Registration Dossier

Administrative data

Description of key information

L-valine did not show adverse effects in two independent 13 week oral toxicity studies up to high doses. All data from the oral route of administration suggest that they sufficiently cover the dermal and inhalative routes.

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
Study period:
2005-04-21 to 2006-01-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Adopted 1998-09-21
Deviations:
yes
Remarks:
In addition to the endpoints specified in the guideline, estrus eycle length and normality and sperm charaeteristies (number, motility and morphology) were evaluated.
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
OJEC, L225, 2001-08-21
Deviations:
yes
Remarks:
In addition to the endpoints specified in the guideline, estrus eycle length and normality and sperm charaeteristies (number, motility and morphology) were evaluated.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar outbred rats (Crl:(WI)WU BR)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: ca. 6 weeks at commencement of treatment period (ca. 4 weeks upon receipt)
- Weight at study initiation: males 142 - 170 g, mean 156 g; females 113 - 136 g, mean 123 g
- Fasting period before study: no fasting as 2 weeks acclimatisation. Fasting only before final examinations or collection of urine and blood
- Housing: All rats were housed in one room except during FOB (food consumption and food conversion efficiency) and motor activity measurement in week 13 when the selected rats were temporarily transferred to a different room. The rats were housed in macrolon cages with wood shavings (Lignocel Type 3/4, Rettenmaier, Rosenberg, Germany) as bedding material and shreds of paper as environmental enrichment (Envirodri, Lillico, Betchworth, UK). The cages and bedding were changed weekly. The cages were divided over the cage racks stratified randomly. The animals were housed in groups of five, separated by sex. On the day of FOB testing and motor activity assessment the animals were temporarily kept singly in macrolon cages. During urine collection, the animals were kept individually in stainless-steel metabolism cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
According to guideline

IN-LIFE DATES: From: 2005-04-20 To: 2005-08-03
Route of administration:
oral: feed
Vehicle:
other: feed
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): 3 times during study period
- Mixing appropriate amounts with (Type of food): RM3 diet
- Storage temperature of food: < -18 °C

VEHICLE
- Justification for use and choice of vehicle (if other than water): Feed was selected in accordance with use of the test substance in animal feed and the potentially resulting oral exposure of humans.
- Concentration in vehicle: o, 0.2, 1, 5 % (w/w)
- Purity: Certificate of analyses proofes acceptable purity
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Principle
The analysis of L-valine is based on a TNO Standard Operating Procedure for the analysis of free amino acids in feed. After addition of norvaline as intemal standard, samples of rodent diet were extracted with an aqueous solution of trichloroacetic acid. The concentration of L-valine was determined using ion chromatography after post-colurnn reaction with ninhydrin reagent (amino acid analyser). Quantitation was obtained by comparing the peak areas of L-valine and norvaline in the sample extracts with those of a calibration solution containing known amounts of L-valine and norvaline.

Validation criteria
According to the study plan, the method should conform to the following criteria:
recovery: the recovery of the test substance from test diet should be between 80 and 110 % at each of the three concentration levels used in the toxicity study;
repeatability: the relative standard deviation in the percentage recovery found upon analysis of five spiked diet samples per concentration level (the same three levels as used in the recovery test) should be less than 10%.

Preparation of validation samples
Validation samples with nominal concentrations of 0.2 %, 1 % and 5 % L-valine, respectively, were prepared, in five-fold, by addition of 0.800 ml, 4.00 ml and 20.00 ml, respectively, of a L-valine stock solution*>, to 5.0 g (accurately weighed) rodent diet. The validation samples and the blank diet (control) were analysed as describedbelow.
*) The L-valine stock solution was prepared by addition of 2.5513 g of the test substance L-valine to a 200 ml volumetric flask, which was brought to volume with 0.1 mol/1 HCI. The test substance was dissolved by placing the mixture in an ultrasonic bath for approximately 20 minutes.

Sample treatment
To 5 g of diet (accurately weighed) a calculated amount of an internal standard solution (norvaline) was added. Subsequently, a volume of trichloroacetic acid (TCA) solution was added to the diet sample and the mixture was stirred for approximately 1 hour and left to settle for 15 minutes. The extract was diluted with lithium citrate buffer solution (0.1 mol/1, pH 2.2), filtered (0.45 µm) and analysed as described below.

Chromatography
The following LC conditions were used:
Analyser : JEOL AminoTac JLC-500N amino acid analyser
Column :4 x 120 mm, tandem packed column
Column temp. : gradient, see table below
Injection volume : 50 p.l
Detection : 570 nm after post-column reaction with ninhydrin reagent
Reaction temp. : 135 °C

Calibration
For calculation of the amount of L-valine in the test substance L-valine and in the validation samples, a calibration solution was prepared containing 21.40 mg valine and 25.81 mg norvaline per liter lithium citrate buffer solution (0.1 mol/1, pH 2.2).


Determination of homogeneity, stability and content of L-valine diet
Homogeneity
The homogeneity of diets containing the test substance L-valine was determined in the first batch of diets prepared for studies on 2 May 2005. Five samples per dose level, taken at different locations in the feed container, were analysed to determine homogeneity. For each dose level, a one way analysis ofvariance (Anova) was performed using place as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the
mean concentrations differ significantly at the five locations in the feed container). The test substance L-valine was considered tobe homogeneously distributed in the diet ifp ~ 0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the five locations is less than 5%.

Stability
The stability of the test substance L-valine in diet under simulated experimental conditions was determined by analysis of the first batch of diets prepared for the studies on 2 May 2005, after storage at room temperature in an open container for 7 days and after storage at < -18 °C in a closed container for 5 weeks. For each dose level, a one way analysis of variance (Anova) was performed using time as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the difference between the results of the first day (t = 0) and the day on which the analysis is repeated, is significant). If p ~ 0.01 and/or if the relative decrease in the mean concentration on storage is smaller than 10 %, the test substance L-valine was considered to be stable in the diet over that period.

Content
The content of the test substance L-valine was determined in diets prepared on 2 May 2005, 2 June 2005 and 1 July 2005. The content of the test substance L-valine in diet was considered to be 'close to intended' if the difference between the mean concentration and the intended concentration is less than 10 %.


Determination of valine in the test substance L-valine
The amount of the active ingredient valine in the test substance L-valine (g/ 100 g) was determined in triplicate, using essentially the same method as described for the diet samples: To 0.2 g test substance (accurately weighed) 8.00 ml internal standard solution was added. Subsequently, 8 ml trichloroacetic acid
solution (20 %) and 34 ml trichloroacetic acid solution (10 %) were added to the sample and the mixture was stirred for approximately 1 hour. 50 µl of this solution was diluted with 10 ml Iithium citrate buffer solution (0.1 mol/1, pH 2.2), filtered (0.45 µm) and analysed as described above.


Results
The criteria for homogeneity were met for all dose Ievels.
The test substance L-valine was considered stable in rodent diet at all dose Ievels upon storage at room temperature for 7 days and upon storage at < -18 °C for 5 weeks.
The concentrations were 'close to intended' (relative difference less than 10 %) for all dose Ievels of the test substance in diet, except for the 1 % dose Ievel prepared on 2 May 2005, for which the criterion for content was just exceeded (relative difference was+ 12 %).
The mean measured amount of L-valine in the test substance L-valine was 98.0 g/ 100 g.
Duration of treatment / exposure:
90 d
Frequency of treatment:
The test substance was administered at constant concentrations in the feed for 13 consecutive weeks. The testing animals were allowed free access to food (and drinking water).
Remarks:
Doses / Concentrations:
0 % = 0 g/kg b.w.*d (control)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.2 % = 0.13 g/kg b.w.*d
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1 % = 0.6 g/kg b.w.*d
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5 % = 3.0 g/kg b.w.*d
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Intended use of the test substance in animal feed and the potentially resulting oral exposure of humans
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily in the morning, on working days additionally in the afternoon

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to first exposure and then weekly including week 12

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to exposure, then weekly, and on the day of necropsy

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: Over periods of 3-4 days. Results expressed in g / animal*d
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, despite not being a water intake study
- Time schedule for examinations: 4-day periods in weeks 1, 6 and 12

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 4 days prior to treatment, last week of treatment period (day 87)
- Dose groups that were examined: Control group and high dose group. Because no treatment-related ocular changes were observed, eye examinations were not extended to the animals of other groups at the end of the treatment period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period, at necropsy
- Anaesthetic used for blood collection: Yes (C02/02)
- Animals fasted: Yes, overnight
- How many animals: 39 = all survivors. (On day 66, one male rat of the low-dose group was killed because of posterior paralysis. The cause was non-treatement related.)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period.
- Animals fasted: Yes (overnight)
- How many animals: All an
imals.
- Parameters: alkaline phosphatase activity (ALP), bilirubin (total), aspartate arninotransferase activity (ASAT), cholesterol (total), alanine aminotransferase activity (ALAT), triglycerides, gamma glutamyl transferase activity (GGT), phospholipids, total protein, calcium (Ca), albumin, sodium (Na), ratio albumin to globulin, potassium (K), urea, chloride (Cl), creatinine, inorganic phosphate, (fasting) glucose

URINALYSIS: Yes
- Time schedule for collection of urine: Once on day 86-87
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, food for 16 h, water for 24 h
- Parameters: appearance, dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen), microscopic examination of the sediment (red blood cells, white blood cells, epithelial cells, amorphaus material, crystals, casts, bacteria, sperrn cells, worrn eggs).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Arena testing weekly. Functional Observational Battery (FOB) tests and spontaneous motor activity measurements towards the end of the study (week 13).
- Dose groups that were examined: 39 = all survivors. (On day 66, one male rat of the low-dose group was killed because of posterior paralysis. The cause was non-treatement related.)
- Battery of functions tested:
Domain Behavioural endpoint
Autonomic Autonomic lacrimation, salivation pupil response to light, palpebral closure, piloerection, defaecation, urination
Neuromuscular gait, mobility, forelimb and hindlimb gripstrength, landing foot splay, righting reflex
Sensorimotor response to tail pinch, click, touch and approach of a visual object
Convulsive clonic and tonic movements
Excitability ease of removal, handling reactivity, arousal, vocalizations
Activity rearing, posture, motor activity
Physiological body temperature

OTHER:
- Renal concentration test: On day 86-87, all surviving rats were deprived of water for 24 hours and of food during the last 16 hours of this period. During the last 16 hours of deprivation, the rats were kept individually in stainless-steel metabolism cages and urine was collected in glass tubes. The concentrating ability of the kidneys was investigated by measuring the volume and density of the individual samples.
- Estrus cycle evaluation: Vaginal smears to evaluate the estrus cycle length and normality were made daily for 3 weeks prior to sacrifice. Smears were made of all females. The smears of the control and high-dose animals were examined microscopically.
- Sperm analyses:
a) Epididymal sperm motility, count and morohology: At scheduled necropsy, epididymal sperm was derived from the left cauda epididymidis
of all surviving males of all groups. In addition, a smear of the sperm solution was prepared and stained for all males. The smears of the control and high-dose males were examined microscopically for morphology.
b) Testicular sperm count: At scheduled necropsy, the left testis of all surviving males of all groups were taken for later determination of the number of homogenization-resistant spermatids. Following removal of the tunica albuginea, the testicular parenchyma was weighed, minced and homogenized. Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS. The daily sperm production was calculated as 'number of spermatozoa
per gram testicular parenchyma/6.1 '. The evaluation of homogenization-resistant spermatids was performed in the control group and high-dose group.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy:
At the end of the treatment period, the animals were killed on 2 consecutive days (males on day 91; females on day 92), in such a sequence that the average time of killing was approximately the same for each group. Prior to killing the rats were fasted ovemight but had free access to water. The animals were killed by exsanguination from the abdominal aorta under C02/02 anaesthesia. Subsequently, they were examined macroscopically for pathological changes. A thorough necropsy was also performed on the low-dose male which was killed on day 66 of the study (because of posterior paralysis ).
Upon microscopic examination, that animal exhibited leukemia with extensive tumour cell infiltration of the liver, spieen, hone marrow and sternum (hone) including surrounding tissues. Expansion of the tumour into hone and surrounding tissues is a possible explanation for the posterior paralysis which was seen in the in-life phase. This finding was not considered tobe treatment-related because it was observed in one low dose animal only and leukemia is known to occur at a low incidence in rats of this strain and age.

Organ weights:
At scheduled necropsy, the following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying. Relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight: adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroid (with parathyroids), liver, uterus.

Tissue preservation:
Samples of total of 46 tissues and organs of all animals killed at scheduled necropsy and of the animal that was killed in moribund condition on day 66, were examined.

HISTOPATHOLOGY: Yes
The tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. Histopathological examination (by light microscopy) was performed on most of the tissues and argans mentioned above of all animals of the control group and of the high-dose group, and of that animal of the low-dose group that was killed in moribund condition during the study. The lungs, liver, kidneys and all gross lesions were
examined microscopically in all rats of all groups.
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.
- Food consumption/efficiency and water intake: one-way analysis of variance (Anova) followed by Least Significant Difference (L.S.D.) tests.
- 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 tests.
- Reticulocytes and relative differential white blood cell counts, semi-quantitative urinary determinations and microscopy ofthe urinary sediment: Kruskal-Wallis nonparametric one-way analysis of variance.
- 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 test (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.
- Estrus cyclicity: Fisher's exact probability test, Anova followed by by Dunnett's multiple comparison
tests (number of cycles per animal), or Kruskal-Wallis non parametric Anova followed by Mann-Whitney U-tests.
- Sperm parameters: Anova followed by Dunnett's multiple comparison tests or Kruskal-Wallis non-parametric Anova followed by Mann-Whitney U-tests
- Histopathological changes: Fisher's exact probability test.
All analyses were two-sided. Group mean differences with an associated probability of less than 0.05 were considered to be statistically significant.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs.
Mortality:
no mortality observed
Description (incidence):
No treatment-related clinical signs.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females: Body weights significantly decreased in high-dose group on day 7.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake was decreased in males and females of the high-dose group throughout the study. Differences with controls reached Ievel of statistical significance at several stages. No toxicological relevance.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Minor effects at high dose group.
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):
No toxicological significance of effects
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related effects.
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no treatrnent-related clinical signs. One male of the low-dose group showed posterior paralysis and was humanely killed on day 66. One female of the lowdose group showed an abdominal nodule from day 23.

BODY WEIGHT AND WEIGHT GAIN
Females: Body weights significantly decreased in high-dose group on day 7.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food intake was decreased in males and females of the high-dose group throughout the study. Differences with controls reached Ievel of statistical significance at several stages.

FOOD EFFICIENCY
No significant effects.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related changes.

HAEMATOLOGY
No treatment-related changes.

CLINICAL CHEMISTRY
Clinical chemistry showed statistically significant increases in alanine aminotransferase activity in females of the high-dose group, and in sodium Ievel in males and females of this group.

URINALYSIS
No treatment-related changes.

NEUROBEHAVIOUR
The neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance.

ORGAN WEIGHTS
The following statistically significant differences between rats of the test groups and the controls were noted:
- The absolute weight and the relative weight of the liver were decreased in males of the high-dose group. This finding was ascribed to the lower body weight in this group.
- The absolute but not the relative weight of the heart was decreased in males of the high-dose group. This finding was ascribed to the lower body weights in this group.
- The absolute weight of the brain was slightly decreased in males of all test groups. The relative brain weight was not affected. Because a dose-response relationship was not evident, no significance was attached to this finding.
- The relative weight of the epididymides was increased in males of the low- and highdose groups. In the absence of a dose-related response, this finding was considered to be fortuitous.
- The relative weight of the thymus was decreased in mal es of the low-dose group. This finding was not confirmed at the higher dose Ievels and was considered to be fortuitous.
No significance was attached to the above changes in organ weight.

GROSS PATHOLOGY
No treatment related effects.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment related effects.

HISTOPATHOLOGY: NEOPLASTIC
The study showed no effects of the test substance on estrus cyclicity, epididymal sperm motility, epididymal sperm count, testicular sperm count (including
daily sperm production) and sperm morphology.
Key result
Dose descriptor:
NOAEL
Effect level:
628 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
food consumption and compound intake
clinical biochemistry
other: reduction in growth
Key result
Dose descriptor:
NOAEL
Effect level:
666 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
food consumption and compound intake
clinical biochemistry
other: reduction in growth
Critical effects observed:
not specified
Conclusions:
A sub-chronic oral toxicity guideline study of L-valine with rats was conducted for 90 days. The feeding of L-valine was not associated with overt signs of toxicity. The only treatment-related effects noted in the high-dose group were relatively mild reductions in growth and food intake, and possibly some elevated clinical chemistry variables (alanine aminotransferase activity and sodium levels ).
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-wk feeding study in rats was placed at a dietary dose of 1% L-valine (equivalent to an overall mean intake of 628 (males) and 666 (females) mg/kg body weight/day).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
628 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP guideline study with Klimisch Code 1.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A sub-chronic oral toxicity guideline study of L-valine with rats was conducted for 90 days. The feeding of L-valine was not associated with overt signs of toxicity. The only treatment-related effects noted in the high-dose group were relatively mild reductions in growth and food intake, and possibly some elevated clinical chemistry variables (alanine aminotransferase activity and sodium levels ).

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-wk feeding study in rats was placed at a dietary dose of 1% L-valine (equivalent to an overall mean intake of 628 (males) and 666 (females) mg/kg body weight/day).

Another sub-chronic oral toxicity guideline study of L-valine with rats was conducted for 90 days. The feeding of L-valine was not associated with overt signs of toxicity. The only treatment-related effects noted in the high-dose group were with females and were diet consumption volume and, resulting from this, a drop in body weight. These changes were recoverable.

The NOAEL was estimated to be 5.0% for males (3.23 ± 0.14 g/kg/day) and 2.5% for females (1.85 ± 0.06 g/kg/day).

For purpose of risk estimation the lowest NOAEL (1% L-valine) provided in the oral toxicity studies is considered as the key level for CSA.

1 % of L-valine are equivalent to 11 times times amount of the estimated mean oral intake for human, which is approximately 4 g/d (Food and Nutrition Board, 2005).

Food and Nutrition Board (2005): Dietary reference intakes for energy, carbohydrate, fiber, fatty acids, cholesterol, protein, and amino acids (macronutrients). Washington DC, National Academic Press

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Key study

Justification for classification or non-classification

L-valine does not cause toxicity when administered in sub-chronic application. Hence, L-valine should not be classified in the following relevant hazard classes of the CLP Regulation 1272/2008

- acute toxicity (CLP Annex I No. 3.1.)

- specific target organ toxicity - single exposure (CLP Annex I No. 3.8.)

- specific target organ toxicity - repeated exposure (CLP Annex I No. 3.9.)

- aspiration hazard (CLP Annex I No. 3.10.).