Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
other: SIDS
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
SIDS for L-Glutamic acid
Author:
OECD
Year:
2013
Report Date:
2013
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Specific details on test material used for the study:
Test material equivalent to submission substance identity:
yes

Test animals

Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Samtako Co., LTD
- Age at study initiation: 8 week old
- Weight at study initiation: 33.2 - 36.4 g
- Assigned to test groups randomly: yes, Random method according to weight range
- Housing: 5 per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days include receipt day
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 3°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 3°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs of lighting duration (lighting up at 8 am-lighting out at 8 pm) and 150-300 Lux of luminous intensity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
(1) Identity: 1% CMC․Na
(2) Lot No.: 120M0216V
(3) Date of receipt : Jan. 25, 2011
(4) Amount : 500 g
(5) Appearance : beige powder
(6) Purity : 99.5%
(7) Storage Condition : Stored at room temperature
(8) Supplier : Sigma-Aldrich
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The volume of dosing was 10 mL/kg per body weight that measured before administration, animals were fixed by holding cervix backside skin method and test substance was via gavage using oral zonde.
Duration of treatment / exposure:
3 d
Frequency of treatment:
once a day
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
(1) Identity : Mitomycin C (MMC)
(2) Lot No. : SLBB7481V
(3) Date of receipt : Jul. 18, 2012
(4) Amount : 2 mg
(5) Appearance : Blue powder
(6) Storage Condition : 2-8 °C
(7) Supplier : Sigma-Aldrich

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary study was performed to select a dose producing signs of toxicity such that higher dose levels would be expected to produce lethality. According to MSDS, the LD50 for test substance is about 30,000 mg/kg in rat, indicating relatively low toxicity. On the basis of OECD TG 474, a limit test as the preliminary study was carried out at one dose level of 2000 mg/kg. The limit test produces no mortality and clinical signs. Also, there was no substantial difference between male and female mice in toxicity. Thus the main test using male mice was conducted in 3 treatment groups, which consisted of high dose (2,000 mg/kg) and 2 additional lower doses divided by factor 2. Each group was classified as below.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- Treatment: 3 times
- Collecting bone marrow cells was carried out at the time, 24 hrs after last administration of test substance.

DETAILS OF SLIDE PREPARATION:
To obtain bone marrow cells, femur extracted from animals were washed and suspended with Fetal bovine serum (FBS) using 23 gauge needle and then centrifuged at 1000 rpm for 5 min. After removing supernatant, precipitated cells were smeared on slide glass, fully dried at room temperature, immerged in methanol for 5 min to fix. Specimens completely fixed and dried were stained with acridine orange (40 ug/mL) and then examined by 1000 x magnification.

METHOD OF ANALYSIS: In the total count of PCE and normochromatic erythrocytes (NCE), approximately 200, the ratio of PCE, as PCE/(PCE+NCE), was calculated. To obtained frequency of micronucleus, the number of MNPCE which were occurred in about 2000 PCE per animal was counted and expressed as [MNPCE/PCE].
Evaluation criteria:
It was judged to be positive when MNPCE was significantly increased in dose-dependent manner or there was a significant increase in the numberof MNPCE at more than one test dose level.
Statistics:
The statistical analysis was performed by SPSS 19.0k program. The statistical significance was tested for the induction of frequency of micronucleus. One-way ANOVA was conducted for the appearance rate of PCE within 5% significance of p-value. Also multiple comparison (Dunnett T test or Duncan test) was tested. ANOVA test was also carried out for a body weight changes within 5% significance of p-value.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Additional information on results:
1) Clinical signThere was no dead animal in all test groups until autopsy, and there was no abnormality by administration of test substance.
2) Body weightThere was no statistically significant change in body weight of each group.
3) Frequency of micronucleus and cytotoxicity (Table 1)
- The average values of PCE/(PCE+NCE) were 37.6±1.67% for negative control, 35.4±0.96%, 35.3±1.30 and 36.8±2.51% for 500, 1,000 and 2,000 mg/kg, respectively. The average rates for all treatment groups were not significantly decreased when compared to that for negative control. In addition, the proportion of immature erythrocytes was not less than 20% of the negative control value recommended by OECD TG 474.
- The mean frequency of MNPCE observed in about 2000 PCE per animal was 0.17±0.057% for negative control, 0.18±0.027%, 0.17±0.027% and 0.20±0.035% for 500, 1,000 and 2,000 mg/kg, respectively. There was no significant difference in an appearance rate of MNPCE in PCE for all treatment groups in contrast with that of a negative control.
- In positive control group, the frequency of MNPCE in PCE was increased significantly in contrast with that of the negative control group, demonstrating the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:
L-Glutamic acid does not have a mutagenic potential in inducing micronucleus formation in the bone marrow cells in mice.