Sodium hydrogen glutamate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 28, 2006 - March 14, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study has been performed according to OECD 416 and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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.a. Guidelines for Reproduction Studies (July 20, 2000)

Deviations:

not specified

Remarks:

This guideline was not available. Compliance with OECD 416 was checked instead.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

The conduct of the study agreed with OECD 416 (2001).

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 administration period showed that the test substance content was 99.0%.
- Storage condition of test material: Room temperature, shading from light, well-closed

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: (F0) 6 wks; (F1) 10 wks
- Weight at study initiation: (F0) Males: 181.4-217.6 g; Females: 133.3-161.1 g
- Fasting period before study: not applicable
- Housing:
Individually in aluminium cages with stainless steel wire mesh fronts and floors.
Dams were housed individually in polycarbonate-econ cages placed on an automatic watering rack, and materials for making a nest (sun flake) were
put in the cages from day 18 of gestation to necropsy.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4-23.7
- Humidity (%): 47.1-82.6%
- Air changes (per hr): 12 times or more
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

An appropriate amount of the basal diet was weighed and put into a powerful mixer. An appropriate amount of test substance was weighed, fractured in a mortar, filtered through a 425 um sieve and added to the basal diet in the powerful auto mixer and blended.
Formulated diets were prepared once every 2 weeks or more, packed in two batches and stored in the storage refrigerator. The formulated diets were used within 12 days in the refrigerator plus 12 days at room temperature after preparation.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until copulation or 21 days had elapsed
- Proof of pregnancy: vaginal plug or sperm in vagina referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

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). Data on homogeneity and stability of the test material in diet at 0.5% w/w and 5% w/w were also generated in study 9957 (258-057) (90-day repeated dose dietary toxicity study in rat). In the present study, homogeneity, stability and accuracy were confirmed. Homogeneity was determined for one batch of diet by analysis of duplicate samples taken from the upper, middle and lower layer of the concentrations of 0.5% and 5% w/w. , Homogeneity was acceptable (RSD was 5.7% or lower for the 0.5% w/w diet and 2.8% or lower for the 5% w./w diet). Stability was determined for one batch of diet by analysis of duplicate samples from the middle layer of the concentrations of 0.5% and 5% w/w following storage conditions representative of those of the diet batches used in the study (12 days in the refrigerator followed by 12 days in the animal room). Stability was acceptable (the mean concentrations after storage were in the range of 91.6% to 109.5% of the initial concentrations). The accuracy (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. The concentrations were acceptable, as at 0.5% w/w, 1.5% w/w and 5% w/w the analyzed concentrations represented 95.7% to 106.4% of the nominal concentrations.

Duration of treatment / exposure:

-Daily dosing of the parental (F0) males and females began when they were 6 weeks old, and dosing was continued for 10 weeks at least before the
mating period (until 15 weeks of age). Dosing was continued as follows: males throughout mating and up to necropsy (before necropsy, fertility or
infertility of the pair was confirmed), and females throughout mating, gestation and lactation up to weaning of their F1 offspring.
- Daily dosing of the F1 males and females began at weaning, and dosing was continued for 10 weeks at least before the mating period. Dosing was
continued as follows: males throughout mating and up to necropsy (before necropsy, fertility or infertility of the pair was confirmed), and females
throughout mating, gestation and lactation until the F2 generation was weaned. The F1 animals were treated in identical concentrations to the F0
animals.

Details on study schedule:

- F0 parental animals not mated until 16 weeks of age, following 10-week dosing.
- F1 parental animals not mated until 13 weeks of age or older, following 10-week or more dosing.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
5% (w/w)
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1.5% (w/w)
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.5% (w/w)
Basis:
nominal in diet

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: In this study, a concentration of 5% (w/w) was selected as a maximum concentration of the test substance in diet because
it was considered an acceptable nutrition-based endpoint for high concentration selection for dietary toxicity studies in rodents. As intermediate and
low concentrations, 1.5% (w/w) and 0.5% (w/w) were selected in a common ratio of about 3.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS (F0 and F1): Yes
- Time schedule: twice a day (a.m. and p.m.)
- Cage side observations : Mortality and abnormalities

DETAILED CLINICAL OBSERVATIONS (F0 and F1): No

BODY WEIGHT (F0 and F1): Yes
- Time schedule for examinations:
First day of dosing and weekly thereafter. Parental males were also weighed on the day of necropsy. Parental females were also weighed on days 0, 7,
14 and 21 of gestation and on days 0, 7, 14 and 21 postpartum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Food consumption was measured on the first day of dosing, weekly thereafter, on days 0, 7, 14 and 21 of gestation and on days 0, 7, 14 and 21 postpartum.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, on a weekly basis.

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

Oestrous cyclicity (parental animals):

Female animals were observed for their estrous cycles for 21 days prior to mating. Estrous cycle observation was continued during mating. The
mean estrous cycle was determined based on the number of days between each standing estrous during the 21-day period prior to mating. The
copulatory intervals (mean number of days until mating) and indices of copulation and fertility were also determined. The F1 males which were not copulated or with confirmed infertility of the pair were mated with females for the further examinations on reproductive functions until copulation
occurred or 21 days had elapsed. Estrous cycle of females for the further examinations on reproductive functions was determined by observing
the vaginal smears from 9 days before mating until the confirmation of copulation.

Sperm parameters (parental animals):

Parameters examined in male parental generations (F0 and F1):
cauda epididymal sperm reserve, sperm motility and sperm morphology.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, to a maximum of 8 pups/litter (4 /sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 and F2] offspring:
number and sex of pups, stillbirths, live births and the incidence of external anomalies. In addition, one male and one female F1 pup were randomly selected at weaning and subjected to functional tests at weaning (motor activity, auditory startle response, pain response, pupil response, righting reflex). They were observed daily from weaning until sexual maturity.

GROSS EXAMINATION OF DEAD PUPS:
yes, for internal anomaly; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Dead animals and dams with total litter death: Dead animals were necropsied immediately after the state was discovered. Dams with total litter death were euthanized by exsanguination from the abdominal aorta under ether anesthesia and necropsied.
- Male animals: All surviving animals after mating (after successful or unsuccessful copulation of the pair had been confirmed)
- Maternal animals: All surviving animals at the time of weaning.
- Unsuccesfully copulated females and females failing to produce a litter naturally: euthanized by exsanguination from the abdominal aorta under ether anesthesia and necropsied.

GROSS NECROPSY
- Gross necropsy consisted of macroscopically examination for any structural abnormalities or pathological changes, with special attention paid to reproductive system organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the following tissues and organs were collected and prepared:
- Dead animals and dams with total litter death: Abnormal parts (lymph node (renal and parapancreatic), mediastinum, incisor tooth, bone (nose), skin, mammary gland, lymph nodes (mesenteric, mandibular), mandibular gland, sublingual glands, sternum, femur, thymus, trachea, lungs (including bronchi), heart, thyroid glands, parathyroid glands, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, spleen, kidneys, adrenal glands, urinary bladder, seminal vesicles, prostate gland, testes, epididymides, ovaries, oviducts, uterus, vagina, eyeballs, Harderian gland, brain, pituitary gland, spinal cord (cervical, thoracic and lumbar parts), skeletal muscle (femoral region), sciatic nerve, aorta.
- Females allowed to deliver naturally and males: histpathology was performed on adrenal glands, vagina, uterus, ovaries, oviducts, testes, epididymis (unilateral), seminal vesicles, coagulation glands, prostate glands, pituitary gland and lesioned tissues from 10 animals of high-dose and control F0 and F1 animals. Body weights and the weights of the following organs were determined and organ to body weight ratios calculated: brain, thymus, heart, lungs, liver, kidneys, spleen, adrenal glands, pituitary gland, thyroid glands, ovaries, uterus (including the cervix), testes, epididymides, seminal vesicles (including the coagulation gland) and the prostate gland (including the urethra).
- Unsuccessfully copulated males and females and females necropsied on day 25 of gestation (F0 and F1): adrenal glands, vagina, uterus, ovaries, oviducts, testes, epididymis (unilateral), seminal vesicles, coagulation glands, prostate glands, pituitary gland and lesioned tissues from all F0 and F1 animals

Postmortem examinations (offspring):

SACRIFICE
- Culled F1 and all F2 pups were necropsied at the time of weaning

GROSS NECROPSY
- Gross necropsy consisted of macroscopically examination for any structural abnormalities or pathological changes.

HISTOPATHOLOGY / ORGAN WEIGTHS
Samples of the following tissues and organs were collected and prepared:
Lesion-bearing tissues (liver, mandibular gland, kidneys, small intestine, prostate gland, eyes, Harderian gland, tail and sternum). Body weight and the weights of the following organs were determined and organ to body weight ratios calculated: brain, thymus, spleen.

Statistics:

Live pups data were presented on the mean per dam as a unit.
Body weight, food consumption, number of implantations, numbers of newborns, live newborns and dead newborns, gestation period, estrous
cycle, copulatory intervals, external differentiation (detachment of the auricles, eruption of the lower incisors and separation of the eyelids), organ weight (absolute weight), organ weight to body weight ratio (relative weight) and number of sperm data were subjected to automatic analysis by
Bartlett's test for homogeneity of variance. Homogeneous data (without a significant difference) were analyzed by Dunnett's multiple comparison
test for the significant differences between the control group and each treatment group. Heterogeneous data (with a significant difference) in
Barlett's test were subjected to the Steel's test for the significant differences between the control group and each treatment group.
The indices of gestation, copulation and fertility, sex ratio and external differentiation data (preputial separation and vaginal opening) were
subjected to X2 test, and the incidence of external anomalies, indices of birth and delivery, survival rate on day 4, 7 or 14, weaning rate, sperm
activity, percentage of sperm with abnormal morphology and percent motile sperm to Mann-Whitney U test.
The incidence of necropsy and microscopic findings was subjected to Fisher's exact test.
Clinical signs and results of functional tests were not analyzed statistically.
The Barlett's test was conducted at the 5% level of significance and the other tests at the two-tailed 5% and 1% levels of significance.

Reproductive indices:

Copulatory interval: Mean number of days until mating
Indices of copulation: (Number of animals with successful mating / number of animals cohabiting) x 100
Fertility: Number of animals with progeny / number of animals with successful mating
Gestation index: Number of females giving birth to live offspring / number of pregnant females x 100

Offspring viability indices:

Birth index: Number of offspring born alive / number of implantations x 100
Delivery index: Number of newborns / number of implantations x 100
Survival rate on day 4: (Number of live offspring on day 4 after birth / number of offspring born alive) x 100
Survival rate on day 7 or 14: (Number of live offspring on day 7 or 14 after birth / number of live offspring on day 4 after birth just after culling) x 100
Weaning rate: (Number of live pups on day 21 after birth / number of live offspring on day 4 after birth just after culling) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: See below under “details on results (parental animals)”.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

Compound intake
The report provided only data on the compound intake in terms of mg/kg bw/day on a weekly basis, calculated from the weekly data on body weight and food consumption. The mean overall compound intake in terms of mg/kg bw/day was not reported. The mean overall compound intake at 1.5 and 5% w/w (dose levels relevant for NOAEL) was calculated by the author of this summary by averaging the weekly data on compound intake during the 10 weeks prior to mating. This estimation is a worst case value, since the compound intake during gestation and lactation is higher than during the period prior to mating. This procedure is recommended by EFSA.
At 1.5% w/w, the compound intake of F0 parental males and females was 939 and 1039 mg/kg bw/day, respectively, and for F1 parental males and females 1305 and 1422 mg/kg bw/day, respectively. At 5% w/w, the compound intake of F0 parental males and females was 3131 and 3496 mg/kg bw/day, respectively, and for F1 parental males and females 4404 and 4618 mg/kg bw/day, respectively.

Other findings.
The results of the study are summarised in the Tables under “Remarks on results including tables and figures” and under "Overall remarks, attachments". Unless indicated differently, the changes are represented either as percentage change relative to the control (e..g -35 = 35% reduction, +10 = 10% increase) or as incidence (e.g. 3/10 represents 3 out of 10 rats). Where a dose relationship was observed, this is indicated in the last column.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

1.5 other: w/w%

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on increased absolute and relative kidney weight. Based on compound intake by F0 parental males and females (worst case), this is equivalent to 939 and 1039 mg/kg bw/day, respectively.

Remarks on result:

other: Generation: F0 and F1 (migrated information)

2

Dose descriptor:

NOAEL

Remarks:

(reproduction)

Effect level:

>= 5 other: % w/w

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects at highest test dose. Based on compound intake by F0 parental males and females (worst case), this is equivalent to ≥3131 and ≥3496 mg/kg bw/day, respectively.

Remarks on result:

other: Generation: F0 and F1 (migrated information)

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

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

The results of the study are summarised in the Tables under “Remarks on results including tables and figures” and under "Overall remarks, attachments". Unless indicated differently, the changes are represented either as percentage change relative to the control (e..g -35 = 35% reduction, +10 = 10% increase) or as incidence (e.g. 3/10 represents 3 out of 10 rats). Where a dose relationship was observed, this is indicated in the last column.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

offspring

Generation:

F1

Effect level:

1.5 other: w/w%

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on reduced absolute and relative spleen weight. Based on compound intake by F0 parental males and females, this is equivalent to 939 and 1039 mg/kg bw/day, respectively.

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Remarks:

offspring

Generation:

F2

Effect level:

>= 5 other: % w/w

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects at highest test dose. Based on compound intake by F0 parental males and females (worst case), this is equivalent to ≥3131 and ≥3496 mg/kg bw/day, respectively.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

The findings in parents and offspring are summarised in table 1 below and in Table 1(ctd) under "Overall remarks (attachments). The splitting of the table was necessitated by constraints of the IUCLID software.

 

Table 1. Summary of findings in 2-generation reproduction study

Dose (% w/w)	0			0.5		1.5		5		DR
Sex	m	f		m	f	m	f	m	f
F0 parental animals
Mortality	none
Clinical signs	no treatment related findings
Body weight
Lactation day 21							-4.5*
Food consumption
Pre-mating day 21-28								+9*
Pre-mating day 42-49								+9*
Necropsy	no treatment related findings
Histopathology	no treatment related findings
Organ weight
Liver (abs)						+9.7*
Liver (rel)						+6.8**
Kidney (abs)								+10**	+9.5**
Kidney (rel)								+6.9**	+14**
Brain (rel)							+6.0*
Thymus (abs)							-22*
Lung (rel)							+8.3**		+8.3**
Heart (rel)									+5.9**
Spleen (rel)					-11*
Reproductive performance
Copulation/fertility	no treatment related findings
Sperm parameters^(A)	no treatment related findings
Effects on F1 offspring
sex ratio (M/F)	0.98			1.11		0.89**		0.94*
sex ratio (M/(M+F))	0.50			0.53		0.47		0.48
Survival
day 4						+6.4*
day 14								+5.9*
day 21								+5.9*
External differentiation^(B)	no treatment related findings
Functional tests	no treatment related findings
Necropsy	no treatment related findings
Histopathology	no treatment related findings
Body weight
After weaning^(F)								-5.2*
Organ weight
Brain (rel)								+7.0*	+6.5**
Thymus (abs)								-10*
Spleen (abs)								-16**	-13**
Spleen (rel)								-11**	-8.6*

DR          dose related

*              Statistically significant difference from control at 5% level.

**            Statistically significant difference from control at 1% level.

(A)          Morphologically, a significantly lower incidence of head abnormalities was noted in the 0.5 (blunt hooks) and 5% w/w (short head) groups, as compared with the control group. These are however no adverse findings.

(B)          The completion index of separation of the eyelids in the 5% w/w group was significantly higher than that in the control group on day 17 after birth, that of vaginal opening was significantly higher in the 1.5% w/w group on days 29 and 20 after birth and in the 5% w/w group on day 36 after birth, and that of preputial separation was significantly lower than that of the control group on day 37 after birth. These findings are considered to be unrelated to the treatment because of their sporadic occurrence.

(F)          After weaning the number of animals was reduced to 49-52 per sex per dose by random selection.

Applicant's summary and conclusion

Conclusions:

In a 2-generation study in rat (dose levels 0, 0.5, 1.5 and 5% w/w) according to OECD 416, the NOAEL for effects on F0 and F1 parental animals was 1.5% w/w, based on increased absolute and relative kidney weight. For F0 parental males and females, this is equivalent to 939 and 1039 mg/kg bw/day, respectively, and for F1 parental males and females to 1305 and 1422 mg/kg bw/day, respectively. The NOAEL for effects on offspring was also 1.5% w/w, based on reduced absolute and relative spleen weight of F1 offspring. The NOAEL for effects on reproduction was ≥5% w/w; based on compound intake by F0 parental males and females (worst case), this is equivalent to ≥3131 and ≥3496 mg/kg bw/day, respectively.