L-isoleucine

Endpoint summary

Description of key information

The feeding of L-isoleucine to rats was not associated with overt signs of toxicity.

The NOAEL determined for L-isoleucine is ca. 600 mg/kg bw/d based on an observed increased

maximum estrus cycle length at higher doses.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Additional endpoints that were evaluated and which are not described in these guidelines: estrus cycle length and normality and sperm characteristics.

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Additional endpoints that were evaluated and which are not described in these guidelines: estrus cycle length and normality and sperm characteristics.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species: Wistar outbred rats Crl:(WI)WU BR
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx.. 4 weeks old
- Initial bodyweight: 139-167g (mean 153g) for males and 120-145g (mean 129g) for females.
- Housing: macrolon cages with wood shavings as bedding material and shreds of paper as environmental enrichment. The animals were housed in groups of five, separated by sex. During urine collection, the animals were kept individually in stainless-steel metabolism cages.
- Diet: Commercial rodent diet: Rat & Mouse No. 3 Breeding Diet, Special Diet Services, Witham, England. Ad libitum.
- Water: tap water. Ad libitum.
- Acclimation period: Upon arrival, the rats were checked for overt signs of ill health and anomalies, and kept in quarantine. During the quarantine period, their microbiological status was checked by the conduct of serology in random samples. After the results of serology turned out to be satisfactory, the quarantine room was cleared for use as experimental room, and the rats were further acclimatized to the conditions in this room until initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 50-70%, occasionally the relative humidity was shortly lower than 50% (min. 40%). Due to room cleaning, the relative humidity exceeded the upper limit during short periods. Additionally, the relative humidity exceeded 70% for unknown reasons during a few to several hours on a number of days. The deviations in relative humidity from the target upper limit were only slight (incidental max. value recorded: 87%), and were not considered relevant.
- Air changes (per hr): approx. 10
- Photoperiod: artificial lighting by fluorescent tubes, 12-hours light / 12-hours dark.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The test substance was incorporated in the RM3 diet. Fresh batches of experimental diets were prepared 3 times during the study. Homogeneity was obtained by mixing in a mechanical blender (Lödige, Germany) for 2 minutes. After preparation, the diets were stored in a freezer (< -18°C) in portions sufficient for 4 days. The feed in the feeders was replaced with fresh portions from the freezer twice a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Immediately after preparation of the diets, samples were taken and analysed or stored in a freezer until analysis. The content of the test substance at each dietary level was examined in all test diets used in the study. The homogeneity of L-isoleucine at each dietary level was examined by analysing five samples per test diet, taken at different locations in the feed container from the first batch of diets prepared. The stability of L-isoleucine under simulated experimental conditions was examined by analysing samples of each diet (of the first batch prepared) after storage in the animal room for 7 days in an open container, or in a freezer (< -18°C) in a closed container for 5 weeks.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously, via the feed

Remarks:

Doses / Concentrations:
0 % w/w
Basis:
other: nominal in diet, based on 94.2% purity of iso-leucine.

Remarks:

Doses / Concentrations:
0.21 % w/w
Basis:
other: nominal in diet, based on 94.2% purity of iso-leucine.

Remarks:

Doses / Concentrations:
1.06 % w/w
Basis:
other: nominal in diet, based on 94.2% purity of iso-leucine.

Remarks:

Doses / Concentrations:
5.32 % w/w
Basis:
other: nominal in diet, based on 94.2% purity of iso-leucine.

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, in the morning + additional observation in the afternoon on week days.
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to first exposure + once weekly during the treatment period up to and including week 12.

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly + fasted body weights were determined on the day of scheduled necropsy for calculation of correct organ to body weight ratios.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was measured per cage, over successive periods of 3 or 4 days (according to the frequency of refreshing the feed in the animal feeders), by weighing the feeders. The results were expressed in grams per animal per day. The intake of the test substance per kg bw per day was calculated from the nominal diet concentration of the test substance, the food consumption and the mean body weight in the pertaining week.

FOOD EFFICIENCY:
The efficiency of food utilisation was calculated over weekly periods and expressed in grams weight gain per gram food consumed.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily in weeks 1, 6 and 12 of the study. Measurements per cage, expressed in g per animal per day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of treatment, and in the last week of treatment
- Dose groups that were examined: first examination: all rats; second examination: control and high-dose group rats
- Methodology: by using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes: CO2/O2
- Animals fasted: Yes, overnight
- How many animals: all surviving animals
- Parameters examined: haemogolobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, mean corpuscular value, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes, overnight
- How many animals: all surviving animals
- Parameters examined: alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase activity, gamma glutamyl transferase activity, total protein, albumin, ratio albumin to globulin, urea, creatinine, (fasting) glucose, total bilirubin, total cholesterol, triglycerides, phospholipids, calcium, sodium, potassium, chloride, inorganic phosphate

URINALYSIS: Yes
- Time schedule for collection of urine: week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, 16 hours
- Parameters examined: volume and density of the urine, appearance dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen), microscopic examination of the sediment (red blood cells, white blood cells, epithelial cells, amorphous material, cyrstals, casts, bacteria, sperm cells, worm eggs)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 13
- Dose groups that were examined: all
- Battery of functions tested: autonomic (lacrimation, salivation, pupil resonse to light, palpebral closure, piloerection, defaecation, urination), neuromuscular (gait, mobility, forelimb and hindlimb gripstrength, landing foot splay, righting reflex), sensorimotor (response to pain pinch, click, touch and approach of visual object), convulsive (clonic and tonic movements), excitability (ease of removal, handling reactivity, arousal, vocalizations), activity (rearing, posture, motor activity), physiological (body temperature).

ESTRUS CYCLE EVALUATION:
- Time schedule for examination: daily vaginal smears were collected about 3 weeks prior to sacrifice (except on 1 specific occasion, when, by mistake no smears were taken)
- How many animals: smears were collected from all female animals. Initiallty only the smears of the control group and the high-dose group were evaluted microscopically. Since a statistically significant increase was observed in the length of the longest cycle, the evaluation was extended to the intermediate-dose group.

SPERM ANALYSIS:
- Time schedule for examination: at scheduled necropsy
- How many animals: samples were prepared from all surving males. Control and high-dose group samples were examined. Due to no observed effects, the samples from the remaining dose groups were not examined.
- Parameters examined: epididymal sperm motility, count and morphology; testicular sperm count.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Methodology: the animals were killed by exsanguination from the abdominal aorta under CO2/O2 anaesthesia. Subsequently, they were examined macroscopically for pathological changes. A thorough necropsy was also performed on the moribund high-dose male which was killed on day 24 of the study.
- The following organs were weighed, and relative organ to body weight ratios were calculated: adrenals, brain, epipidymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroid (with parathyroids), uterus.

HISTOPATHOLOGY: Yes
- Methodology: tissues to be examined were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. Histopathological examination was performed by light microscopy.
- How many animals: samples were preapred from all animals, examinations were carried out on all animals of the control group and of the high-dose group. Lungs, liver, kidneys and gross lesions were examined microscopically in all rats of all groups.
- The following samples were prepared: adrenals, aorta, axillary lymph nodes, brain, caecum, cervical lymph node (not examined), colon, duodenum, epididymides, exorbital lachrymal glands (not examined), eyes, femur joint (not examined), GALT, heart, ileum, jejunum, kidneys, liver, lungs, mammary gland (females), mesenteric lymph nodes, nerve-peripheral (sciatic), oesophagus, ovaries, pancreas, parathyroid, parotid salivary glands, pituitary, prostate, rectum, seminal vesicals + coagulating glands (not examined), skeletal mucsle (thight), skin (flank), spinal cord, spleen, sternum with bone marrow, stomach, sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, trachea/bronchi, urinary bladder, uterus (with cervix), vagina (not examined)

Statistics:

The statistical procedures used to evaluate the results were as follows:
- Body weights: one-way analysis of covariance using pre-exposure (day 0) weights as the covariate. When group means were significantly different (p<0.05), individual pair-wise comparisons were made using Dunnett’s multiple comparison method.
- Food consumtion/efficiency and water intake: one-way analysis of variance (Anova) followed by Least Significant Difference (L.S.D.) tests (experimental unit was the cage).
- Red blood cell and clotting potential variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, volume and density of the urine, organ weights and terminal body weights: Anova followed by Dunnett’s multiple comparison method. Independent from the results of Anova, the homogeneity of variances was tested by Bartlett’s test. There were no parameters for which the variances differed significantly (p<0.001).
- Reticulocytes and relative differential white blood cell counts, semi-quantitative urinary determinations and microscopy of the urinary sediment: Kruskal-Wallis nonparametric one-way analysis of variance. When this analysis yielded a significant difference, pair-wise comparisons between the control- and treatment groups were made by means of Mann-Whitney U-tests.
- Functional Observational Battery: Anova followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric Anova followed by multiple comparison tests (rank order data), or Pearson chi-square (categorical data).
- Motor activity data: repeated measures analysis of variance and one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).

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

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

HAEMATOLOGY
Red blood cell count and haemoglobin concentration were slightly, though statistically significantly increased in males of the high-dose group. Thrombocyte count was statistically significantly decreased in females of this group. The statistically significantly increased thrombocyte count in mid-dose males was considered a fortuitous finding. There were no treatment-related differences between the groups in total or differential white blood cell counts.

NECROPSY OF HIGH-DOSE MALE THAT WAS HUMANELY KILLED ON DAY 24
One high-dose male was killed intercurrently because of emaciation and ataxia. Although many histopathological changes were observed in various organ systems, a clear cause of the conditional decline of this rat could not be established. Because comparable findings were not observed in any other rat, the disease of this single high-dose rat was not considered to be treatment-related.

ESTRUS CYCLE EVALUATION
The mean length of the longest cycle was statistically significantly increased in the high-dose group. No effect was seen on the number of cycles per animal, the number of females with a prolonged estrus period, or the number of acyclic females.

Dose descriptor:

NOAEL

Effect level:

1.06 other: % w/w in the diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Observations related to haematological changes and changes in the mean length of the longest estrus cycle in animals from the highest dose group.

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Observations related to haematological changes and changes in the mean length of the longest estrus cycle in animals from the highest dose group.

Critical effects observed:

not specified

Conclusions:

The feeding of L-isoleucine to rats was not associated with overt signs of toxicity. The only treatment-related effect noted was an increased maximum estrus cycle length in the high-dose group. Because the test substance did not induce any noticeable changes in the mid-dose group, the NOAEL in the present 13 week feeding study in rats was placed at 1.0% of L-isoleucine in the diet, equivalent to an overall intake of 0.6 g L-isoleucine / kg bw / d.

Executive summary:

The oral toxicity of the test substance L-isoleucine was examined in a sub-chronic (13 week) toxicity study with 4 groups of 10 male and 10 female Wistar rats. The test substance was incorporated in the feed at constant levels of 0% (control), 0.2%, 1.0% and 5.0% of the active ingredient L-isoleucine (equal to 0.21%, 1.06% and 5.31% of the test substance L-isoleucine, based on 94.2% purity). These dietary levels were equivalent to overall intakes of approx. 0.1, 0.6 and 3 g of L-isoleucine/kg bw/day, in the low-, mid- and high-dose group, respectively.

 

There were no treatment-related clinical signs. One high-dose male was humanely killed on day 24 of the study for reasons unrelated to treatment. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. Opthalmoscopic examination did not reveal any treatment-related changes. There were no noticeable differences in mean body weights, food intake, food conversion efficiency or water intake between the groups.

 

Haematology was conducted in blood obtained from all rats at scheduled necropsy. Red blood cell count and haemoglobin concentration were slightly increased in males of the high-dose group. Thrombocyte count was decreased in females of this group. There were no significant changes in total or differential white blood cell counts.

 

Clinical chemistry, conducted in plasma obtained from all rats at scheduled necropsy, did not show any treatment-related changes. Urinalysis, conducted in all rats in week 13 of the study, did not reveal any treatment-related changes in renal concentrating ability, semi-quantitative (dipstick) urinary measurements or in microscopy of the urinary sediment. Vaginal smears to evaluate the estrus cycle length and normality were made daily during 3 weeks prior to sacrifice. The mean length of the longest cycle was statistically significantly increased in the high-dose group. No effects was seen on the number of cycles per animal, the number of females with a prolonged estrus period or the number of acyclic females. No effect was observed on epididymal sperm motility, epididymal sperm count, testicular sperm count (including daily sperm production) or sperm morphology.

 

Organ weights, macroscopic examination at necropsy, and microscopic examination of organs and tissues did not reveal any adverse effects.

 

It was concluded that the feeding of L-isoleucine was not associated with overt signs of toxicity. Because the test substance did not induce any noticeable changes in the mid-dose group, the no-observed-adverse-effect level (NOAEL) in the present 13 week feeding study in rats was placed at 1.0% of L-isoleucine in the diet (equivalent to an overall intake of 0.6 g L-isoleucine/kg bw/day).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Four reliable repeated dose toxicity studies are available for L-isoleucine, all four assessed the toxicity of L-isoleucine via oral administration.

In the key study (Lina and Joncker, 2006), the oral toxicity of the test substance L-isoleucine was examined in a sub-chronic (13 week) toxicity study with 4 groups of 10 male and 10 female Wistar rats, according to GLP and OECD guidelines. The test substance was incorporated in the feed at constant levels of 0% (control), 0.2%, 1.0% and 5.0% of the active ingredient L-isoleucine. These dietary levels were equivalent to overall intakes of approx. 0.1, 0.6 and 3 g of L-isoleucine/kg bw/day, in the low-, mid- and high-dose group, respectively.

The feeding of L-isoleucine was not associated with overt signs of toxicity. The only treatment-related effect noted was an increased maximum estrus cycle length in the high-dose group. Because the test substance did not induce any noticeable changes in the mid-dose group, the no-observed-adverse-effect level (NOAEL) in the present 13 week feeding study in rats was placed at 1.0% of L-isoleucine in the diet (equivalent to an overall intake of 0.6 g L-isoleucine/kg bw/day).

 

In the supporting study by Tsubuki et al. (2004), Sprague-Dawley rats were exposed to 1.25, 2.50 or 5.00% w/w of L-isoleucine via the diet for a period of 13 weeks. No adverse effects were observed at any tested dose level. Based on the food consumption of the animals, an NOAEL of 1570 mg/kg bw/d (males) and 1650 mg/kg bw/d (females) was calculated.

In the supporting study by Kawabe et al. (1996), groups of 10 male and 10 female Fisher F344 rats were fed a powdered diet for 13 weeks, containing nominal levels of 1.25, 2.5, 5.0 and 8.0% of L-isoleucine. Actual ingested doses are not determined. The investigation demonstrated that treatment with L-isoleucine at doses of 8.0 and 5.0% in the diet induces statistically significant changes of biochemical parameters in F344 rats. The results indicate a 2.5% level of L-isoleucine in the diet to be a no-effect level (NOEL).

In the supporting study of Varga et al. (2006) 10 males and 10 females were fed two L-isoleucine products, manufactured by different methods, at a dose of 2,000 mg/kg bw for 28 days following OECD TG 407. The oral administration of L-isoleucine 1 and 2 was well tolerated and did not cause lethality or toxic clinical symptoms during. Body weight, body weight gain and food and water intake were similar to the control group. No changes in heamatological or clinical chemistry were observed. Urinary parameters were not affected. No treatment related differences were found in organ weights or gross and histopathological investigations. Therefore, the NOAEL in rats was concluded to be higher than 2,000 mg/kg.

The NOAEL derived in the key study was retained to be used for the hazard assessment and classification and labelling. As this NOAEL can also be used to derive the relevant dermal and/or inhalatory DNELs, and is sufficient for assessing the classification and labelling, further testing of the dermal and inhalatory routes is not required.

Justification for classification or non-classification

No adverse effects were observed upon oral administration of L-isoleucine at a dose of 600 mg/kg bw/d during 90 days. As a consequence, classification of L-isoleucine for Specific Target Organ Toxicity - Repeated Exposure is not required in accordance with the criteria described in Annex I to regulation 1272/2008 (CLP).

Likewise, classification with risk phrase R48 is not required according to the criteria described in Annex VI to Directive 67/548 (DSD).