Glutamic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

May 18, 2006 - August 21, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Based on structural similarity, common metabolic pathway, environmental fate, comparable physico-chemical and (eco)toxicological properties, it is considered justified to use this study also for this substance. See the attached background document for detailed information. This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: US Food and Drug Administration Redbook 2000: Toxicological Principles for the Safety Assessment of Food Ingredients (IV.C.4b. Subchronic Toxicity Studies with Non-Rodents, November 2003)

Deviations:

not specified

Remarks:

This guideline was not available during evaluation.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

no

Principles of method if other than guideline:

The report stated that the study was performed according to US Food and Drug Administration Redbook 2000: Toxicological Principles for the Safety Assessment of Food Ingredients (IV.C.4b. Subchronic Toxicity Studies with Non-Rodents, November 2003). The study conduct also complied with OECD 409.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium hydrogen glutamate

EC Number:

205-538-1

EC Name:

Sodium hydrogen glutamate

Cas Number:

142-47-2

Molecular formula:

C5H8NNaO4

IUPAC Name:

Sodium 2-amino-4-carboxybutanoate

Constituent 2

Reference substance name:

6106-04-3

Cas Number:

6106-04-3

IUPAC Name:

6106-04-3

Details on test material:

- Name of test material (as cited in study report): Monosodium L-Glutamate Monohydrate Produced by a New Method
- Substance type: hydrated form.
- Physical state: Solid, white crystal
- Stability under test conditions: stable. The analysis of the test substance after the experiment phase showed that the test substance content was 100.2%.
- Storage condition of test material: Room temperature, shading from light, well-closed

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products Inc. through LSG Corporation
- Age at study initiation: Approximately 6 months.
- Weight at study initiation: males 7.9-9.0 kg, females 6.9-8.9 kg
- Fasting period before study: not applicable
- Housing: Individually in aluminium cages
- Diet (e.g. ad libitum): approximately 250 g per day
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 26 days before the start of treatment
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.0 – 24.9
- Humidity (%): 51 - 97%
- Air changes (per hr): 8 or more
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: capsule

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

METHOD OF ADMINISTRATION:
The test substance was constituted into the required dosage units by encapsulation into gelatin capsules (2 capsules per dose administration) and administered to animals daily. Test substance amounts were based on the most recent body weight, but for days 1 to 7 of dosing, they were based on the body weight at the time of group assignment.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

The administration period was from 90 to 92 days.

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1500 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
500 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

4

Control animals:

other: yes, two empty gelatin capsules per dose administration

Details on study design:

A concentration of 5% (w/w) was considered an acceptable nutrition-based endpoint for high dose selection for dietary mixture studies. Therefore, the dose of 1500 mg/kg/day, roughly equivalent to 5% (w/w) which was predicted not to produce any nutritional disturbance, was selected to be the highest dose for this study. The mid- and low-dose were selected using a common spacing factor of about 3.

Positive control:

No positive control.

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day (a.m. and p.m.) form day -7 of dosing until necropsy

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly form day -7 of dosing until necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: before daily feeding on days -7, -1, 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 and 91 of dosing

FOOD CONSUMPTION: Daily
- The mean daily food consumption (g/day) were calculated by computer.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on days -2, 14, 43 and 90 of dosing
- Animals fasted: Yes, approximately 18-hours
- How many animals: all animals
- Parameters examined: White blood cell count, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cell count), Red blood cells count, Reticulocyte ratio, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelet count, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on days -2, 14, 43 and 90 of dosing
- Animals fasted: Yes, approximately 18-hours
- How many animals: all animals
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Total Bilirubin, Creatinine, Glucose, Cholesterol, Blood urea nitrogen, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphorus, glutamyl transpeptidase, total bile acid, cholinesterase, triglyceride

BONE MARROW EXAMINATION:
- Time schedule: at the time of necropsy
- How many animals: all animals
- Bone marrow examination was however not conducted because no abnormal conditions such as anemia were detected hematologically

SERUM PROTEIN ELECTROPHORESIS: YES
- Time schedule for collection of blood: on days -2, 14, 43 and 90 of dosing
- Animals fasted: Yes, approximately 18-hours
- How many animals: all animals
- The concentration (g/dL) of each fraction (albumin, alpha1, alpha2, beta, gamma) was calculated based on the ratio of each fraction and the total protein concentration determined in the examination of blood chemistry

URINALYSIS: Yes
- Time schedule for collection of urine samples: fresh (within 3 hours after excretion) and 24-hour urine samples on days -7 and -6, 29 and 30, 57 and 58, and 85 and 86 of dosing
- How many animals: all surviving animals
- Parameters examined: pH, occult blood, ketone bodies, glucose, protein, bilirubin, urobilinogen

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule examination: conducted on all animals (both eyes) on days -6, 41 and 86 of dosing
- Parameters examined: overall appearance and corneal light reflex, pupil dilatation, anterior part of the eyeballs, optic media, fundus oculi

Sacrifice and pathology:

ORGAN WEIGHTS:
- The following organs from animals intented for the scheduled necropsy were weighed:
Heart, liver (including gallbladder), spleen, kidneys (right and left), adrenal glands (right and left), prostate, testes (right and left), ovaries (right and left), brain (excluding olfactory bulb), pituitary gland, lungs, pancreas, mandibular glands (right and left, including sublingual gland), thymus, thyroid glands (right and left including parathyroid glands), glands), uterus and epididymides (right and left).

GROSS POST-MORTEM EXAMINATION: Yes
- The external surface and orifices were observed, and the organs and tissues in the abdominal, thoracic, pelvic, and cranial cavities were examined during necropsy.

HISTOPATHOLOGY: Yes
- Histopathological specimen preparation of the organs and tissues from all animals was conducted

Statistics:

- The following data were analyzed statistically by Dunnett's test for the significance of difference of mean values between the control group and each dosage group: body weight, food consumption, measurements of hematology, blood coagulation, blood chemistry, serum protein electrophoresis, urinalysis (volume, specific gravity), organ weight and organ weight to body weight ratio.
- Significant differences were analyzed at the 5 and 1% levels of significance with a two-sided analysis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS:
- Clinical signs in the control group were limited to vomiting of yellowish-white foamy substances in 2 males for 1 or 2 days, loose stools in 1 male and 2 females for 1 or 3 days, and mucous stools in 1 female for 1 day.
- Clinical signs in the low-dose group included vomiting of compounds containing the test substance in 1 male and 1 female for 1 day each, vomiting of yellowish-white foamy substances in 1 male and 1 female for 2 or 3 days, and loose stools in 3 males for 1 to 8 days.
- Clinical signs in the intermediate-dose group included vomiting of compounds containing the test substance in 2 males and 1 female for 1 or 2
days, vomiting of yellowish-white foamy substances in 3 males and 4 females for 1 or 2 days, vomiting of undigested food in 4 males and 4 females
for 1 to 9 days, and loose stools in 1 male and 2 females for 1 to 5 days.
- Clinical signs in the high-dose group consisted of vomiting of compounds containing the test substance in 4 males and 4 females for 17 to 43
days, vomiting of yellowish-white foamy substances in 4 males and 4 females for 31 to 58 days, vomiting of undigested food in 4 males and 4 females
for 9 to 29 days, salivation in 4 males and 4 females for 1 to 74 days, loose stools in 3 males and 4 females for 1 to 9 days, liquid stools in 2 males
and 2 females for 1 day each, and mucous stools in 1 female for 1 day. Vomiting tended to occur in the afternoons, salivation at the time of dosing.
- In addition, edema, purulence, crust, and loss of hair on the mandible or buccae were observed in 1 female from week 8 to week 12 of dosing.
- The detailed clinical observations revealed no abnormal signs.

FOOD CONSUMPTION:
- Significant increases in food consumption were seen in the low-dose females on day 1 of dosing, low- and intermediate-dose males on day 27 or 43 of dosing, and intermediate-dose males on day 58 of dosing, while in the high-dose animals food consumption was unaffected by treatment. Based
on the lack of dose dependency, the increased food consumption in the low- and intermediate-dose groups was not considered to be toxicologically
significant. Although significant decreases in food consumption were seen in the high-dose males on day 31 of dosing and inversely significant
increases in the high-dose females on day 43 of dosing, these changes were isolated and were therefore considered to be of no toxicological
significance.

HAEMATOLOGY:
- [Day 14 of dosing]: The mean corpuscular hemoglobin concentration in the low-dose females was significantly decreased as compared with the
control group. Since no reduction in mean corpuscular hemoglobin concentration was observed in the high-dose group, the change in the low-dose
females was not considered to be toxicologically significant.
- [Day 43 of dosing]: A reduction in the mean corpuscular hemoglobin was seen in the high-dose males. This change was significant as compared
with the control group but small (6.5% reduction) and transient, and therefore not considered to be toxicologically significant.
Significant changes also included elevation of white blood cell counts, neutrophil counts, and monocyte counts in the high-dose females. These changes were, however, not considered to be toxicologically significant because they were transient and associated with the values of one animal which showed clinical signs including edema, purulence, crust, and loss of hair on the mandible or buccae.
- [Day 90 of dosing]: The eosinophil ratio and count in the low-dose males were significantly elevated as compared with the control group. Since no such elevation was observed in the high-dose group, the changes in the low -dose males were not considered to be toxicologically significant.

CLINICAL CHEMISTRY:
- [Day 14 of dosing]: Significant increases in total protein concentration as compared with the control group were noted in the intermediate- and
high-dose females. These changes were not concluded to be toxicologically significant because they were small (6.4% increase at both the mid- and high-dose) and transient.
- [Day 43 of dosing]: The total cholesterol concentrations in the high-dose females and the total bile acid concentrations in the low-dose females were significantly elevated as compared with the control group. These changes were not concluded to be toxicologically significant because they were transient and (in case of total bile acid) a dose-response relationship was absent.
- [Day 90 of dosing]: No statistically significant differences were seen in any of the clinical chemistry parameters examined between the control group and any of the low-, intermediate-, or high-dose groups.

URINALYSIS:
- [Days 29 and 30 of dosing]: Significant increases in total sodium excretion as compared with the control group were noted in the intermediate- and high-dose females.Significant changes also included decreases of potassium and chloride concentrations in the high-dose males; these decreases in concentration were not considered to be toxicologically significant because no concomitant changes in total excretion of potassium and chloride were seen.
- [Days 57 and 58 of dosing]: Significant increases in total sodium excretion as compared with the control group were noted in the high-dose males
and females. Significant changes also included decreases of potassium concentrations in the intermediate-dose females and chloride decreases in the high-dose males and intermediate-dose females; these decreases in concentration were not considered to be toxicologically significant because no concomitant changes in total excretion of potassium and chloride were seen.
- [Days 85 and 86 of dosing]: The sodium concentrations for the high-dose males and females were significantly elevated as compared with the
control group, accompanied by significant increases in total excretion for the high-dose males and females as well as the intermediate-dose females. Significant changes also included decreases of chloride concentrations in the intermediate- and high-dose females (reductions of 42-52%, statistically significant at the mid-dose only) and urinary volume increases in the intermediate-dose females. The decrease of the chloride concentration was not considered to be toxicologically significant because no concomitant change in total excretion of chloride was seen. Likewise, the volume increase was not considered to be toxicologically significant because no such trend was observed in the high-dose group.

OPHTHALMOLOGY:
- All of the observed findings were spontaneous and there was no evidence of a treatment related effect.

ORGAN WEIGHTS:
- Significant increases in both the absolute weight (+100%) and relative weight (+100%) of the thymus, as compared with the control group, were noted in the high-dose females.
- Significant increases in absolute adrenal weight were also observed in the low-dose males, but not considered to be toxicologically significant
because they were unilateral and a dose-relationship was absent.

GROSS PATHOLOGY:
- All findings observed in all groups including the control group were considered to be spontaneous lesions, because these findings are frequently observed in age-matched, non-treated beagle dogs.

HISTOPATHOLOGY:
- All findings observed in all groups were considered to be spontaneous lesions, because these findings are frequently observed in age-matched, non-treated beagle dogs.

SERUM PROTEIN ELECTROPHORESIS:
- [Day 14 of dosing]: The gamma globulin concentrations in the intermediate-dose females were significantly elevated as compared with the control
group. Since no elevation of gamma globulin concentration was observed in the high-dose group, the change in the intermediate-dose females was
not considered to be toxicologically significant.
- [Day 43 of dosing]: Alpha2 globulin ratios and alpha2 globulin concentrations were significantly elevated in mid- and high-dose females (+14% to +33%, dose-related, statistically significant except for alpha2 globulin ratios mid-dose females). Gamma globulin ratios and gamma globulin concentrations were also significantly elevated in mid- and high-dose females (+26% to +34%, not dose-related, statistically significant). The increased alpha2 globulin and gamma globulin levels were accompanied by significantly reduced albumin ratios (-13%) in high-dose females and significantly reduced A/G ratios (-16% and -24%, statistically significant at high-dose only) in mid- and high-dose females. The change in these parameters however was transient (observed on day 43 only, not on day 14 and 90), and not accompanied by any finding during the study indicative of inflammatory reactions, which may be associated with increased Alpha2 globulin and gamma-globulin levels. These changes are therefore concluded not to be toxicologically significant.
- [Day 90 of dosing]: No statistically significant differences were seen in any of the serum protein electrophoresis parameters examinded between the control group and any of the low-, intermediate-, or high-dose groups.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 1 500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No adverse effects of oral administration of monosodium L-glutamate monohydrate produced by a new method were seen in any of the observations, measurements or examinations in this study.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The results obtained with the substance Monosodium glutamate are suitable for the regulatory purpose of REACH for the substance L-Glutamic acid:
In a study according to OECD 409, no adverse effects of oral administration of 150, 500 or 1500 mg/kg/day monosodium L-glutamate monohydrate produced by a new method to male and female beagle dogs for 90 days were seen in any of the observations, measurements, or examinations in this study. In conclusion, the no observed adverse effect level was 1500 mg/kg/day or more for both sexes under the conditions of this study.