Registration Dossier

Administrative data

Description of key information

No carcinogenicity was observed in rats in a 104-week carcinogenicity conducted in a study equivalent to OECD Guideline 453.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from SEP 17 1997 to MAR 18 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies) performed by Japanese governmental Hatano Research Institute. 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.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:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
GLP compliance:
not specified
Remarks:
performed by Japanese governmental Hatano Research Institute
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratorie Inc., Atsugi Breeding Center, SPF, F344/DuCrj)
- Age at study initiation: 4 weeks
- Weight at study initiation: mean 57g
- Fasting period before study: acclimmatization pre-feeding 15 days
- Housing: autoclaved metal mesh cage (220w x 270d x 190h mm)
- Diet (e.g. ad libitum): CRF-1 (Oriental Yeast Co., Ltd.)
- Water (e.g. ad libitum): tap water (Hadano city waterworks department)
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS: controlled
- Temperature (°C): 24 +-1
- Humidity (%): 50-65%
- Air changes (per hr): 15x per hour
- Photoperiod (hrs dark / hrs light): 12-hour (7-19 o'clock)
Route of administration:
oral: feed
Vehicle:
other: CRF-1 solid feed
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): once every four weeks, stability tested (5-8th week, 45-48th, 85-88th week). Betaine content: 103.2%-108.6% and water content 1.75-4.26%.
- Mixing appropriate amounts with (Type of food): agitation
- Storage temperature of food: 15 Celsius, light-resistant container
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
once every four weeks, stability tested (5-8th week, 45-48th, 85-88th week). Betaine contentt: 103.2-108%
Duration of treatment / exposure:
52 / 104 weeks
Frequency of treatment:
ad libitum
Remarks:
Doses / Concentrations:
0, 1, 2.3, 5%
Basis:
nominal in diet
chronic (52-week) study
Remarks:
Doses / Concentrations:
0, 1, 5%
Basis:
nominal in diet
carcinogenicity (104-week) study
No. of animals per sex per dose:
Chronic (52-week) study: 10 rats/sex/dose
Carcinogenicity (104-week) study: 25 rats/sex/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: A preliminary test was performed. Fifteen, 5-week-old male rats, divided into 3 groups consisting of 5 rats each, were fed diet ad libitum containing 0, 1, or 5% test substance. The rats fed at 5% showed significant decrease in feed intake at the 1st week, and temporary decrease in feed intake was also observed at 1%, though they did not affect the weight increase. From these results, doses selected for the chronic test were 1, 2.3, and 5% and doses for the carcinogenicity study were 1 and 5%.
Positive control:
Not required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (2-13th week), once every four weeks (13-104th week)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY: No data

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

OPHTHALMOSCOPIC EXAMINATION: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: autopsy, abdominal aorta
- Anaesthetic used for blood collection: Yes, pentobarbital sodium (EDTA-2K anticoagulant)
- Animals fasted: No data
- How many animals: All
- Parameters checked: red blood count (RBC), white blood count (WBC), haemoglobin content (Hb), mean corpuscular volume (MCV), haematocrit value (Ht), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count, and differential white blood count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: autopsy
- Animals fasted: No data
- How many animals: All
- Parameters checked: total protein level, albumin level, blood urea nitrogen (BUN), creatinine level, total cholesterol level, alkaline phosphatase activity (ALP), GOT activity (GOT), GPT activity (GPT), gamma-GTP activity (gamma-GTP), total bilirubin level, inorganic phosphorus level, calcium level, A/G ratio, sodium level, potassium level, chlorine level

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals underwent necropsy at the completion of administration/observation period. Gross examination included brain, heart, lung, liver, spleen kidney, testis or ovary weight, body weight ratio
HISTOPATHOLOGY: Yes; Histological examination was conducted on the brain, spinal cord, heart, aorta, trachea, bronchus, lung, liver, kidney, bladder, tongue, esophagus, stomach, duodenum, jejunum, ileum, colon, rectum, testis, epididymis, prostate, seminal vesicle, preputial glands, ovary, uterus, vagina, clitoral gland, submaxillary, sublingual, pituitary, thyroid, parathyroid, adrenal, femoral marrow, sternal bone marrow, thymus, mesenteric lymph node, mandibular lymph node, eye, harderian gland, skin, mammary gland, nasal cavity, and tissues with grossly-visible change
Other examinations:
No
Statistics:
The mean and standard deviation of body weight, feed intake, hematological test, blood biochemistry and organ weight in each group were calculated. Significant differences from the control group were analyzed by student's t-test if the variance was equal in F-test and by Aspin-Welch t-test if the variance was unequal. Significant difference in tumor incidence between betaine groups and control group in carcinogenicity test was examined by Fisher's exact probability test, and nonneoplastic lesions graded in chronic toxicity test and carcinogenicity test were analyzed by Mann-Whitney test at 5% and 1% significance level. Difference in survival time between betaine groups and control group was examined by log-rank test.
Clinical signs:
no effects observed
Mortality:
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):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
decrease in mean corpuscular volume (MCV) and mean corpuscular hemogolobin (MHC), whereas platelet count tended to increase. Blood biochemistry chronic toxicity test showed some minor effects.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increase in liver and kidney weight (f/m) in high-dose goups
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Up to 5% in oral feed no significant changes between study groups.

CLINICAL SIGNS AND MORTALITY: A number of rats were killed in a moribund state or died during the test period (8, 7, and 4 at 0, 1, and 5% in males respectively; 6, 11, and 6 at 0, 1, and 5% in females, respectively). There was no significant change in the performance status attributable to the test substance and there was no difference in survival rate between test substance treated and control groups.

BODY WEIGHT AND WEIGHT GAIN: Body weight gradually increased in all groups. Body weight was significantly lower on a temporary basis in males at 5% at the 2nd through 5th week and in females at 5% at the 2nd through 3rd week. Body weight was higher in male at 5% during the 17th week and at 1% around the 7th week and later. Thereafter, values were significantly higher in test substance groups on a sporadic basis. Also, body weight was higher in females at 5% at the 10th week and later.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Feed intake gradually decreased at the 3rd through to 7th week in all male and female groups including controls. At the 1st week through to 7th week, feed intake in male and femal groups was lower in the order of 0, 1, and 5% groups. Feed intake was lower on a sporadic basis in male and females given the test substance at the 13th week and later. Dry intake of test substance per body weight (kg) was 1389-4659 and 1804-4337 mg/kg in males and females, respectively, at 5% and 307-997 and 379-915 mg/kg in males and females, respectively, at 1%.
HAEMATOLOGY: Significant increase in platelet count was observed in males at 1% and in males and females at 5%, and platelet count tended to increase with dose in males. There was a significant decrease in mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in females at 5%

ORGAN WEIGHTS: Significantly increased absolute and relative kidney weight was observed in male at 5%, and absolute kidney weight was increased in males at 1% and females at 5%. In the iver, significant increased in absolute weight and relative weigh was observed in females at 5%, increased relative weight in females at 1%, and increased absolute weight in males at 1%. In addition, significant decrease in relative weight of the heart and significant increase in absolute lung weight was observed in females at 5%.

GROSS PATHOLOGY: THe incidence of size reduction of seminal vesicles and increased testis size tended to decrease in males at 5%, while incidence of light-colored spots on the liver tended to increase in females at 5%. Tumorous lesions, nodular transformations, or spontaneous were observed in various organs of test substance and control groups.

HISTOPATHOLOGY: NON-NEOPLASTIC: In the kidney, basophilic renal tubule, cast, lymphocytic infiltration, and brown pigmentation were observed in males and females, but the degree of change of basophilic renal tubule and cast was slightly greater in males at 5%. In the liver, biliary hyperplasia with fibrosis was observed in males and females, but the degree of change was mild in many cases in females at 5%.

HISTOPATHOLOGY: NEOPLASTIC: Among those that died or were sacrificed in extremis during the administration period, the first tumour (interstitial cell tumour of the testis) in males was found at 1% in the 74th week and in females (medulloblastoma in the kidney and its lung metastasis) in the 50th week. There was no difference between test substance and control groups with respect to survival. Various tumours were observed in all groups, with no significant difference in tumour incidence between test substance and control groups.

HISTORICAL CONTROL DATA (if applicable): recorded and observed phenomena are common in Fischer 344-rats
Relevance of carcinogenic effects / potential:
No carcinogenic effects or potential exhibited in any testing.
Dose descriptor:
NOAEL
Effect level:
> 5 other: % in feed
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no carcinogenicity at the highest dose tested
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Conclusions:
Betaine does not cause carcinogenic effects up to 5% in oral feed (500 times the expected exposure to human via oral route).

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

Rats were administered the test substance at doses up to 5% in feed for 52 or 104-weeks. No significant changes were observed at any dose level. The test substance did not cause carcinogenic effects at doses up to 5% in feed (500 times the expected exposure to humans via oral exposure).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Betaine is not carcinogenic. Betaine has been extensively tested in various settings via oral route in acute, sub-acute, sub-chronic and chronic animal studies as well as nutritional studies in humans. Taking into account the well-known metabolism of betaine and the results of a well-conducted carcinogenicity study, betaine has not caused any phenomena that would raise a concern for carcinogenicity.

Due to high tolerance (5% in feed) effect levels could not be determined.

Carcinogenicity is not cause a concern vial the dermal route based on experiences within the cosmetic industry, where betaine is abundantly used in preparations due to its anti-irritant properties. Carcinogenicity via inhalation is not a concern since the physico-chemical properties rule out the possibility of toxicity via inhalation.

Justification for classification or non-classification

Not classified according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 based on extensive studies including a carcinogenicity study, betaine has not indicated any properties that would raise a concern for carcinogenicity.