N,N-bis(carboxymethyl)-L-glutamic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

May-July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and reported GLP study; this is a repeat study of the study carried out in 2008 (Beekhuizen, 2009) at the request of the French authorities (see letter of French authorities attached in section 13).

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate

EC Number:

257-573-7

EC Name:

Tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate

Cas Number:

51981-21-6

IUPAC Name:

tetrasodium 2-[bis(carboxylatomethyl)amino]pentanedioate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Description: white powder
Batch: FC-C 10943
Purity: 87.3wt% as GLDA-Na4
Test substance storage: at room temperature in the dark
Stability under storage conditions: stable
Expiry date: 19 April 2015

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Test System: female albino rabbits, New Zealand White (NZW) strain (SPFQuality), from a non-inbred laboratory colony. Nulliparous, non-pregnant
and untreated females were used at initiation of the study. Stock male NZW rabbits were used for mating with the females. These males were
adult and proven fertile. After mating these males were placed back in their stock and might be used for future studies.
Rationale: this species and strain of rabbit has been recognized as appropriate for developmental toxicity studies. WIL Research Europe B.V. has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Source: Charles River, Chatillon sur Chalaronne, France.
Number of animals: F0-generation: 88 females, F1-generation: 697 fetuses.
Age at delivery: females were approximately 18 weeks.
Acclimatization: at least 5 days prior to pairing.
Conditions: environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were recorded in the raw data; these variations were
considered to have had no effect on the outcome of the study.
Accommodation: females were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm). Shelters were removed for females that were excessively soiling their shelters.
Diet: free access to pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy). In addition, pressed hay (BMI,
Helmond, the Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study
period.
Water: free access to tap-water.
Diet and water evaluation for contamination and/or nutrients was performed according to facility standard procedures. There were no contaminant levels found that could interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Method: oral gavage, using a plastic catheter attached to a plastic disposable syringe. Formulations were placed on a magnetic stirrer during dosing.
Frequency: once daily for 7 days per week, approximately the same time each day with a maximum of 4 and a half hours difference between the earliest and latest dose on these days.
Exposure period: from Day 7 to Day 28 post-coitum, inclusive.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest recorded body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (25 May 2012), according to a validated method (Project 483244) using an HPLC-UV method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all
concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

From 8.00 am onwards, one female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating (which usually occurred within 5 minutes). This day was designated Day 0 post-coitum. Twenty-four male rabbits were used for mating with the 88 female rabbits.

Duration of treatment / exposure:

From Day 7 to Day 28 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 4 and a half hours difference between the earliest and
latest dose on these days.

Duration of test:

Animals surviving to scheduled necropsy were euthanised on Day 29 post-coitum.

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were based on a prenatal developmental toxicity study in NZW rabbits (Project 487520) in which doses of 30, 100 and 300 mg/kg body weight/day were tested. No fetal malformations or developmental variations were noted in this study. As such the developmental NOAEL for GLDA was established to be at least 300 mg/ kg bw/day. An increased number of non-pregnant females was, however, noted in all treated groups compared to the control group but these numbers were within the normal range or just below the historical control data and still considered normal. A dose response relationship was not present. Based on the reduced number of pregnancies the French Authorities, however, concluded that this study was of too low quality and should be repeated with at least one dose level lower than 30 mg/kg bw/day. In consultation with the sponsor, dose levels of 20, 75 and 300 mg/kg were selected.

Examinations

Maternal examinations:

Mortality / Viability: at least twice daily.
Clinical signs: at least once daily from Day 0 post-coitum onwards. The time of onset, degree and duration was recorded. All symptoms were graded according to fixed scales: Maximum grade 1: grade 0 = absent, grade 1 = present; Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate,
grade 3 = severe, grade 4 = very severe.
Cage debris was examined to detect abortion or premature birth
Body weights: Days 0, 4, 7, 10, 13, 16, 20, 23, 26, 29 post-coitum.
Food consumption: Days 0-4, 4-7, 7-10, 10-13, 13-16, 16-20, 20-23, 23-26 and 26-29 postcoitum.
Water consumption: subjective appraisal was maintained during the study until 11 June 2012, and a quantitative assessment was introduced from 12 June 2012 onwards based on a possible treatment related effect.
External, thoracic and abdominal examinations were performed for the detection of macroscopic abnormalities. All abnormalities were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

Ovaries and uterine content:

Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
Animals found dead or sacrificed before planned necropsy, were subjected to relevant examinations of the ovaries and uterine horns.

Fetal examinations:

External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.
External:
Each viable fetus was examined in detail, sexed and weighed. All live fetuses were euthanized by subcutaneous injection of 0.1 mL pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands) in the area between the scapulas. Nonviable fetuses (the degree of autolysis was
minimal or absent) were examined, crown-rump length measured and weighed. The crown-rump length of late resorptions (advanced degree of autolysis) was measured, the degree of autolysis recorded, a gross external examination performed (if possible).
Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. The sex of all fetuses was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development. The sex of all fetuses was determined by internal examination. The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution (Klinipath, Duiven, The Netherlands). Tissues were then transferred to a 70% aqueous ethanol solution for subsequent processing and soft-tissue examination using the Wilson sectioning technique. After examination, the tissues were stored in 10% buffered formalin. The heads from the remaining one-half of the fetuses in each litter were examined by a mid-coronal slice. All carcasses, including the carcasses without heads, were eviscerated and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.
Skeletal:
The eviscerated fetuses from Groups 1 and 4, following fixation in alcohol, were macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. All specimens were archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to- one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

Indices:

For each litter the following calculations were performed:

Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) x 100
number of corpora lutea

Post-implantation loss (%) = (number of implantation sites - number of live fetuses) x 100
number of implantation sites

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal toxicity was noted at 300 mg/kg. At this dose level, clinical signs consisted of increased incidences of dark faeces, diarrhoea and reduced faeces production. Food and water consumption was reduced. In addition, body weight gain was decreased, with several animals showing a transient body weight loss.
In animals treated with 75 mg/kg bw, dark faeces, diarrhea, reduced faeces production and slightly reduced food and water intake were also observed but because these changes were very limited and in view of the absence of more severe effects such as changes in body weight gain, these effects were not considered to be toxicologically relevant.
No maternal toxicity was observed in the 20 mg/kg dose group.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

3

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (actual dose received)

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:
No treatment related effect on litter size was noted. The mean number of viable fetuses per litter was 9.9, 8.3, 8.8 and 8.3 for Groups 1, 2, 3 and 4,
respectively.

There were no treatment-related effects on the sex ratio of the fetuses.

No treatment related effect on fetal body weights were noted.

The mean fetal body weights (sexes combined) were 38.4, 41.4, 40.4 and 37.4 gram, respectively.

There were no treatment-related effects on fetal external morphology. External malformations were observed in fetus no. A024-09 at 20 mg/kg GLDA and in control fetus no. A006-08. The fetus in the low dose group had an omphalocele, whereby several loops of intestine and a portion of liver protruded through a defect in the abdominal wall at the umbilicus. The control fetus also had an omphalocele and in addition hyperextension of both hind limbs and absence of the anus and tail. There were no other fetuses with external malformations, and external developmental variations were not observed in this study.

There were no treatment-related effects on fetal visceral morphology. Visceral malformations were observed in 3(3), 5(5), 3(2) and 6(5) fetuses (litters) in the control, 20, 75 and 300 mg/kg groups, respectively. The most frequently observed malformation was absent lung lobe and in all cases, the accessory lung lobe was missing. In the control, 20, 75 and 300 mg/kg groups, respectively 1(1), 4(4), 2(2) and 5(4) fetuses (litters) were affected, resulting in mean litter incidences of respectively 0.6%, 2.2%, 1.0% and 3.2% per litter. The fetuses involved were nos. A010-09 (control group), A024-09, A033-02, A042-11, A043-08 (20 mg/kg group), A046-07, A057-04 (75 mg/kg group), A070-08, A072-03,05, A077-08 and A078-07 (300 mg/kg group). Of these fetuses, no. A024-09 also had an omphalocele externally, which was described in the paragraph above. Other visceral malformations in test substance-treated groups were observed in fetus no. A028-06 at 20 mg/kg (abnormal liver lobation), fetus no. A057-07 at 75 mg/kg (diaphragm cyst) and in fetus no. A068-01 at 300 mg/kg (atretic pulmonary trunk, dilated aortic arch and atrial septum defect). Besides, visceral malformations were observed in two control fetuses, namely in fetus no. A004-15 (persistent truncus arteriosus) and in fetus no. A006-08 (absent urinary bladder). The latter fetus also had multiple external malformations, which were described above. Visceral developmental variations observed in the test substance-treated groups were malpositioned left carotid, partially undescended thymus horn, small gallbladder, liver cyst, hemorrhagic thymus, retrocaval ureter, absent renal papilla, supernumerary artery, supernumerary spleen, small lung lobe, ovary cyst, bilobed gallbladder, liver appendix and discolored thymus. All but the last four findings were also observed in control fetuses.

There were no treatment-related effects on fetal skeletal morphology. Skeletal malformations were observed in 6(4) and 4(3) fetuses (litters) in the control and 300 mg/kg groups, respectively. Two different malformations were observed at 300 mg/kg. Three fetuses (nos. A085-01,02 and A087-01) from two litters had a vertebral anomaly with or without associated rib anomaly and one fetus (no. A088-01) had fused sternebrae. The same findings were also seen in
control fetuses, namely a vertebral anomaly in fetus no. A013-10 and fused sternebrae in fetus nos. A006-11, A020-01,05 and A022-07. Except for a case of spina bifida (that could not be detected externally) in control fetus no. A006-08, which also had malformations externally and viscerally, there were no other fetuses with skeletal malformations. Skeletal developmental variations observed in the high GLDA dose group were 13th full ribs, 13th rudimentary ribs, unossified sternebrae nos. 5 and/or 6, caudal shift of pelvic girdle, unossified line in skull bone, unossified metacarpals, 7th cervical rudimentary ribs, supernumerary sternal ossification sites, slightly to moderately malaligned sternebrae, wide sternebra, reduced ossification of vertebral centrum, supernumerary sternebra and 7th cervical full ribs. All but the last four findings were also observed in control fetuses.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results in this prenatal developmental toxicity study, the maternal No Observed Adverse Effect Level (NOAEL) was established as 75 mg/kg body weight/day. The developmental NOAEL was at least 300 mg/kg body weight/day.

Executive summary:

The study was based on the following guidelines: 1) OECD 414, Prenatal Developmental Toxicity Study, January 2001. 2) EC No 440/2008 B.31: "Prenatal Developmental Toxicity Study", May 2008. 3) OPPTS 870.3700, Prenatal Developmental Toxicity Study, August 1998.

Rationale for dose levels: Dose levels were based on a prenatal developmental toxicity study in New Zealand White (NZW) rabbits (Project 487520) in which doses of 30, 100 and 300 mg/kg were tested. No fetal malformations or developmental variations were noted in this study. As such the developmental NOAEL for GLDA was established to be at least 300 mg/kg. An increased number of non-pregnant females was, however, noted in all treated groups compared to the control group but these numbers were within the normal range or just below the historical control data and still considered normal. A dose response relationship was not present. Based on the reduced number of pregnancies the French Authorities, however, concluded that this study should be repeated with at least one dose level lower than 30 mg/kg. In consultation with the sponsor, dose levels of 20, 75 and 300 mg/kg were selected.

Study outline: Eighty-eight mated female NZW rabbits were assigned to four dose groups. The test item was administered once daily by gavage from Days 7 to 28 post-coitum at doses of 20, 75 and 300 mg/kg (Groups 2, 3 and 4 respectively). The rabbits of the control group received the vehicle, elix water, alone. Females were checked daily for the presence of clinical signs. Body weights and food consumption were determined at periodic intervals. Based on a possible treatment related effect, water consumption was determined daily from 12 June 2012 onwards. Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability. All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, all fetuses were dissected and examined for visceral anomalies (all Groups) and subsequently fixed in 96% aqueous alcohol and stained with Alizarin Red S for skeletal examinations (Groups 1 and 4).

Results: Accuracy, homogeneity and stability of formulations were demonstrated by analyses. Maternal toxicity was noted at 300 mg/kg. At this dose level, clinical signs consisted of increased incidences of dark faeces, diarrhoea and reduced faeces production. Food and water consumption was reduced. In addition, body weight gain was decreased, with several animals showing a transient body weight loss. In animals treated with 75 mg/kg bw, dark faeces, diarrhea, reduced faeces production and slightly reduced food and water intake were also observed but because these changes were very limited and in view of the absence of more severe effects such as changes in body weight gain, these effects were not considered to be toxicologically relevant. No maternal toxicity was observed in the 20 mg/kg dose group. No developmental toxicity was observed in the 20, 75 and 300 mg/kg/day groups.

Conclusion: Based on the results in this prenatal developmental toxicity study, the maternal No Observed Effect Level (NOEL) for GLDA was established as 20 mg/kg body weight/day; the No Observed Adverse Effect Level (NOAEL) was established as 75 mg/kg body weight/day. The developmental NOAEL was at least 300 mg/kg body weight/day.