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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
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:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 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.
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
gavage
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
gavage
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
gavage
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
Historical control data:
See attachment

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (no. 38) at 20 mg/kg was found dead on Day 22 post-coitum. One day before, blood was noted in the catheter after dosing. Macroscopic examination revealed lung abnormalities (large dark red focus and perforation) indicating a gavage error.
In addition, one female (no. 15) of the control group had to be euthanized before planned necropsy as she aborted on Day 21 post-coitum.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg, body weight gains were reduced when compared to the control group. This was statistically significant on Days 10-26 post-coitum. A transient body weight loss was noted for several animals of this dose group. There were no statistically significant changes in (absolute) body weight or (for uterus) corrected body weight gain in any of the groups
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after allowance for body weight was decreased on Days 7-23 post-coitum at 300 mg/kg. A compensatory increase in food consumption was noted during the remainder of the treatment period. At 75 mg/kg, food consumption was slightly reduced on Days 7-20, however not statistically significant. Food intake was not affected at 20 mg/kg.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg, water consumption was decreased when compared to the control group. This was statistically significant for five occasions. A trend towards lower water consumption was also noted at 75 mg/kg, statistically significant for three occasions.
At 20 mg/kg, no changes in water consumption were noted.
The statistically significantly increased water consumption noted on Days 28-29 post-coitum for high dose animals was considered to be due to a slightly low control value, and therefore considered to be an incidental finding.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

The female (no. 38) at 20 mg/kg that was found dead on Day 22 post-coitum showed a large (30x25 mm) dark red focus and perforation of the right caudal lung lobe, indicating a gavage error.
The incidence of other findings among control and treated animals was within the background range of findings that are encountered among rabbits of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One female (no. 15) of the control group aborted on Day 21 post-coitum.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No significant differences were observed between control and treated groups regarding pre- and postimplantation loss (see tables attached).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No significant differences were observed between control and treated groups regarding total litter loss (see tables attached).
Early or late resorptions:
no effects observed
Description (incidence and severity):
No significant differences were observed between control and treated groups regarding early or late resorptions (see tables attached).
Dead fetuses:
no effects observed
Description (incidence and severity):
No significant differences were observed between control and treated groups regarding dead fetuses (see tables attached).
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were three non-pregnant animals (nos. 3, 7, 16) in the control group, one (no. 32) in the low dose group and three (nos. 79, 81, 82) in the high dose group. One control animal (no. 15) showed an abortion on Day 21 post-coitum.
Sufficient litters were available for evaluation of fetal development: 18, 20, 22 and 19 litters in the control, 20, 75 and 300 mg/kg groups, respectively.
Other effects:
no effects observed
Details on maternal toxic effects:
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)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
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.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
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.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on fetal external morphology.

External malformations were observed in one fetus of the 20 mg/kg GLDA group and in control group fetus. 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.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on fetal visceral morphology.

Visceral examination revealed absence of the accessory lung lobe in 1(1), 4(4), 2(2) and 5(4) fetuses (litters) of the control, 20, 75 and 300 mg/kg groups, respectively, resulting in mean litter incidences of respectively 0.6%, 2.2%, 1.0% and 3.2% per litter. The incidences for all treated groups were higher than in the concurrent control group, but were within or near the historical control data range (0.0% - 3.1% per litter). Therefore, and due to the absence of a dose response relationship, the higher incidences of the absent accessory lung lobe in treated groups were not considered to be treatment-related (see tables attached).
In each GLDA dose group, there was one other viscerally malformed fetus. The malformations in these fetuses occurred singly and were not related to the malformations in other fetuses and therefore were not considered to be toxicologically relevant. All visceral variations observed in the GLDA-treated groups occurred at similar frequencies in the control group, occurred infrequently and/or occurred without dose-relationship (see tables attached)
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
There were no treatment-related embryotoxic / teratogenic effects.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of developmental toxicity at the highest dose tested

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

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