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Description of key information

L-isoleucine is not carcinogenic in F344 rats.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
See below for a detailed description of the method.
GLP compliance:
not specified
Specific details on test material used for the study:
- Analytical purity: 100.3%
- Stability under test conditions: stable as determined by analysis of L-isoleucine content in diets after 3 months
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc., Atsugi, Japan
- Age at study initiation: 5 weeks
- Weight at study initiation: ca. 100g, based on graph
- Fasting period before study: not reported
- Housing: plastic cages (5 rats/cage of the same sex) on hardwood chip bedding
- Diet (e.g. ad libitum): Oriental CRF-1 basal diet, Oriental Yeast Co., Ltd., Tokyo, Japan; ad libitum
- Water: ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): Fluorescent light in a 12-h light/dark cycle
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Diet preparation was conducted by Oriental Yeast Co., Ltd.
The content of L-isoleucine in the prepared diets was analyzed after 3 months storage at room temperature and after 6 months under refrigeration, and the amino acid was confirmed to be stable over these periods.

Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
Continuously, via the diet.
Post exposure period:
None
Remarks:
Doses / Concentrations:
2.5%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5.0%
Basis:
nominal in diet
No. of animals per sex per dose:
50 males and 50 females per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on preliminary 13-week toxicity study (Kawabe et al, 1996)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 14 weeks and once every 4 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food intake was measured over a 2-day period before each weighing.

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes: ether
- Animals fasted: Yes: overnight
- How many animals: all
- Parameters examined: red blood cell counts (RBC), white blood cell counts (WBC), measurement of hemoglobin (HB) concentration, hematocrit values (HT) and platelet counts (PLATELET) were performed (Sysmex model CC-180A; Sysmex Co., Ltd., Tokyo, Japan). Blood smears were used to check for leukemia.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes: overnight
- How many animals: all
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), total cholesterol (T-CHO), triglycerides (TG), total protein (TP), inorganic phosphate (IP), calcium (CA), magnesium (MG), sodium (NA), potassium (K) and chlorine (CL).

URINALYSIS: Yes
During weeks 26, 52, 78 and 104 fresh urine samples were obtained from 10 rats in each group. Urinary volume was measured by weighing, and samples were analyzed semi-quantitatively with test strips (Multistix, Miles-Sankyo Co., Ltd., Tokyo, Japan) for protein, glucose, bilirubin, ketones, occult blood and urobilinogen.
The levels of urinary electrolytes [magnesium (MG), calcium (CA), inorganic phosphate (IP), sodium (NA), potassium (K) and chlorine (CL)] were determined using a Hitachi-Biochemical Automatic Analyzer 7040E (Hitachi Ltd., Tokyo, Japan). Urine pH was measured separately with fresh urine specimens using a pH meter (F-8DP, Horiba Ltd., Kyoto, Japan).

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
SACRIFICE
After 104 weeks, all surviving rats were deprived of food, but not water, overnight and then killed by exsanguination from the abdominal aorta under ether anesthesia.

GROSS PATHOLOGY: Yes
A gross pathological examination was made during autopsy and the brain, liver, kidneys, spleen, heart, adrenals and testes or ovaries were weighed. Samples of these organs and of the aorta, mandibular lymph nodes, mesenteric lymph nodes, bone marrow, thymus, pituitary, thyroids, parathyroids, nasal cavity, trachea, lungs, tongue, salivary glands, esophagus, stomach, small intestine, large intestine, pancreas, urinary bladder, prostate, epididymides, seminal vesicles, mammary gland (female only), uterus, vagina, femur, sternum, musculature, skin/subcutis, ear/Zymbal's gland, eyes, Harderian gland, spinal cord, sciatic nerve, and any other tissues with an abnormal appearance were in fixed 10% buffered formalin.

HISTOPATHOLOGY: Yes
Tissues were routinely embedded in paraffin, sectioned and stained with hematoxylin and eosin. A full histopathological examination was performed on the
control and 5.0% groups, and rats that died or killed when they become moribund during the experiment in the 2.5% group. For surviving rats of the 2.5% group, limited organs such as spleen, lung, liver, kidneys, pituitary, testes, urinary bladder, uterus, vagina, ovaries, skin/subcutis (mammary glands, female only) and gross lesions were examined histopathologically.
Statistics:
The body weight, hematology, clinical chemistry and organ weight data were subjected to analysis of variance, and differences between means were tested with the Students t-test. The incidences of histopathologic lesions were analyzed using the Fishers exact probability test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related changes in physical appearance or general behavior were observed in any of the rats throughout the study. At the end of the study, the survival rates of females receiving 0%, 2.5% and 5.0% L-isoleucine were 86%, 82% and 74%, respectively, and those of males were 84%, 86% and 76%. There were
no significant differences in the survival rate between controls and treated animals at the end of the 104 week period of the experiment.

BODY WEIGHT AND WEIGHT GAIN
Significant elevation for body weight was observed in female rats given 5.0% L-isoleucine as compared with the control group from week 42 to 94 (excluded week 46). Also in male rats, significant elevation of body weight was observed in rats given 2.5% L-isoleucine at week 3 and from week 78 to 98 (excluded week 86), and in rats given 5.0% L-isoleucine from week 2 to 4 and at week 78 and 82.

FOOD CONSUMPTION
Food consumption was similar in both control and treated groups. The average L-isoleucine intakes for females and males were 1142.4 and 924.7 mg/
kg body weight/day, respectively, in the 2.5% group, and 2188.6 and 1788.2 mg/kg body weight/day, respectively, in the 5.0% group.

HAEMATOLOGY
No significant differences between groups were evident in any hematological parameters.

CLINICAL CHEMISTRY
Significantly increased CA in females receiving 5.0% L-isoleucine and decreased NA in females receiving 2.5% and 5.0% L-isoleucine were found as compared with the control group values. In male rats, the levels of ALT, T-CHO, IP and NA in rats given 5.0% and MG in rats given 2.5% and 5.0% were significantly increased as compared with the control group. In other parameters, significant differences were noted, but there was no toxicological significance.

URINALYSIS
No biologically significant differences in urinalysis values at weeks 26, 52, 78 and 104 were noted between control and treatment groups.

ORGAN WEIGHTS
Relative kidney weights for male rats given 5.0% L-isoleucine were significantly increased as compared with the control group values. A significant decrease in the testes weights was observed in the 2.5% and 5.0% male groups.

GROSS PATHOLOGY
No grossly visible changes related to L-isoleucine treatment were found in either sex.

HISTOPATHOLOGY: NON-NEOPLASTIC
As a non-neoplastic lesion, atrophy of the testes in the 5.0% males was significantly increased as compared with the control value, but there was no dose dependence. No treatment-related differences in other findings, including in the liver or kidney, were observed in either sex.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
The incidence of c-cell adenomas of the thyroid in the 5.0% male group was significantly decreased as compared with the control group values. Incidences of
benign or malignant tumors were otherwise comparable between the treated groups and controls of either sex.

In conclusion, the present investigation demonstrated no carcinogenic potential in either sex of F344 rats fed L-isoleucine (up to a 5.0% dietary level) for 104 weeks.

Conclusions:
L-isoleucine is not carcinogenic in F344 rats.
Executive summary:

The carcinogenic potential of L-isoleucine, used as a food fortifier, was examined in both sexes of F344 rats. Groups of 50 female and 50 male animals were given diet containing L-isoleucine at concentrations of 0%, 2.5% or 5.0%.

The average L-isoleucine intakes for females and males were 1142.4 and 924.7 mg/kg body weight/day, respectively, in the 2.5% group, and 2188.6 and 1788.2 mg/kg body weight/day, respectively, in the 5.0% group.

No toxicologically relevant treatment-related changes in the survival rate, general condition, body weight, food consumption, urinalysis, hematology or clinical chemistry data and organ weights were noted. Detailed histopathological examination revealed no treatment-related increase in the incidences of any nonneoplastic or neoplastic lesions. The results indicate that L-isoleucine is not carcinogenic in F344 rats of either sex.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
rat

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

No adverse effects were observed upon oral administration of L-isoleucine during 104 weeks. As a consequence, classification of L-isoleucine for carcinogenicity is not required in accordance with the criteria described in Annex I to regulation 1272/2008 (CLP).

Likewise, classification for carcinogenicity is not required according to the criteria described in Annex VI to Directive 67/548 (DSD).

Additional information

The carcinogenic potential of L-isoleucine, used as a food fortifier, was examined in both sexes of F344 rats. Groups of 50 female and 50 male animals were given diet containing L-isoleucine at concentrations of 0%, 2.5% or 5.0%.

The average L-isoleucine intakes for females and males were 1142.4 and 924.7 mg/kg body weight/day, respectively, in the 2.5% group, and 2188.6 and 1788.2 mg/kg body weight/day, respectively, in the 5.0% group.

No toxicologically relevant treatment-related changes in the survival rate, general condition, body weight, food consumption, urinalysis, hematology or clinical chemistry data and organ weights were noted. Detailed histopathological examination revealed no treatment-related increase in the incidences of any nonneoplastic or neoplastic lesions. The results indicate that L-isoleucine is not carcinogenic in F344 rats of either sex.