Arginine

Endpoint summary

Description of key information

L-arginine 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:

1995-03-28 to 1996-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

other: MHW/PAB/ERD-1 notification No. 24 (September 11, 1989), entitled “Guidelines for toxicity studies of drugs,” and MHW/PAB/NDD notification No. 88 (August 10, 1993), entitled “Amendments on guidelines of a single and repeated dose toxicity studies"

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 6 weeks
- Weight at study initiation: The body weight range at the start of administration was 183 to 218 g for males and 138 to 174 g for females.
- Fasting period before study: not given. Crj: CD (SD) SPF rats were purchased at 4 weeks of age and subjected to about 2-week quarantine and acclimation before study.
- Housing: The animals were individually housed in the metal mesh cages (W254 × D350 × H170 mm: Lead Engineering Co., Ltd.) under the following environmental conditions: temperature; 23 ± 3°C, relative humidity; 50 ± 20%, frequency of ventilation; 11 to 13 times/hr and lighting: 12 hours per day.
- Diet (e.g. ad libitum): ad libitum. During the administration period: food mixed with the test material or basal food (CRF-1); during the quarantine and acclimation period and the recover period: basal food (CRF-1)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
According to guideline

Route of administration:

oral: feed

Vehicle:

other: powder CRF-1 (sterilized by irradiation, Oriental Yeast Co., Ltd.)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): The preparation was performed at least once every 4 weeks.
- Mixing appropriate amounts with (Type of food): The premix food was prepared by mixing the required amount of the test material in each concentration with about 10-fold amount of basal food (powder CRF-1 sterilized by irradiation: Oriental Yeast Co., Ltd.). The stipulated amount of basal food was added to each premix food to make the designated concentration of the test material. The concentrations of the test substance were 1.25%, 2.5% and 5.0%.
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): basal food (CRF-1), because the test substance is solid.
- Concentration in vehicle: 1.25%, 2.5% and 5.0%.
- Amount of vehicle (if gavage): not available
- Lot/batch no. (if required): Lot Nos.: 950113, 950208, 950309, 950411 and 950510
- Purity: not available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and uniformity of the test material were examined in the food mixed with the test material used at week 1 of administration by an amino acid analyzer method before the commencement of administration. Also the concentrations of the test material were examined in the mixed food used at the last week of administration by an amino acid analyzer method before use.
Method:
About 0.5 g of the sample was weight exactly. 0.1 N hydrochloric acid was added to the sample to make 100 mL exactly. Then the solution was stirred for 30 min and filtered through a disc filter (type 25, German Science). 5 mL of the filtrate was taken exactly, and a certain amount of an internal standard, DL-norleucine and distilled water was added to make 50 mL exactly to obtain a sample solution.
The sample solution was determined according to the standard analytical method with an amino acid analyzer L-8500 (Hitachi).

Duration of treatment / exposure:

13 weeks.
In addition, the 0% (control) and 5.0% concentration groups (6 animals per sex per group) were provided to a 5-week recovery period to investigate the reversibility of toxic changes.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0 % (control) = 0 mg/kg b.w.*d
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
1.25 % = 752.2 mg/kg b.w.*d (male) or 909.9 mg/kg b.w.*d (female)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
2.5 % = 1552.0 mg/kg b.w.*d (male) or 1802.9 mg/kg b.w.*d (female)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
5.0 % = 3130.9 mg/kg b.w.*d (male) or 3565.1 mg/kg b.w.*d (female)
Basis:
nominal in diet

No. of animals per sex per dose:

12 (concentrations: 1.25 % and 2.5 %) or 18 (concentrations: 0 % and 5.0 %)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Food containing 5.0% of the test material, about 200 times the estimated maximum oral dose in humans, was given to the high dose group. Food containing 2.5% and 1.5% of the test material was given to the middle and low dose groups, respectively.
- Rationale for animal assignment (if not random):
The animals were selected on the basis of the body weight gain during the quarantine and acclimation period after excluding the animals that showed abnormalities in the clinical signs and ophthalmological examination, and assigned to each group to make the mean body weight in each group almost equivalent by stratifying on the basis of body weight on the grouping day (3 days before the start of administration) by the combination of a block placement method and random extraction method (the group composition is prepared by the block placement method, and the dose and the individual number in each group are given to each animal randomly) with a computer program.
- Post-exposure recovery period: 5 weeks for recovery groups

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical signs and behaviors in all the animals were observed twice a day (in the morning and afternoon except once in the morning on
holidays) during the administration period, and once a day (in the morning) during the recovery period. All the animals were weighed at a certain time (8 a.m. to 0:30 p.m.) twice every week.

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

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to exposure, then over periods of 3-4 days until day 91

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, 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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, water intake was examined
- Time schedule for examinations: 1-day water intake was measured once before the start of administration and once every week.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the quarantine and acclimation period, at week 13 of administration and at week 5 of recovery
- Dose groups that were examined: During the quarantine and acclimation period: all the animals were examined. At week 13 of administration and at week 5 of recovery 6 males and 6 females in each group (animals for the recovery study in the groups with the recovery group) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the next day of the last day of the administration period and of the recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 11 or 12 per concentration
- Parameters: Red blood cell (RBC), Hemoglobin (Hb), Hematocrit (Ht), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Reticulocyte, Platelet, White blood cells (WBC), differential leukocyte counts, Prothrombin time (PT), Activated partial thromboplastin time (APTT), Fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the next day of the last day of the administration period and of the recovery period
- Animals fasted: Yes
- How many animals: 11 or 12 per concentration
- Parameters: GOT, GPT, LDH, AIP, Total cholesterol (T⋅ cho), Triglyceride (TG), Phospholipid (PL), Total bilirubin (T⋅ bilirubin), Blood glucose (Glucose), Urea nitrogen (BUN), Creatinine, Sodium (Na), Potassium (K), Chlorine (Cl), Calcium (Ca), Inorganic phosphorus (P), Total protein (TP), albumin-globulin ratio (A/G)

URINALYSIS: Yes
- Time schedule for collection of urine: at weeks 5 and 13 of administration and at week 5 of recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters: pH, Protein, Ketone body, Glucose, Occult blood, Bilirubin, Urobilinogen, Color, Sediments, Urine volume, Specific gravity (S.G.), Sodium (Na), Potassium (K), Chlorine (Cl)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The numerical data in each item examined were tested by Bartlett’s test for homogeneity of variance. When the variances were homogeneous, a one-way analysis of variance was carried out. When the significant difference was observed, Dunnett test was performed to analyze the difference of the mean values between the control group and each treated group. When the variances were not homogeneous, the rank sum analysis of Kruskal-Wallis was carried out. When the significant difference was observed, Dunnett test was performed to analyze the difference of the mean rank sum between the control group and each treated group. Significance of the differences was estimated at 5 and 1% (both sides) levels of probability.

Clinical signs:

no effects observed

Description (incidence and severity):

No dose-related change in the clinical signs was observed. Two dead animals (animals sacrificed at moribund), produced by an accident.

Mortality:

no mortality observed

Description (incidence):

No dose-related change in the clinical signs was observed. Two dead animals (animals sacrificed at moribund), produced by an accident.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No dose-related effects were observed.

Food efficiency:

no effects observed

Description (incidence and severity):

No dose-related effects were observed.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

Only some changes in urine volume were observed.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period decrease in the relative weight was observed in the salivary glands in the females of the 5.0% group, and in the kidney in females of each treated group. These changes were considered toxicologically meaningless.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- Dead animals (animals sacrificed at moribund):
1) Administration period: Two animals, 1 male in the control group and 1 male in the 2.5% group, were sacrificed at moribund. These were produced by an accident.
2) Recovery period: No death was observed throughout the recovery period.

- Survivors:
1) Administration period: No dose-related change in the clinical signs was observed.
2) Recovery period: No abnormality was observed in any animal.

BODY WEIGHT AND WEIGHT GAIN
Throughout the administration and recovery period, the body weight change in each treated group was the same as that in the control group without significant difference between them.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
1) Administration period: Males: There was not significant change in mean food consumption throughout the administration period. Females: Significantly high values were observed transiently (days 38 and 73 of administration) in the 1.25 and 2.5% groups, but there was no dose-dependency in these changes.
2) Recovery period: There was no significant difference in mean food consumption throughout the recovery period.

FOOD EFFICIENCY
1) Administration period: In males, there was no significant difference in food efficiency between each treated group and the control group. In females, the significantly low value was observed at week 7 of administration in the 1.25% group, and the significantly high value at week 3 of administration in the 2.5% group. There was, however, no dose-dependency in these changes. There was no significant difference in mean food efficiency throughout the administration period.
2) Recovery period: In males, there was no significant difference in food efficiency between the 5.0% group and the control group. In females, the significantly low value (decreased by 64% compared with mean control value) was observed at week 2 of recovery in the 5.0% group, but this change was transient. There was no significant difference in mean food efficiency throughout the recovery period.

WATER INTAKE
1) Administration period: In males, the significantly high values (increased by 25 to 41% compared with mean control value) were sporadically observed throughout the administration period in the 5.0% group. The significantly high values (increased by 21 to 28% compared with mean control value) were observed
at the early phase of the administration period (week 1 or 2 of administration) also in the 1.25 and 2.5% groups, and thereafter these groups showed a little higher values, though there was no significant difference compared with the control group.
In females, the values in the 5.0% group tended to be higher throughout the administration period, and at week 7 of administration, the significantly high value (increased by 28% compared with mean control value) was observed. Although the significantly high value was observed at week 2 of administration in the 1.25% group, there was no dose-dependency in this change.
2) Recovery period: There was no significantly difference in water intake between the 5.0% group and the control group.

OPHTHALMOSCOPIC EXAMINATION
No abnormality was observed in any animal at week 13 of administration and at week 5 of recovery.

HAEMATOLOGY
In the examination both at the end of the administration period and of the recovery period, there was no significant difference in any parameter between each treated group and the control group.

CLINICAL CHEMISTRY
In the examination both at the end of the administration period and of the recovery period, there was no significant difference in any parameter between each treated group and the control group.

URINALYSIS
1) At week 5 of administration: In the qualitative parameters, there was no marked difference in any parameter between the control group and each treated group. In the quantitative parameters, the significant increase in urine volume (increased by 79% and 116% compared with mean control value) was
observed in males and females of the 5.0% group.
2) At week 13 of administration: In the qualitative parameters, there was no marked difference in any parameter between the control group and each treated group. In the quantitative parameters, the significant increase in urine volume (increased by 46 to 98% compared with mean control value) was observed in males of the 2.5% group and in males and females of the 5.0% group, and the significantly low value (decreased by 29% compared with mean control value on the basis of the value obtained by subtracting 1 from observed value) in males of the 5.0% group.
3) At week 5 of recovery: In the qualitative and quantitative parameters, there was no marked difference in any parameter between the control group and the 5.0% group.

ORGAN WEIGHTS
1) At the end of the administration period: In the salivary glands, the significant decrease in the relative weight (unilateral, decreased by 11% compared with mean control value) was observed in females of the 5.0% group, and the absolute weight also showed the low value (unilateral, decreased by 6% compared with mean control value). In the kidney, the significant decrease or a tendency toward a decrease in the relative weight (bilateral, decreased by 7 to 10% compared
with mean control group on the basis of the bilateral weight) was observed in females of each treated group, and the absolute weight also showed the low values (decreased by 3 to 5% compared with mean control value on the basis of the bilateral weight).
2) At the end of the recovery period: There was no significant difference in the weight of any organ and tissue between the 5.0% group and the control group.

GROSS PATHOLOGY
- Dead animals:
In two males sacrificed at moribund during the administration period, the following changes were observed. These changes were, however, produced incidentally by the accident.
External appearance: reddish brown smudge of peri-nasal area (1 male of the 2.5% group)
Thymus: disseminated dark red spots (1 male of the control group)
Lung: dark red patch (1 male of the 2.5% group) and disseminated black brown patch (1 male of the control group)
Other tissues or organs: fracture of maxilla (1 male of the 2.5% group) and malocclusion (1 male of the control group and 1male of the 2.5% group)

- Survivors:
1) At the end of the administration period: The following changes were observed, but these were considered incidental, judging from the occurrence situation.
External appearance: focal dark red of peripheral region of cornea in eye ball (unilateral, 1 male of the 1.25% group)
Thyroid: enlargement (unilateral, 1 male of the 1.25% group)
Stomach: dark red spot in the glandular stomach (1 female of the 2.5% and 1 female of the 5.0% group)
2) At the end of the recovery period: The following changes were observed, but these were considered incidental, judging from the occurrence situation.
Liver: yellowish white patch (1 male of the control group)
Testis: atrophy (bilateral, 1 male of the control group)

HISTOPATHOLOGY: NON-NEOPLASTIC
- Dead animals:
In 2 males sacrificed at moribund during the administration period, the following changes were observed. These changes were, however, produced incidentally by the accident.
Lung: Slight or moderate focal hemorrhage was observed in 2 males.
Tongue: Slight ulcer was observed in 1 male of the control group.
Thymus: Slight hemorrhage was observed in 1 male of the control group.
Other gross lesion: Slight fracture of maxilla was observed in 1 male of the 2.5% group.

- Survivors:
The following changes were observed, but these were considered incidental, judging from the occurrence situation and pathological characteristics.
1) At the end of the administration period:
Heart: Slight focal myocarditis was observed in 2 males of the control group.
Stomach: Slight erosion in the glandular stomach was observed in 1 female of the 5.0% group (the animal showed dark red spot in the glandular stomach in the macroscopical observation).
Liver: Altered cell focus was observed in 1 male of the control group.
Prostate: Slight interstitial cellular infiltration was observed in 3 males of the 5.0% group.
Macroscopically abnormal sites: Focal dark red of peripheral region of cornea in eye ball (1 male of the 1.25% group) was histopathologically slight hemorrhage in posterior chamber. Enlargement of the thyroid (1 male of the 1.25% group) was histopathologically slight hypertrophy of follicular cells. Dark red spot in the glandular stomach (1 female of the 2.5% group) was histopathologically slight erosion in the glandular stomach.
2) At the end of the recovery period:
Heart: Slight focal myocarditis was observed in 1 male of the control group and in 1 male of the 5.0% group.
Liver: Slight focal vacuolation of hepatocytes was observed in 1 male of the control group and altered cell focus in 1 female of the 5.0% group.
Testis: Moderate atrophy of somniferous tubules was observed in 1 male of the control group.

OTHER FINDINGS
There was no abnormality in other organs and tissues examined such as brain, pituitary, spinal cord, sciatic nerve, thoracic aorta, trachea, esophagus, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands (submandibular gland and sublingual gland), pancreas, kidney, adrenal, spleen, mesenteric lymph node, cervical lymph node, urinary bladder, epididymis, seminal vesicle, ovary, uterus, vagina, mammary gland, skin, optic nerve, Harderian gland, bone and bone marrow (sternum and femur) and muscle of thigh.

Dose descriptor:

NOAEL

Effect level:

3 130.9 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: 5% dose level

Dose descriptor:

NOAEL

Effect level:

3 565.1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: 5% dose level

Critical effects observed:

not specified

Conclusions:

A 13-week oral toxicity study of L-Arginine with 5 week recovery study was conducted.
No dose-related death was observed throughout the administration and recovery periods. Furthermore, no dose-related changes were observed in the clinical signs, body weight, food consumption, food efficiency, ophthalmological examination, hematological examination, blood chemical examination, necropsy and histopathological examination.
Therefore, the no-observed-adverse-effect level (NOAEL) was set at a dietary dose of 5.0 % L-arginine (equivalent to an overall mean intake of 3130.9 (males) and 3565.1 (females) mg/kg body weight/day).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 130.9 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:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

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:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

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:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

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:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A 13-week oral toxicity guideline study of L-arginine with rats with 5 week recovery study was conducted. No dose-related death was observed throughout the administration and recovery periods. Furthermore, no dose-related changes were observed in the clinical signs, body weight, food consumption, food efficiency, ophthalmological examination, hematological examination, blood chemical examination, necropsy and histopathological examination. The only treatment-related effects noted were at the end of the administration period a slight decrease in the relative weight in the salivary glands in the females of the 5.0% group, and in the kidney in females of each treated group. But these changes were considered toxicologically meaningless.

Therefore, the no-observed-adverse-effect level (NOAEL) was set at a dietary dose of 5.0 % L-arginine (equivalent to an overall mean intake of 3130.9 (males) and 3565.1 (females) mg/kg body weight/day).

Another sub-chronic oral toxicity guideline study of L-arginine with rats was conducted for 90 days. The feeding of L-arginine was not associated with overt signs of toxicity. The NOAEL was estimated to be 5.0% for males (3.3 ± 0.1 g/kg/day) and 5.0% for females (3.9 ± 0.2 g/kg/day) and thus is in the same range as the NOAEL in the key study.

For purpose of risk estimation the lowest NOAEL 3130.9 mg/kg body weight/day (5% L-arginine) provided in the key study is considered as the key level for CSA.

The mean human daily intake of arginine for all life stage and gender groups from food and supplements is approximately 4.2 g/d (Food and Nutrition Board, 2005). A dietary dose of 5.0% L-arginine is equivalent to about 50 times amount of the mean daily oral intake for humans.

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-arginine does not cause toxicity when administered in sub-chronic application. Hence, L-arginine 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.).