Sodium hydrogen glutamate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 19, 2006 - July 20, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: According to GLP and comparable to guideline study with restrictions.

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, Center for Food Safety and Applied Nutrition Redbook 2000: Toxicological Principles for the Safety Assessment of Food Ingredients IV.C.9.b. Guidelines for Developmental Studies (July 20, 2000)

Deviations:

not specified

Remarks:

This guideline was not available. Compliance with OECD 414 was checked instead.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See below.

Principles of method if other than guideline:

The study design agreed with OECD 414 (2001), with the following deviation. The scheme for dose administration (day 6 through 20 of presumed gestation) does not agree with OECD 414 (2001), which states day 5 post mating to the day prior to scheduled caesarian section (i.e. day 5 through 20 of presumed gestation).

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 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: 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 98.5%.
- Storage condition of test material: Room temperature, shading from light, sealing

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi breeding center of Charles River Laboratories Japan, Inc.
- Age at study initiation: 11 or 12 weeks
- Weight at study initiation: body weight pregnant females ranged from 233 to 292 g on day 0 of gestation
- Housing: individually in aluminium cages with stainless steel wire mesh fronts and floors
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.9 to 23.1 degrees
- Humidity (%): 54.2 to 63.5%
- Air changes (per hr): 12 times or more
- Photoperiod (hrs dark / hrs light): 12 hours (light on: 7 a.m., light off: 7 p.m.)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
Appropriate amounts of the test substance and the basal diet were accurately weighed and mixed in a mortar and filtered through a sieve. This
mixture was added to the required amount of diet and mixed in a powerful auto mixer for 40 minutes.
Formulated diets were prepared once a week and were stored in the storage refrigerator. The feeders with basal diet for the control animals or the
formulated diets were exchanged every week. The formulated diets were used within 16 days after preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For method description and pre-test validation see section 8 Analytical methods Ajinomoto 9956 (258-056). For data on homogeneity and stability of the test material in diet at 0.5% w/w and 5% w/w, see study 9957 (258-057) (90-day repeated dose dietary toxicity study in rat). The concentration of the test substance was determined by analysis of duplicate samples taken from the center of the first batch diet prepared for the present study. At 0.5% w/w, 1.5% w/w and 5% w/w, respectively, the analyzed concentrations represented 99.9%, 100.3% and 98.0% of the nominal concentrations.

Details on mating procedure:

Female animals were observed for estrus cycle for at least 7 days and those in a suitable condition for mating were selected.One female rat at 11 or 12 weeks of age was caged with 1 male rat at 11 or 12 weeks of age overnight, and checked the next morning for the presence of sperm in the vaginal smear. The day when sperm was confirmed was designated as day 0 of gestation.

Duration of treatment / exposure:

The test substance was administered from day 6 to 20 of gestation.

Duration of test:

20 Days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
5% (w/w)
Basis:
nominal in diet
Based on the mean feed consumption during the treatment period, the mean compound intake was 3019 mg a.s./kg bw/day.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1.5% (w/w)
Basis:
nominal in diet
Based on the mean feed consumption during the treatment period, the mean compound intake was 898 mg a.s./kg bw/day.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.5% (w/w)
Basis:
nominal in diet
Based on the mean feed consumption during the treatment period, the mean compound intake was 302 mg a.s./kg bw/day.

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: the concentration of 5 w/w% was considered an acceptable nutrition-based endpoint for high dose selection for dietary
mixture studies. Therefore, the concentration of 5 w/w% which was predicted not to produce any nutritional disturbance was selected for this
study. The middle and low concentrations for this study were selected to be 1.5 w/w% and 0.5 w/w% in a common ratio of about 3.
- Rationale for animal assignment (if not random): the successfully copulated females were assigned to the study groups on the day of copulation by
using the system package software for safety study on the basis of the body weight on day 0 of gestation. Animals copulated on the same day were
uniformly assigned to the groups.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 2 times a day (am and pm) during the administration period and once a day during the rest of the experimental period

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The weight of food was measured on days 0, 6, 13 and 20 of gestation. The mean daily food consumption was calculated using the weight of
supplied and remained diets.
- The test substance intake (mg/kg/day) was calculated during day 6 to 20 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: organs and tissues in the abdominal, thoracic, pelvic and cranial cavities.
- The heart, lungs, liver, kidneys, spleen, adrenal glands, ovaries and gravid uterus were weighed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Placentas of live fetuses weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Other: number of live and dead fetuses

Fetal examinations:

- External examinations: Yes: all live fetuses
- Soft tissue examinations: Yes:half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

The following data were recorded and analyzed statistically using an automatic data processing system. Fetus data were analyzed statistically on
the mean per dam as a unit.
The Bartlett's test was conducted at the 5% level of significance and the other tests at the two-tailed 5% and 1% levels of significance.

Dunnett's multiple comparison test
- Body weight, body weight gain, food consumption, number of corpora lutea graviditatis, number of implantations, number of live fetuses,
number of resorption and dead fetuses, fetal weight, placental weight, gravid uterus weight, relative uterus weight and degree of ossification
(sacrococcygeal vertebra) data were subjected to automatic analysis by Bartlett's test for homogeneity of variance following a decision tree
approach. Homogeneous data were analyzed by Dunnett's multiple comparison test for the significant differences between the control group and
each treatment group. Heterogeneous data in Bartlett's test were subjected to the Steel's test for the significant differences between the control
group and each treatment group.

Mann-Whitney U test
- Implantation rate, live fetus rate, resorption and dead fetus rate, sex ratio, incidence of external anomalies, incidence of visceral anomalies,
incidence of skeletal variation, incidence of skeletal malformation, incidence of incomplete ossification and degree of ossification (excluding
sacrococcygeal vertebra) were subjected to Mann-Whitney U test.

Fisher's exact test
- The incidence of necropsy findings was subjected to Fisher's exact test.

- Clinical signs were not analyzed statistically.

Indices:

- Implantation rate: (number of implantations/number of corpora lutea graviditatis) x 100
- Live fetus rate: (number of live fetuses/number of implantations) x 100
- Resorption and dead fetuses rate: (number of dead embryos and fetuses/number of implantations) x 100
- The incidence of external abnormalities per dam: (number of abnormal fetuses/number of fetuses observed) x 100
- Sex ratio per dam: [surviving male fetuses/(surviving male fetuses + surviving female fetuses)] x 100
- The incidence of visceral abnormality: (number of abnormal fetuses/number of fetuses observed) x 100
- Incidence of skeletal variation: (number of fetuses with variation/number of fetuses observed) x 100
- Incidence of skeletal malformation: (number of fetuses with malformation/number of fetuses observed) x 100
- Incidence of incomplete ossification per dam: (number of fetuses with incomplete ossification/number of fetuses observed) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No clinical signs were observed and no mortalities occurred. There were no treatment related effects on body weight, body weight gain and food consumption. Necropsy revealed no treatment related findings and the weights of the selected organs of the treated groups were comparable to those of the control group.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEC

Effect level:

>= 5 other: 5% w/w in diet

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEC

Effect level:

>= 5 other: % w/w in diet

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
CESAREAN SECTION
There were no biologically relevant and statistically significant differences in the number of corpora lutea graviditatis, number of implantations, number of live fetuses, sex ratio, number of resorption and dead fetuses, live fetal weight or placental weight between the control group and any of the treatment groups.

EXTERNAL EXAMINATION
The only finding was absent eye bulge observed in 1 fetus in the 5 w/w% group.

VISCERAL EXAMINATION
- Visceral abnormalities were observed in 22 (14.2%), 14 (9.8%), 13 (7.2%) and 17 (11.0%) fetuses in the control, 0.5, 1.5 and 5 w/w% groups, respectively. Thymic remnant in the neck, persistent left umbilical artery, anophthalmia, malpositioned thyroid gland, persistent atrioventricular canal, right-sided aortic arch, retroesophageal subclavian, abnormal lung lobation, misshapen liver, dilated renal pelvis, dilated ureter, and supernumerary carotid were sporadically observed. There were no biologically relevant and statistically significant differences in the incidence of these visceral anomalies between the control group and any of the treatment groups.

SKELETAL EXAMINATION
- Skeletal malformations were observed in 18 (11.0%), 18 (11.3%), 28 (14.3%) and 9 (5.8%) fetuses in the control, 0.5, 1.5 and 5 w/w% groups, respectively. Branched rib cartilage, short rib, detached rib cartilage, misshapen sternebra, misaligned cervical arch, misshapen cervical arch, absent lumbar vertebra,supernumerary lumbar vertebra, splitting of the thoracic vertebral body, fused vertebral plate, and fused transverse process of the cervical and thoracic vertebra were sporadically observed. Skeletal variations were observed in 76 (45.0%), 60 (38.5%), 79 (41.0%), and 64 (41.3%) fetuses in the control, 0.5, 1.5 and 5 w/w% groups, respectively. Cervical rib, full supernumerary rib, short supernumerary rib, discontinuous rib cartilage, dumbbell-shaped cartilage of the thoracic centrum, spilt cartilage of the thoracic centrum, misshapen spinous process of a thoracic vertebra, misshapen transverse process of a lumbar vertebra and noncombined transverse process of the sacral vertebra were observed. There were no biologically relevant and statistically significant differences in the incidence of these skeletal anomalies and variations between the control group and any of the treatment groups.
- The sites of retarded ossification included incomplete ossification of the hyoid, unossified hyoid, incomplete ossification of the interparietal, incomplete ossification of the parietal, bipartite ossification of the supraoccipital, incomplete ossification of the supraoccipital, incomplete ossification of the cervical arch, bipartite ossification of the thoracic centrum, dumbbell ossification of the thoracic centrum, incomplete ossification of the thoracic centrum, unossified thoracic centrum, bipartite ossification of the lumbar centrum, dumbbell ossification of the lumbar centrum, incomplete ossification of the lumbar centrum, incomplete ossification of the ischium, incomplete ossification of the pubis and unossified pubis. There were no biologically relevant and statistically significant differences in the incidence of these findings between the control and any of the treatment groups.

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

>= 5 other: w/w% in diet

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a study according to OECD 414 (deviation: administration from day 6-20 of gestation instead of day 5-20), no adverse effects of administration of monosodium L-glutamate monohydrate produced by a new method in the diet at 0.5, 1.5 and 5% w/w (equivalent 302, 898 and 3019 mg a.s./kg bw/day), no treatment related adverse effects were seen on the health of the dams and on all of the reproductive and foetal parameters examined. In conclusion, the NOAEL for maternal and developmental toxicity was ≥5% w/w in the diet equivalent to ≥3019 mg a.s./kg bw/day.