Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Specific details on test material used for the study:
- Name of test material: L-Glutamic acid
- Molecular weight: 147.13
- Physical state: White powder
- Lot No.: BCBF3951V
- Storage condition of test material: Room temperature
- Supplier: Sigma-Aldrich

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
[TEST ANIMALS]
- Source: ORIENTBIO INC., Korea
- Sex, number, age and body weight range at receipt: Male, 56 rats, 7 weeks old, 191.3~210.5 g. Female, 56 rats, 7 weeks old, 124.1~180.4 g
- Sex, number, age and body weight range at the start of administration: Male, 56 rats, 8 weeks old, 249.6-294.0 g. Female, 56 rats, 8 weeks old, 158.3~214.1 g
- Housing:
* Quarantine and acclimatization: The five animals per cage were housed in stainless steel cages (270W x 500D x 200H (mm)).
* Administration: The two animals per cage were housed in stainless steel cages (270W x 500D x 200H (mm)).
* Mating period: One female to one male were housed in stainless steel cages (270W x 500D x 200H (mm)).
* Gestation and lactation period: Maternal rats and fetuses were housed individually in polycarbonate cage (250W x 400D x 200H (mm)).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 7 days

[ENVIRONMENTAL CONDITIONS]
- Temperature (°C): 20.7-22.7 degree C
- Relative humidity (%): 45.7-53.2%
- Air changes (per hr): 10-15 clean, fresh, filtered air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle, 8AM to 8PM via automated timer at 150~300 Lux

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.1% Tween 80 in 1% CMC-Na
Details on exposure:
[PREPARATION OF DOSING SOLUTION]
Test substance was measured according to the dose levels and dissolved in 0.1% Tween 80 in 1% CMC-Na. The formulations of 25, 50 and 100 mg/mL were prepared daily. The test article suspension was freshly prepared right before the administration.
[DIET PREPARATION]
Certified rodent diet [r-ray sterilized EP pellet, Cargill Agri Purina, Inc., Lot No : KSN120504, KSN120710] was given ad libitum.
[VEHLCLE]
- Name: 0.1% Tween 80 in 1% CMC-Na
- Supplier: Sigma-Aldrich (Tween 80), Sigma (CMC-Na)
- Lot No.: MKBG3475V (Tween 80), SLBB5612V (CMC-Na), 89L4K21, 07L4K21, C1L4K21, 26L6K21, M3L3K21 (distilled water)
Details on mating procedure:
Normally, 1:1 (one male to one female) mating were used in this study. Every morning the females were examined for the presence of sperm was detected. Females which did not delivery were sacrificed at 26 days following mating.
Duration of treatment / exposure:
- Male: Two weeks before mating to the end of the mating period, for at least 28 or more days
- Female: Two weeks before mating to day 3 of lactation including the mating and gestation period
Frequency of treatment:
once a day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- G1: 0 mg/kg/day, male 14 rats, female 14 rats
- G2: 250 mg/kg/day, male 14 rats, female 14 rats
- G3: 500 mg/kg/day, male 14 rats, female 14 rats
- G4: 1000 mg/kg/day, male 14 rats, female 14 rats
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
[Clinical sign]
All animals were monitored once a day during the experimental period. Clinical signs including mortality, moribundity, motility, general appearance and autonomic activity were recorded with the date and duration.
[Measurement of body weights]
Individual body weights of males were measured once a week during the study. In females, body weights were measured once a week during pre-mating period. during pregnancy and lactation period, body weights were measured as follows;
- Pregnant animals: Day 0, 3, 6, 9, 12, 15, 18, and 20 of gestation
- Lactation animals: Day 0 and 4 post-partum

[Food consumption]
During pre-mating, pregnancy and lactation period, rats were supplied with a certain amount of diet on days of body weight measurement and food consumption were recorded for the animals on the following days. Food consumption during mating period was not measured. In addition, on day 4 of lactation a diet was supplied on day 3 of lactation and food consumption was recorded on the following day.

[Mating evaluation]
- Mating index (%)=(No. of animals with successful copulation/No. of mated animals)x100
- Fertility index (%)=(No. of impregnating animals/No. of animals with successful copulation)x100
- Pregnancy index (%)=(No. of pregnant animals/No. of animals with evidence of mating)x100

[Observation during the lactation period]
Nursing behaviors of dams were observed.
Litter observations:
[Observation on parturition date]
At the parturition date, gestation length, live and dead pups, sex and external anomalies of live pups were observed and recorded.
- Delivery index: (No. of dams with live newborns/No. of pregnant dams)x100
- Sex ration: No. of live male pups/No. of live female pups
- Viability ratio at day 4 post-partum: (No. of live pups on day 4 post-partum/No. of live neonates at birth)x100
- Body weights of live pups on day 0 and day 4 post-partum
[Observation during the lactation period]
Viability of pups were observed.
Postmortem examinations (parental animals):
[Necropsy]
At scheduled termination, all live males and females were necropsied after repeated dosing for at least 28 or more days and at day 3 of lactation, respectively. Animals were anesthetized with isoflurane and then terminated by exsanguinating the abdominal examination and identification of all abnormal findings.
- External surface and all orifices
- Cranial cavity
- External surface of brain and spinal cord
- Nasal cavity and paranasal sinus
- Thoracic, abdominal and pelvic cavities and their viscera
- Cervical tissues and organs
At necropsy, the numbers of corpora lutea and implantation sites were counted in all female animals. The following data were calculated.
- Pre-implantation loss=((No. of corpora lutea-No. of implantation)/No. of corpora lutea)x100
- Post-implantation loss=((No. of implantation-No. of live fetuses)/No. of implantation)x100

[Organ weights]
Absolute organ weights were measured and their relative organ weights (organ-to body weight ratios) were calculated from the terminal body weight for the following organs of all live animals when they sacrificed.
- Liver
- Kidneys L/R
- Spleen
- Adrenal gland L/R
- Brain- Pituitary gland
- Lung
- Heart
- Testes
- Epididymides
- Seminal vesicles
- Prostate gland
- Ovaries
- Uterus
- Vagina
- Cervix
[Histopathology examination]
Gross finding organ and testes, epididymides, seminal vesicles, prostate gland, ovaries, uterus, vagina, cervix were embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and examined microscopically in the vehicle control and 1000 mg/kg bw/day groups.
Postmortem examinations (offspring):
[Necropsy]
All live pups were sacrificed while anesthetized with Ether and all pups including dead offspring were necropsied with special attention to all vital organs.
Statistics:
The data will be analyzed for homogeneity of variance using Levene's test, and the data were subjected to one-way analysis of variance (ANOVA). If these tests showed statistical significance, the data were analyzed by the multiple comparison procedure of Dunnett's T3 or Scheffe. The difference was considered statistically significant when p<0.05 or 0.01. Statistical analyses were performed with the SPSS Statistical Analysis Systems (SPSS ver. 19, SPSS Korea Data Solution Co., Ltd.).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical sign was observed in any groups. Cannibalism was observed in 1, 1 and 1 female of the 250, 500 and 1000 mg/kg bw/day groups, respectively.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant changes in body weights of the treatment group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No change in food consumption was observed in the treatment group.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in reproductive organs. At histopathological examination of males, interstitial inflammatory cell infiltration of prostate in vehicle control and 1000 mg/kg bw/day was not considered to be treatment-related, since its severity was minimal and its incidence was low. Extramedullary hematopoiesis in liver and alveolar foamy macrophage in lung were observed in the vehicle control. In females, there was no abnormal finding in any groups.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
[Other: Analysis of the concentration of L-glutamic acid in the amniotic fluid]
In DRF test, caesarean section was performed at Day 15 of pregnancy. According to the results of the analysis of the concentration of L-glutamic acid in the amniotic fluid of fetuses, no treatment-related concentration changes were observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in estrus cycle among the groups. and no treatment-related changes were observed in the mating, fertility and pregnancy indices.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No statistically significant differences were seen in the following parameters examined: gestation period, the number of corpora lutea and implantation, delivery index, the number of live and dead pups, pre-implantation loss, post-implantation loss, sex ratio, viability ratio, number of neonates with external anomalies, and body weights of pups on post-natal day 0 and day 4.

Details on results (F1)

No dose-related changes in clinical signs, body weight, viability index, external malformations and sex ratios were noted in pups

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female

Applicant's summary and conclusion

Conclusions:
The NOAEL for parental toxicity of L-glutamic acid was 1000 mg/kg bw/day and the NOAEL for reproductive toxicity was 1000 mg/kg bw/day, the highest dose tested. Based on these results, L-glutamic acid has not shown any potential for reproductive toxicity.