Sodium hydrogen glutamate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 26 - November 30, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study has been performed according to OECD guidelines and to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Guidelines for Designation of Food Additives and for Revision of Standards for Use of Food Additives" (Japanese Ministry of Health and Welfare, Eika No. 29, March 22, 1996)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guideline for Genotoxicity Tests on Drugs" (Pharmaceutical and Medical Safety Bureau, Japanese Ministry of Health and Welfare, Notification Iyakushin No. 1604, November 1, 1999)

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

US Food and Drug Administration Redbook 2000: Toxicological Principles for the Safety of Food Ingredients IV.C.l.d. Mammalian Erythrocyte Micronucleus Test"(July 7, 2000). The study conduct also complied with OECD 474.

GLP compliance:

yes

Type of assay:

micronucleus assay

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 hydrogen 2-aminopentanedioate

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: white powder
- Stability under test conditions: stable
- Storage condition of test material: At room temperature under shading from light and well-closed conditions

Test animals

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 9 weeks old
- Weight at study initiation: 28.9-33.7 g
- Housing: Individually housed in TPX synthetic resin cages containing paper clean as bedding material

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22.5-24.5°C
- Humidity (%): 46.0-71.0%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Water for injection JP

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
-The test article was weighed and dissolved in the vehicle to prepare the dosing formulation of the highest concentration (200 mg/mL) and then
serially diluted with the vehicle to desired concentrations.
-Concentrations of the dosing formulations prepared were as follows.
For the preliminary toxicity test: 50, 100, 150 and 200 mg/ml, for the micronucleus test: 50, 100 and 200 mg/ml,
-The dosing formulations in the preliminary toxicity test were prepared just before use. Those in the micronucleus test were stored in tight
containers under cooling (actual range: 3 to 5°C) and shading conditions, and used within 3 days after preparation.

Duration of treatment / exposure:

- In the preliminary toxicity test, the test article was orally administered once daily for 3 days at an interval of 24 hours.
- In the micronucleus test, the negative control substance and the test article were orally administered once daily for 3 days at an interval of 24 hours

Frequency of treatment:

3 days at an interval of 24 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
2000 mg/kg/day
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
500 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Route of administration: oral (once)
- Doses / concentrations: concentration 5 mg/ml, doses 50 mg/kg

Examinations

Tissues and cell types examined:

Frequency of micronucleated polychromatic erythrocytes (MNPCEs).
Proportion of polychromatic erythrocytes in the total erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In the preliminary toxicity test, the MTD was estimated as 2000 mg/kg/day or more in both sexes. As a remarkable difference in toxicity was not
observed between sexes, only male mice were used for the micronucleus test and the highest dose was fixed at 2000 mg/kg/day.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The negative control substance and dosing formulations of the test article were orally administered once daily for 3 days at an interval of 24 hours.
That of the positive control substance was orally administered once. The specimens of bone marrow cells for microscopic observation were prepared about 24 hours after the last administration.

DETAILS OF SLIDE PREPARATION:
Mice were euthanized by cervical dislocation and then femora of both sides were removed from the mice. Both edges of the femur were cut off, and
bone marrow cells were flushed out with about 0.6 mL of fetal bovine serum into a centrifuge tube followed by centrifugation at about 200 x g for
5 minutes to remove the supernatant, Following pipetting, the bone marrow cells were smeared on clean glass slides which were marked with study
number, animal number and slide number. Three slides per mouse were prepared. The slides were dried at room temperature and then fixed with
methanol for 5 minutes. All slides were coded by sticking labels with study number, code number and slide number according to the code number
table and stored at room temperature until observation.
Each specimen was stained with a few drops of 40 ug/ml, Acridine Orange solution in 1/15 mol/L Sorensen phosphate buffer just before observation,covered with a cover glass.

METHOD OF ANALYSIS:
The slides were observed by fluorescence microscopy set up for blue excitation with absorption filter (510 nm) using a 100-fold objective lens and a
10-fold eyepiece lens. Two thousand polychromatic erythrocytes (PCEs) per mouse (1000 PCEs per person) were observed by two persons and the
number of micronucleated polychromatic erythrocytes (MNPCEs) was recorded. One thousand erythrocytes (ERYs) per mouse (500 ERYs per person) were observed by two persons and the number of PCEs was recorded.

Evaluation criteria:

The effect of the test article on proliferation of bone marrow cells was assessed from the results of statistical analysis, dose-dependency and the
historical data of the negative control groups.

Statistics:

A) Frequency of MNPCEs
It was ascertained whether the mean frequencies of MNPCEs in the negative and positive control groups were included in the fluctuated range of their historical control (Mean ± 3 S.D.).
The frequency of MNPCEs in the negative control and dosing groups or positive control group was statistically analyzed by Fisher's exact probability
test (one-tailed) at 0.05 and 0.01 levels with a Bonferroni correction considering the multiplicity of the data. Furthermore, the dose (in logarithms)
dependency of the frequencies of MNPCEs was analyzed by the trend test of Cochran-Armitage (one-tailed) at 0.05 and 0.01 levels.
B) Proportion ofPCEs in ERYs
For the proportion of PCEs in the total ERYs, the homogeneity of variances in each group except for the positive control group was analyzed by
Bartlett's test at 0.05 level. As the variances were homogeneous, the difference in the mean between the negative control and the dosing groups was
analyzed by Dunnett's test (two-tailed) at 0.05 and 0.01 levels. The homogeneity of variances in the negative and positive control groups was
analyzed by F-test at 0.05 level. As the variances were homogeneous, the difference in the mean between the negative and positive control groups
was analyzed by Student's t-test (two-tailed) at 0.05 and 0.01 levels.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: test was carried out using male and female mice at doses of 500, 1000, 1500 and 2000 mg/kg/day. The test article was orally
administered once daily for 3 days at an interval of 24 hours.
- Solubility: 740 mg/mL, 25°C
- Clinical signs of toxicity in test animals: neither change in general condition nor death was observed in male and female mice.
- Rationale for exposure: Oral route was chosen because the test article is intended for oral administration in clinical use.
- Harvest times: 24 hours after the last administration

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): the frequency of micronucleated polychromatic erythrocytes (MNPCEs) did not increase
significantly in any test article group as compared with that in the negative control group, while a significant increase of the frequency of MNPCEs wasobserved in the positive control group.
- Ratio of PCE/NCE (for Micronucleus assay): The proportion of polychromatic erythrocytes in the total erythrocytes in any test article group and the positive control group was not significantly different from that of the negative control group.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that monosodium L-glutamate monohydrate produced by a new method has no clastogenic activity in bone marrow cells of mice
(negative) under the conditions of this study.