L-Glutamine, L-alanyl-

Endpoint summary

Description of key information

Subacute oral toxicity (OECD 407), 28 days, rat (m, f): NOAEL systemic = 1000 mg/kg bw/day

Subchronic oral toxicity (OECD 408), 90 days, rat: NOAEL systemic = 3129 mg/kg bw/day (males) and 3601 mg/kg bw/day (females)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-06-03 to 1991-08-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1981

Deviations:

yes

Remarks:

only 2 doses tested

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1984

Deviations:

yes

Remarks:

only 2 doses tested

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Bor:WISW (SPFCpb)

Details on species / strain selection:

The test system was selected based on international recommendations. The rat is the preferred rodent species for toxicity testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann Versuchstierzucht GmbH & Co.KG, Borchen, Germany
- Age at study initiation: 7 weeks (males); 8 weeks (females)
- Weight at study initiation: 145 - 195 g (males); 135 - 166 g (females)
- Housing: individually in Macrolon cages, type II
- Diet: standart diet, ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 (for short periods up to 25 °C)
- Humidity (%): 40 - 70 (for short periods up to 80%)
- Photoperiod: 6 a.m. - 6 p.m. CET artificial lighting; 6 p.m. - 6 a.m. CET natural light-dark-rhythm

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The solutions for administration with different substance concentrations according to the intended doses were prepared daily.

VEHICLE
- Test substance concentration in vehicle: 46.4 mg/mL (low-dose group, 215 mg/kg bw/day), 215 mg/mL (high-dose group, 1000 mg/kg bw/day)
- Administration volume: 4.64 mL/kg bw
- Purity: The tap water quality is regularly monitored by Stadtwerke Bielefeld (municipal water provider)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance was determined in aqueous samples at the beginning and at the end of the study by HPLC.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

215 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main study:
Control group: 5
Low-dose group (215 mg/kg bw/day): 5
HIgh-dose group (1000 mg/kg bw/day): 5

Satellite groups:
Control group 5
HIgh-dose group (1000 mg/kg bw/day): 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected on the basis of a previous dose finding study
- Post-exposure recovery period in satellite groups: 6 weeks

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Occurence of toxicity symptoms: daily
Mortality: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Reflexes: once a week, starting with pretest period
Examination of eyes, hearing and teeth: prior to first substance administration and in test week 4

BODY WEIGHT: Yes
- Time schedule for examinations: once a week, starting with pretest period

FOOD CONSUMPTION: Yes
- Time schedule for examinations: once a week, starting with pretest period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: test week 4
- How many animals: all animals
- Anaesthetic: CO2
- Parameters checked: Erythrocytes (RBC), hematocrit (Hct), hemoglobin (Hb), leucocytes (VBC), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), thrombocytes (Platelets), and differential leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: test week 4
- How many animals: all animals
- Parameters checked: Alanine aminotransferase (ALAT), albumin, alkaline phosphatase (AKP), aspartate aminotransferase (ASAT), blood urea, calcium (Ca), chloride (Cl), cholinesterase (CHE), creatine kinase (CK), creatinine, gamma-glutamyltransferase (Gamma-GT), glucose, glutamate dehydrogenase (GLOB), inorganic phosphate (P), potassium (K), sodium (Na), total bilirubin (Tot.Bili), total cholesterol (Chol), total protein (Tot. Prot), and triglycerides (Triglyc)

URINALYSIS: Yes
- Time schedule for collection of urine: test week 4
- How many animals: all animals
- Parameters checked: Bilirubin, glucose, hemoglobin/erythrocytes, ketones, leucocytes, nitrite, osmolality, pH-value, protein, and urobilinogen. Microscopical examinations of the urine sediment were done in animals whose urine state showed pathological changes in hemoglobin/erythrocytes, leucocytes, and protein.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals were subjected to full gross necropsy, examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents.
- Organs/tissues preserved: All gross lesions, adrenal glands (r/1), bone marrow (as present in sternum), bone marrow smear (from femur), brain (cerebrum, cerebellum, brain stem), cecum, colon, duodenum, heart, ileum, jejunum, kidneys (r/1), liver, lungs (including main bronchi), ovaries (r/1), rectum, spleen, stomach, and testes (r/1).
- Organ weights: Adrenals (r/1), brain, heart, kidneys (r/1), liver, ovaries (r/1), spleen, and testes (r/1).

HISTOPATHOLOGY: Yes

Statistics:

For food consumption, body weights, and organ weights the DUNNETT-Test was used.
For values of hematological and clinical chemistry examinations the DUNNET-Test was used in case of normal distribution, otherwise the STEEL-Test was employed.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Alopecia in 1/5 control male and 2/5 control female animals (3 weeks after termination of treatment). Not considered to be related to the test substance.

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:

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:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute organ weights showed no significant differences between the control and treatment groups. In relative organ weights minimal alterations of statistical significance in the weight of kidneys (left, decrease in low dose males week 5, slight increase in high dose females week 5) and testes (left, high dose males week 11) were recorded, but these marginal variations are well within the normal range of the species and they are considered to be incidental findings.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Slight or marked alopecia in 2/5 animals and one rat each showed a reddish fluid in the urinary bladder, a yellowish nodule in the epididymis or a nodule in one horn of the uterus. All findings are considered as incidental changes known to occur in this species.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Only minimal changes in livers, kidneys, lungs, epididymis and stomach were observed. All findings were considered as spontaneous changes.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Critical effects observed:

no

Conclusions:

The administration of 215 and 1000 mg/kg bw/day test substance resulted in no observable effects. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) is considered to be 1000 mg/kg bw/day in the rat.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

limited information on results, materials & methods, and raw data

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Japan, Inc., Japan
- Age at study initiation: 5 weeks
- Weight at study initiation: 187 - 217 g (males); 143 - 170 g (females)
- Housing: individually in stainless steel cages
- Diet: powdered CRF-1, Oriental Yeast Co., Ltd., ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

634.5 mg/kg bw/day (nominal)

Remarks:

males, calculated from food intake, corresponding to 1% in diet

Dose / conc.:

1 860 mg/kg bw/day (nominal)

Remarks:

males, calculated from food intake, corresponding to 3% in diet

Dose / conc.:

3 129 mg/kg bw/day (nominal)

Remarks:

males, calculated from food intake, corresponding to 5% in diet

Dose / conc.:

726.5 mg/kg bw/day (nominal)

Remarks:

females, calculated from food intake, corresponding to 1% in diet

Dose / conc.:

2 161 mg/kg bw/day (nominal)

Remarks:

females, calculated from food intake, corresponding to 3% in diet

Dose / conc.:

3 601 mg/kg bw/day (nominal)

Remarks:

females, calculated from food intake, corresponding to 5% in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on 14 days dose-range finding study

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: On dosing days and on day of necropsy, following overnight fasting.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: On dosing days, following overnight fasting.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: Yes
- 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: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: week 13 (Days 88 – 89)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: and dose groups examined: All animals during the acclimation period and in 5 rats/sex/group in week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: not specified
- Animals fasted: not specified
- How many animals: all animals
- Parameters checked: RBC, Hb, Ht, MCV, MCH, MCHC, reticulocyte percentage, platelet count, WBC, differential WBC percentage, PT, APTT, and fibrinogen.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: not specified
- How many animals: all animals
- Parameters checked: AST, ALT, LDH, cGTP, AP, total cholesterol, triglycerides, phospholipids, total bilirubin, glucose, BUN, creatinine, Na, K, Cl, Ca, P, total protein, albumin, A/G ratio, and protein fractions.

URINALYSIS: Yes
- Time schedule for collection of urine: week 13 (Days 88 – 89)
- Metabolism cages used for collection of urine: No
- Animals fasted: Not specified
- Parameters checked: pH, protein, ketones, glucose, occult blood, bilirubin, urobilinogen, color, urinary sediment, volume, osmolality, Na, K, Cl, and water intake.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, opening of the cephalic, thoracic and abdominal cavities for observation of all major organs.
- Absolute and relative weights of the following organs recorded: adrenals, brain, pituitary, thyroid, thymus, spleen, heart, lung, salivary glands, liver, kidney, testis, prostate, seminal vesicle, ovary and uterus.

HISTOPATHOLOGY: Yes
- Organs and tissues investigated: adrenals, brain, pituitary, thyroid, thymus, spleen, heart, lung, salivary glands, liver, kidney, testis, prostate, seminal vesicle, ovary, uterus, spinal cord, sciatic nerve, eyes, optic nerves, harderian glands, parathyroids, mandibular lymph node, mesenteric lymph node, thoracic aorta, trachea, tongue, oesophagus, stomach, duodenum, ileum, jejunum, cecum, colon, rectum, submandibular and sublingual glands, pancreas, urinary bladder, epididymides, vagina, oviduct, mammary gland, sternum, femur, femoral skeletal muscle, skin, nasal cavity, and Zymbal’s glands.

Statistics:

Body weight, food consumption, feed efficiency, quantitative urinalysis data, organ weight data, and the results of the haematological and clinical chemistry evaluations were subject to calculation of the group mean with standard deviations. For the control group and the test substance groups, data were analysed for homogeneity of variance by Bartlett’s test (p = 1%, two-tailed). Homogenous data were then evaluated by Dunnett’s test (p = 1% and 5%, two-tailed) while heterogeneous data were analysed through the Dunnett-type mean rank test (p = 1% and 5%, two-tailed).

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:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

634.5 mg/kg bw/day (males): significantly higher value for AST
3129 mg/kg bw/day (males): increased total cholesterol concentration
Both observations were considered not to be of toxicological significance since the magnitude of change was small, did not show dose-response relationships, and did not occur in the females.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

534.5 mg/kg bw/day (males): reduced absolute thymus weight, increased absolute adrenal (paired)
3129 mg/kg bw/day (males): reduced absolute thymus weight
None of these organ weight differences were concluded to be biologically significant since they did not show a dose response, occurred only in one sex, were minor in nature, and did not correlate with any relevant histopathological changes.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

3129 mg/kg bw/day (males): one or more of seminiferous tubular atrophy, degeneration of the spermatogenic cells, vacuolisation of the Sertoli cells in the testis, with associated hypospermia, or intraductal cell debris in the epididymis of 3/10 animals.

These findings were of the same nature and severity of normally occurring spontaneous changes observed in this age and strain of rat. In all cases the findings were graded as either ‘‘minimal’’ or ‘‘mild’’. The testing laboratory historical control data range noted average incidence rates for seminiferous tubule atrophy of 3.54% (21/593) in 19-week-old rats with a range of 0 – 20%. Severity was concentrated in the minimal to mild range, however, a few of the historical controls were reported to show moderate (2/593) or severe atrophy (1/593) of the seminiferous tubules. Similarly, both the incidence and severity of ‘‘degeneration of spermatogenic cell’’ and ‘‘vacuolization of Sertoli cell’’ in the high-dose group were within that reported in the historical control data range for 19-week-old rats at the testing laboratory. In fact, the incidence of each of these observations was up to 60% and 40%, respectively. Also, the findings in the epididymis that follow from the testicular findings (i.e., they occurred in the same 3 rats) are likewise similar in incidence and severity to that found in the historical control data range for controls from the same laboratory. Finally, it has been noted in the literature that there is a significant incidence of testicular lesions, including testicular degeneration/atrophy, in young Sprague–Dawley rats (i.e., from 13-week studies), with overall mean control incidence rates of 2.5% and 9.4% in oral and inhalation studies, with corresponding ranges of 0.0 to 5.9% and 2.5 – 26.7%. Historical control ranges can vary significantly depending upon the source of the rats and on the perspective of the reviewing pathologist. Overall, there was no clear dose-response relationship that could be established between test item treatment and the testicular changes since none of these changes were observed in the mid-dose group (1860 mg/kg bw/day). In addition, the changes noted in the present study were at the upper end of the historical control range for the age and strain of rat used at the laboratory.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

3 129 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: overall effects

Remarks on result:

other: highest dose tested (5% in diet)

Dose descriptor:

NOAEL

Effect level:

3 601 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: overall effects

Remarks on result:

other: highest dose tested (5% in diet)

Critical effects observed:

no

Conclusions:

The NOAEL was considered to be 5% in the diet, the highest test concentration, corresponding to 3601 mg/kg bw/day in females and 3129 mg/kg bw/day in males.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable (Klimisch score 1 or 2) studies with the registered substance. The selected studies are thus sufficient to fulfil the standard information requirements set out in Annex VIII, Item 8.5, of Regulation (EC) No. 1907/2006 (REACH).

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

Two studies are available on oral repeated dose toxicity.

In the first study, the subacute toxicity was investigated in a 28-day toxicity study in rats according to OECD guideline 407 and compliant with GLP (92-0150-DNT). The test substance was dissolved in water and administered by oral gavage to male and female rats once daily for a period of 28 days. Two dose levels were selected based on the results of a range-finding study: 5 rats/sex were administered 215 mg/kg bw/day and 10 rats/sex received 1000 mg/kg bw/day or the corresponding vehicle. In addition, 5 rats/sex were included as satellite animals for the vehicle control and for the high-dose group. Satellite animals were treated in an identical manner and observed for a 6-week recovery period after termination of treatment. Signs of systemic toxicity and mortality were recorded daily, while individual body weights and food consumption were recorded at weekly intervals. After 4 weeks of treatment, blood was collected from all animals for haematological and clinical chemistry analysis, and urinalysis was performed. After 4 weeks of treatment (main groups) or after a recovery period of 6 weeks (satellite groups) all animals were subjected to macroscopical examination, weighing of selected organs and histopathological examination.

There were no deaths, and no clinical signs of toxicity which were attributed to treatment were observed. Incidences of alopecia were noted in animals of the control group only. The Body weight gain and food consumption were unaffected by treatment. In addition, there were no test substance-related changes in the hematology, clinical chemistry- and urinary parameters.

Gross necropsy results did not show abnormal findings that could be attributed to treatment. Findings noted in single animals only, in the control or a treatment group comprised: alopecia, reddish fluid in the urinary bladder, yellowish nodules in epididymides or nodules in the horn of the uterus. The observations were considered to be incidental and not related to treatment. The organ weight analysis showed statistically significant alterations such as a decreased relative kidney weight in males and a slightly increased relative kidney weight in females of the high-dose group and a slight increase in testes weight in males of the high dose group. The observations were marginal and within the normal range of variation observed in rats of the strain and age and therefore considered to be spontaneous and not attributed to treatment.

Based on the experimental results the NOAEL for systemic toxicity was 1000 mg/kg bw/day in male and female rats.

The oral repeated dose toxicity assessed in subchronic toxicity that was published (Oda_et_al_2008). The study was performed similar to OECD guideline 408 and in compliance with GLP. The test item was incorporated into the diet and administered via the feed for a period of 90 days. Groups of 10 male and 10 female Sprague-Dawley rats received test item doses of 1, 3 or 5% (corresponding to 634.5, 1860 and 3129 mg/kg bw/day for males and 726.5, 2161 and 3601 mg/kg bw/day for females, respectively) and a control group received the plain diet.

There was no mortality and no clinical signs of toxicity were observed during the study period. The body weight gain, food consumption and food efficiency were unaffected by treatment. The results of the ophthalmologic examination were similar between the treatment groups and control. The analysis of hematology-, clinical chemistry- and urinary parameters revealed no treatment-related findings.

The macroscopic examinations revealed no treatment-related changes in either sex or treatment group. A few sporadic statistically significant differences between treated and control groups were identified for both absolute and relative organ weights. The absolute weight of the thymus was reduced in males of the 634.5 and 3129 mg/kg bw/day dose groups. An increased absolute weight of the adrenal (paired) was reported in the low-dose males. Changes in relative organ weights included decreased thymus weight in the males of the low- and high-dose groups and increased adrenal weight in the low-dose males. None of these organ weight differences were concluded to be biologically significant since they did not show a dose-response, occurred only in one sex, were minor in nature, and did not correlate with any relevant histopathological changes.

Histopathological examinations revealed no lesions attributable to treatment with the test substance. The lesions identified were as expected for this age and strain of rat and occurred with essentially the same incidence between the treated and control groups. The only exception was the finding of one or more of seminiferous tubular atrophy, degeneration of the spermatogenic cells, vacuolization of the Sertoli cells in the testis, with associated hypospermia, or intraductal cell debris in the epididymis of 3/10 males of the high-dose group. These findings were of the same nature and severity of normally occurring spontaneous changes observed in this age and strain of rat. In all cases the findings were graded as either ‘minimal’ or ‘mild’.

Based on the experimental results the NOAEL for systemic toxicity was  5% in the diet, corresponding to 3129 mg/kg bw/day in male and 3601 mg/kg bw/day in female rats.

Justification for classification or non-classification

The available data on repeated dose toxicity following oral administration do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.

No classification for repeated dose toxicity is warranted according to the criteria of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.