(2S)-5-amino-2-[(aminoacetyl)amino]-5-oxopentanoic acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-01-25 - 2022-05-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-(2-aminoacetamido)-4-carbamoylbutanoic acid

EC Number:

700-144-0

Cas Number:

13115-71-4

Molecular formula:

C7H13N3O4

IUPAC Name:

(2S)-2-(2-aminoacetamido)-4-carbamoylbutanoic acid

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Wistar) rats

Details on species / strain selection:

The rat is the preferred rodent species for reproduction toxicity testing. Wistar rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, from SPF colony
- Age at study initiation: 10 weeks old at start, 12 weeks old at mating
- Weight at study initiation: Males: 359-430 g, females: 216-258 g
- Fasting period before study: none
- Housing: group-housed, up to 2 animals of the same sex and dose group/cage, with the exception of the mating and gestation, delivery, lactation period, when they were paired or individually housed (with pups), respectively.
- Diet: ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance” (Batch numbers: 536 84829, Expiry dates: 31 May 2022) produced by ssniff Spezialdiäten GmbH, ad libitum
- Water (e.g. ad libitum): tap water from the municipal supply, as for human consumption from a 500-mL bottle, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 507 room: 19.9-25.9℃ (target range: 19-25°C), 609 room: 20.6-22.6℃ (target range: 19-25°C)
- Humidity (%): 507 room: 24-72% (target range: 30-70%), 609 room: 26-71% (target range: 30-70%)
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 2022-01-25 - 2022-04-04

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The oral route was selected as it is one of the possible routes of human exposure. The way of test item and vehicle control item administration was in compliance with the relevant OECD No. 422 guideline.

PREPARATION OF DOSING SOLUTIONS:
Water content of the supplied product was taken into consideration during sample preparation (correction factor of 1.089 was applied).
According to stability assessment results of the analytical method validation study, the test item had 7 days stability on room temperature, therefore the formulations were prepared 2-5 days interval. The formulations were stirred with a magnetic stirrer during the preparation and 10-15 minutes before the treatment.

VEHICLE
- Concentration in vehicle: 12.5 mg/mL (low dose), 37.5 mg/L (mid dose), 125 mg/mL (high dose)
- Amount of vehicle (if gavage): 8 mL/kg bw
- Lot/batch no. (if required): 2109-8099 / 2201-8186

Details on mating procedure:

- mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage
- sperm positive females were caged individually

Analytical verification of doses or concentrations:

yes

Remarks:

Analysis of the formulations for concentration and homogeneity of test item was performed an appropriate validated HPLC analytical method in the Analytical Department of the Test Facility.

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period).
Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing).

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

equivalent to 108.9 mg/kg bw/day (test item, monohydrate form)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

equivalent to 326.7 mg/kg bw/day (test item, monohydrate form)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

equivalent to 1089 mg/kg bw/day (test item, monohydrate form)

No. of animals per sex per dose:

12 animals/sex/groups, 4 groups

Control animals:

yes, concurrent vehicle

Details on study design:

The test system and the number of animals used in the study were in compliance with the relevant OECD TG 422. The minimum number of animals was used, corresponding to the regulatory guidelines being followed, but taking into consideration the scientific reliability of the collected information.
The dose levels were selected based on the results of a Dose Range Finding (DRF) study (Study code: 21/255-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
In the DRF study there were no clinical symptoms observed. There were no significant effects on the body weight, body weight gain, food consumption, clinical pathology parameters or organ weights on any of the dose groups. No effects were observed at necropsy. It was considered that the dose level of 1000 mg/kg bw/day was suitable as the high dose for the subsequent OECD 422 study. Lower doses were spaced with a factor of approximately 3.

Positive control:

Not examined

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the start of the pre-exposure period then weekly (on Day -14) and once before the first exposure on Day 0 (to allow for within-subject comparisons), then weekly (in the morning (am), before treatment) and before necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during the pre-exposure period, on Day 0 for randomisation purposes, and afterwards weekly, on the day before termination and at termination

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals: 7 males and 7 females/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy
- Animals fasted: Yes (overnight)
- How many animals: 7 males and 7 females/group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week (males on Day 22 am, females on PPD5-8 am)
- Dose groups that were examined: Five males and five females/group randomly selected
- Battery of functions tested: Functional Observational Battery and locomotor activity measurement

THYROID HORMONE ANALYSIS
- up to two pups per litter on PND4 (females if possible), all dams and two pups per litter on PPD 14 (females) / PND13 (pups), all adult males at termination

FOOD CONSUMPTION
- determined by weighing the non-consumed diet with a precision of 1 g at weekly (on a body weight measurements day)
- measured for parent females on Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20, on PPD (Post-partum Day) 0, 4, 7, 10 and 13

WATER CONSUMPTION: No

OBSERVATION OF THE DELIVERY PROCESS AND NURSING INSTINCT
- duration of gestation was recorded and was calculated from Day 0 of pregnancy
- dams were observed to record whether they formed a nest from the bedding material and cover their new-borns or not

Oestrous cyclicity (parental animals):

- monitored by vaginal smears daily during the pre-exposure period before the treatments starts and from the beginning of the treatment period until evidence of mating (during the pre-mating and mating periods)
- additionally, vaginal smears were prepared and examined for each surviving female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs

Sperm parameters (parental animals):

- special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure

Litter observations:

- duration of gestation was recorded and was calculated from Day 0 of pregnancy
- dams were observed to record whether they formed a nest from the bedding material and cover their new-borns or not
- efficiency of suckling was observed by the presence of milk in the pups' stomach
- each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities
- any abnormal behaviour of the offspring was recorded
- live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0), and on PND4 and PND13, with accuracy of 0.01 g
- all the litters were checked and recorded daily for the number of viable and dead pups; clinical signs and any abnormal behaviour or appearance of the pups (external abnormalities) were also recorded on each day
- pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible
- on PND 4, litters were culled to yield 5 males and 5 females per litter (or as nearly as possible). Pups to be culled within each litter were selected random. In litters of insufficient size where the number of males or female pups was less than 5, adjustment of the selection process was made to assure 10 pups were retained. Culling was not performed on litter sizes less (or equal) than 10. All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities.
- anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND0)
- number of nipples/areolae in male pups was recorded on PND13
- Detailed gross external examination (particular attention paid to reproductive development, genitals) on PND13 for all pups
- one male and one female pup per litter (if possible) were previously selected for culling for blood sampling on PND4
- all pups were necropsied on PND13

Postmortem examinations (parental animals):

METHOD OF EUTHANASIA
- at study termination or in the case of early termination due to moribund animals, euthanasia was performed under pentobarbital anaesthesia followed by exsanguination
- Euthanimal 40% (400 mg/mL sodium pentobarbital solution) was used by intraperitoneal injection

UNSCHEDULED EUTHANASIA
- gross necropsy was performed on each animal irrespective of the date of death
- no organ weight measurement was performed, but tissue or organ samples were retained for that animal

SCHEDULED EUTHANASIA
- surviving animals were euthanized at termination
- after blood sampling, gross necropsy was performed on each animal
- weight of selected organs were measured, and selected organs and tissues were retained

CLINICAL PATHOLOGY
- all animals including the randomly selected animals for blood sampling were fasted (overnight period of food deprivation, in case of females this happened after the litter had been culled)
- blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy
- for terminal blood sampling all randomly selected animals (7 males and 7 females/group), 3 samples were taken from each animal: one for haematology (in 1.2-mL tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry

BLOOD SAMPLING FOR THYROID HORMONE ANALYSIS
- blood samples were taken by venepuncture (using vena sublingualis) into tubes containing K3-EDTA as anticoagulant as follows:
• from all dams on PPD 14
• from all adult males at termination
- the collected pup blood (serum) samples were pooled by litter; serum T4 levels determination was made in males’ serum samples
- the timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days (on the morning of the necropsy day)

NECROPSY
- Gross necropsy was performed on each adult animal irrespective of the date of death
- after exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities was opened, and the appearance of the tissues and organs were observed macroscopically
- special attention was paid to the organs of the reproductive system
- number of implantation sites and of corpora lutea was recorded in the females

ORGAN WEIGHTS
- body weight and weight of the following organs of all adult male and female animals/group was determined:
• with a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
• with a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids (trimming and weighing after fixation)
- testes and epididymides were weighed individually
- individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights
- relative organ weight (to body and brain weight) was calculated and reported

TISSUE COLLECTION AND PRESERVATION
- weighed organs and all organs showing macroscopic lesions of all adult animals were preserved (including tissues from equivalent control group animals)
- the eyes with the optic nerve, testes and epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution

HISPATHOLOGY AND MICROSCOPIC EVALUATION
- retained tissues and organs required for histopathology (below) were embedded in paraffin wax; sections were cut at 4-6 µm by microtome and transferred to slides
- tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope
- For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group)
• one animal euthanized pre-terminally during the study in Low dose group
• all macroscopic findings (abnormalities), except of minor order from all animals,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups
- special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure
- detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma
- special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow, and blood smears if examined)
- special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity

THYROID HORMONE ANALYSIS
- at the first instance, samples for the adult males were assessed for T4 levels
- for the assessment of T4 hormone measurement, a competitive immunoassay method was used

Postmortem examinations (offspring):

BLOOD SAMPLING FOR THYROID HORMONE ANALYSIS
- blood samples were taken by venepuncture decapitation into tubes containing K3-EDTA as anticoagulant as follows:
• from up to two pups per litter on PND4 (females if possible),
• from two pups per litter on PND13
- the collected pup blood (serum) samples were pooled by litter; serum T4 levels determination was made in PND13 pups’ serum samples
- the timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days (on the morning of the necropsy day)

NECROPSY
- dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities, with special attention to reproductive organs
- presence of nipples/areolae in the PND13 male pups was also recorded

THYROID HORMONE ANALYSIS
- at the first instance, samples for the PND13 pups for T4 levels
- for the assessment of T4 hormone measurement, a competitive immunoassay method was used

Statistics:

The statistical evaluation of data (labelled as † in the lists below) was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS 9.3 (when using Provantis).

Reproductive indices:

- Female mating index
- Female fertility index
- Gestation index
- Male mating index
- Male fertility index

Offspring viability indices:

- Survival Index of pups on postnatal Days 0, 4 and 13
- Sex ratio % on postnatal Days 0, 4 and 13

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related clinical symptoms were recorded in any of the dose groups.
Red discharge around left eye was recorded for one Mid dose male from Day 13 to Day 20. The longevity of the symptom was 8 days.
Fur thin was observed for one Low dose female and one Mid dose male from Day 49 to Day 54 and from Day 14 to Day 20, respectively. The longevity of the symptom was 6 and 7 days, respectively.
Nodule around the urogenital area was observed for one Mid dose female from Day 45 to Day 51. The longevity of the symptom was 7 days.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One Low dose female was preterminally euthanized on Day 40 of the study due to clinically observed prolapse of the vagina. Piloerection, red discharge from vagina and vaginal prolapse were recorded for this animal on Day 39 and/or Day 40. This was unrelated to test item treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item related adverse effect on food consumption was seen in any test item treated group (males and females).
Statistically significantly increased (by 7.6%, p>0.05) food consumption was observed in the High dose male group from Day 0 to Day 7. Considering the whole period, the food consumption in the dose groups was similar to the Control (male and female).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly decreased prothrombin time (p<0.05) was observed in the High dose male group. Statistically significantly decreased relative reticulocyte (p<0.05) was observed in the Low dose female group. Data were within the historical control range hence it was not considered as a test item related effect.

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Description (incidence and severity):

Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item-related findings were seen in examined males and females from the High dose groups (1000 mg/kg bw/day). The observed microscopic changes were without meaningful differences in the severity and incidence between Control and High dose animals. All changes were considered as a common background or incidental.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Pre-exposure period:
Each female selected for the study showed acceptable cycles (mean cycle length of 4.00 days was observed in the Control, Low and High dose groups and 4.33 in the Mid dose group) before starting the treatment period. For one Mid dose female (#3507) prolonged dioestrus (irregular cycle) was observed.

Exposure period (pre-mating and mating periods):
No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 4.00, 3.98, 4.00 and 4.00 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded in one case for the Control and for the Low dose groups, respectively. This fact was considered as being an occasional finding, not being a test item related effect.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14.

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

Based on the anhydrous form. The equivalent NOEL for the monohydrate form (test item) is 1089 mg/kg bw/day.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

Based on the anhydrous form. The equivalent NOEL for the monohydrate form (test item) is 1089 mg/kg bw/day.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Cold to touch was observed for 4 Control, 1 Low dose and one High dose pups. Cyanosis was observed for 4 Control, 2 Low dose and one High dose pups. Kink in tail was observed for one Control pup. Fur thin was observed for 10 Low dose pups. Tail tip was observed for one Mid dose pup.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item effect on mortality or survival of the pups (F1 generation).
Statistically significantly decreased number of post-natal mortality (PND0-13) was observed (p<0.05) in the Mid and High dose groups. The values were within the historical control range of the rat strain used, and decreased mortality is not an adverse effect. These differences were considered to be incidental and unrelated to test item exposure.
There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in the other dose groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related differences in the offspring body weights or weight gains in Low, Mid and High dose groups when compared to the controls. The measured values were within the range commonly recorded for this strain and age.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There was no nipples/areolae presence seen in any of the male pups on PND13.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The thyroid gland weight (absolute and body related) in the Mid dose male group was increased compared to the control but without significance.
The absolute thyroid gland weight for the PND13 pups was not statistically significantly different from the control group in any of the dose group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related changes were observed in macroscopically examined pups.

Histopathological findings:

not examined

Description (incidence and severity):

No histopathological examination was performed on pups (F1 generation).

Other effects:

no effects observed

Description (incidence and severity):

There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development.

There were no test item related effects on the T4 hormone level and the thyroid gland weight for any of the adult males and PND13 pups.
The thyroid hormone level for the PND13 pups was not statistically significantly different from the control group in any of the dose group.
The measurement of the thyroid hormone levels in the PND4 pups and adult females, and histology of pup thyroids were not performed as it was not deemed necessary by the Study Director.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Evidence of suckling was recorded for all live born pups in the study except the pups of one Control dam (#1505).

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

Based on the anhydrous form. The equivalent NOEL for the monohydrate form (test item) is 1089 mg/kg bw/day.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The daily administration of Glycyl-L-Glutamine anhydrous (CAS: 13115-71-4; EC: 700-144-0) by oral gavage to Wistar rats at dose levels up to 1000 mg/kg bw/day in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 did not result in any toxicological relevant finding. Thus, the overall NOEL of this study is 1000 mg/kg bw/day covering systemic and reproductive toxicity of the parenteral generation as well as development and survival of the F1 generation.