boy weights and food intakes were measured twice weekly during the study. Clinical observations were recorded for all animals prior to the start of the study and once a week thereafter for as long as they remained on the study. After an overnight fast, the animals were weighed and subsequently killed by exsanguination. The blood samples collected were examined for a range of haematological parameters. Plasma separated from the blood collected at necropsy was examined for prothrombin and activated partial thromboplastin time. Serum separated from the blood collected at necropsy was examined for a range of clinical chemistry characteristics. At necropsy, the weights of the kidneys, liver, and ovaries were recorded in the carcass containing all other tissues were preserved. After retaining sections of the liver for histopathological and fat staining, the remaining samples were frozen and retained at -80°C for possible biochemical examination. The livers from all animals were processed and stained with haematoxylin and eosin and further samples were frozen and stained with Oil Red O for the presence of fat and examined microscopically.

Using methods developed at the testing laboratory, the stability of the test substance in rodent diet over a 35-day period and homogeneity of mixing were established and the diet prepared for feeding to rats in this present study were prepared using identical methods to those previously used. The diets for use in this study were prepared on a single occasion and analysed for test substance content, the results of which indicated that the diets were acceptable for use, as containing within ±10% of their nominal concentrations of test substance.

Two animals were killed in extremis during the study due to their poor general condition. One treated with test substance at a level of 1% was killed on study day 8 and was found to have a fluid filled thoracic cavity and one control rat on day 28 was found to have hydrocephalus (fluid in the cranial cavity). These findings were not related to treatment. There were no other adverse observations of condition or behaviour seen throughout the treatment or reversal periods.

The body weight of female rats given the test substance at the top dose level of 5% were consistently approximately 5% lower than control weights during the 28-day treatment period, but some recovery was seen during the reversal period. No consistent marked changes were see in food intakes or food conversion efficiencies and the calculated overall intakes of the test substance over the 28-day treatment period of the study were 0, 1146.6, 2297.6, and 5770.6 mg/kg/day for the 0, 1, 2, and 5% treatment groups, respectively.

At necropsy of female rats on day 28, a treatment-related increase in the incidence of “pale” livers was seen in animals of the 2 and 5% treated groups. This observation had largely disappeared by necropsy on day 56. A dose-related increase in liver weight was also observed at necropsy on day 28, again a finding which had disappeared by day 56. There was also some evidence of an increase in relative kidney weight of rats treated with 2 and 5%, but only after the 28-day reversal period. This effect was slight and was not seen in the previous 90-day study and is of doubtful toxicological significance.

The haematological examination at day 28 indicated a dose-0related decrease in mean cell volume and mean cell haemoglobin, together with an increase in platelet counts. There was also some evidence for shorter prothrombin times and an increase in fibrinogen levels, but these changes were not strictly related to treatment. By day 56, the original effect on mean cell volume remained marked while the other previously effected parameters were similar to control values. White cell populations appeared to be unaffected by treatment.

Serum chemistry examination at necropsy on day 28 indicated slight, but dose related increases in gamma glutamyltransferase and alkaline phosphatase levels. Serum urea levels were also elevated but the pattern of increase was not strictly related to the dose given. There was also evidence of a dose-related decrease in calcium and alanine aminotransferase levels. By necropsy on day 56, although the previously seen changes in serum enzyme levels had largely disappeared, serum urea levels remained elevated.

At necropsy on day 28, a high incidence of pale livers was seen for the 2 and 5% treatment groups. After the 28-day reversal period, this gross hepatic change was only seen in a few animals, including a control. Microscopic histopathological examination of the livers revealed a treatment-related increase in incidence and degree of hepatocellular vacuolation. The Oil Red O stain of frozen liver samples confirmed the presence of fat within the vacuoles. Although the vacuolation was still present in a few animals of the 2 and 5% treatment groups after the reversal period, the incidence and severity was similar to controls.

In summary, it is concluded that the 28-day treatment period caused similar hepatic changes to those seen in the previous study over a 90-day period. The changes seen in this study were, in general, largely reversible over the 28-day recovery period.