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

Endpoint summary

Description of key information

In a 14-day Dose Range Finding (DRF) toxicity study where Glycyl-L-Glutamine was administered by oral gavage to Wistar rats for 14 consecutive days, at 1000 mg/kg bw/day there were no specific adverse effects identified.

The daily administration of Glycyl-L-Glutamine anhydrous 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.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2021-11-24 - 2022-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

This study is a non-GLP dose range finding study for the OECD TG 422 study.

Principles of method if other than guideline:

The objective of the study was to obtain preliminary information on the toxic potential of the test item Glycyl-L-Glutamine monohydrate administered daily to Wistar rats by oral gavage at three dose levels for 14 consecutive days. The results helped to determine the dose levels for a subsequent OECD TG 422 study.

GLP compliance:

no

Limit test:

no

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: 8 weeks old at start
- Weight at study initiation: Males: 303-349 g, females: 228-240 g
- Fasting period before study: none
- Housing: group housed (2 animals/sex/group per cage)
- 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: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 – 22.7 °C (target: 19-25 °C)
- Humidity (%): 26 - 62% (target: 30-70%)
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 2021-11-24 - 2021-12-08

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is one of the possible routes of human exposure.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Water content of the supplied product was taken into consideration during sample preparation (correction factor of 1.089 was applied).
The test item was formulated in the selected vehicle (distilled water), as a visibly stable
homogenous solution at the appropriate concentrations according to the dose level and volume
selected in the Pharmacy of the Test Facility. The formulations were stirred with a magnetic
stirrer from the preparation until completion of each treatment.
Formulations were prepared fresh every day prior to administration to animals.

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:

14 days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

equivalent to 32.67 mg/kg bw/day (test item, monohydrate form)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

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

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

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

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:

4 males / 4 females (Control, 300 mg/kg bw/day, 1000 mg/kg bw/day)
4 males / 0 females (30 mg/kg bw/day, 100 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Details on study design:

Four male and four female animals per group were treated daily for 14 consecutive days in
order to obtain preliminary information on the potential toxicity of the test item following
repeated administration at 4 dose levels. The control group was treated with the vehicle only
(distilled water). All animals were terminated one day after the 14th dose (on Day 14).
A constant dose volume of 8 mL/kg bw was administered daily to all animals by oral gavage
using a disposable (plastic) gavage tube. The individual volume of the treatment was based on
the most recent individual body weight of the animals.

Positive control:

No

Observations and examinations performed and frequency:

Mortality/Morbidity Checks:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

Cage Side Observations:
General (routine) clinical observations were made twice daily, once in the morning, before treatment (am) and once in the afternoon, after the treatment (pm). No general observation was made in the morning on those days when detailed clinical observation was scheduled.

Detailed Clinical Observations:
Detailed clinical observations were made on all animals outside the home cage in a standard arena prior to the first treatment on Day 0 (to allow for within-subject comparisons) and daily thereafter. It was performed after treatment (1-2 hours after dosing as no peak period of effects was observed on Day 0 after dosing).

Body weights:
Body weight of each animal was recorded with a precision of 1 g at randomisation (on the first day of treatment (Day 0, prior to start of treatment)), then at least twice weekly, including on Day 13 (last treatment day) and prior to necropsy (scheduled necropsy, on Day 14 (fasted)).

Food consumption:
The determination of food consumption was performed for all groups at least twice weekly. The remaining, non-consumed food was weighed with a precision of 1 g. Daily food consumption was calculated for reporting purposes.

Sacrifice and pathology:

At the end of the treatment period, prior to scheduled necropsy on Day 14, clinical pathology investigations (haematology and clinical chemistry) were conducted in all animals (after an overnight period of food deprivation).

Blood samples for clinical pathology evaluation were collected immediately prior to the scheduled necropsy, by heart puncture under pentobarbital anaesthesia. Two blood samples were collected; one for haematology (in 0.5 mL tubes with K3-EDTA, 1.6 mg/mL blood) and one to obtain serum (in 1.5 mL tubes with no anticoagulant) for clinical chemistry.

Method of Euthanasia:
At study termination, euthanasia was performed under pentobarbital anaesthesia by exsanguination.
Euthanimal 40% (400 mg/mL sodium pentobarbital solution) supplied by Alfasan Nederland BV, The Netherlands was used by intraperitoneal injection.

Scheduled Euthanasia:
All animals were euthanized at termination on Day 14. Gross necropsy was performed on each animal. Weight of selected organs was measured and selected organs and tissues were retained.

Necropsy:
After exsanguination, the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

Organ weights:
Brain, spleen, kidneys, testes, prostate, liver, thymus, epididymes, adrenals were trimmed of fat and weighed in all animals. Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated.

Tissue Collection and Preservation:
On completion of the full macroscopic examination any tissues showing macroscopic abnormality were preserved. In case of all males the testis and epididymes were preserved.

Statistics:

The statistical evaluation of data was performed using the statistical program package of SAS 9.3 (when using Provantis).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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 increased (p<0.05) MCH concentration was observed in the Mid dose male group and red cell distribution width was observed (p<0.05) in the High dose female group. Statistically significantly decreased mean cell haemoglobin (p<0.05) was observed in the Mid dose female group. All the data were within the historical control range and no clear treatment related trends were observed, therefore these were not considered as test item related effects.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly decreased urea concentration was observed in treated male animals, when compared to Control group, but were within the historical control range, therefore it was not considered as an adverse test item related effect.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly decreased brain related weight of the kidney was measured in the High dose female group. The kidney weights were within the historical control range and the organs appeared to be normal. This finding alone is not considered to be an indication of an adverse effect.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

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

Sex:

male/female

Key result

Critical effects observed:

no

Conclusions:

In conclusion, based on this 14-day Dose Range Finding (DRF) toxicity study where Glycyl-L-Glutamine monohydrate was administered by oral gavage to Wistar rats for 14 consecutive days, at 1000 mg/kg bw/day there were no specific adverse effects identified.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

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

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

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

Route of administration:

oral: gavage

Details on route of administration:

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.

Vehicle:

water

Details on oral exposure:

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

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

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

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

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

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

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

Observations and examinations performed and frequency:

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

OESTRUS CYCLE MONITORING
- 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

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

OBSERVATION OF THE DELIVERY PROCESS, OFFSPRING 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
- 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

Sacrifice and pathology:

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 in case of adult animals) or decapitation (in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
• from up to two pups per litter on PND4 (females if possible),
• from all dams and two pups per litter on PPD 14 (females) / PND13 (pups),
• from all adult males at termination
- the collected pup blood (serum) samples were pooled by litter; serum T4 levels determination was made in PND13 pups’ and 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
- 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

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 PND13 pups and adult males were assessed 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).

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

Description (incidence and severity):

There were no statistically significant differences in the terminal body weights of treated animals when compared to Controls.

Parental Males:
No treatment-related effects were observed in any of the dose groups.
The absolute and relative to body weights of the spleen decreased statistically significantly (by -18.3% and -19.1%, respectively, p<0.01) in the Low dose group compared to Controls, with no dose response and no histological relevance. This was considered to be unrelated to treatment.
The absolute and relative (to body and brain) weights of the thymus increased statistically significantly (by 24.7%, 20.8% and 25.1%, respectively, p<0.05) in the Mid dose group compared to Controls with no dose response and without histological relevance. The data were within the historical control range, these statistical differences were not considered as an effect of the test item.

Parental Females:
There were no statistically significant differences among groups in the weights of the organs when compared to Controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

PARENTAL ANIMALS:
There was no evidence of test item-related findings at necropsy. All changes were incidental or a common background.

F1 GENERATION:
No test item-related changes were observed in macroscopically examined pups.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

PARENTAL ANIMALS:
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.

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

Histopathological findings: neoplastic:

no effects observed

Other effects:

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.
There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding was recorded for F1 pups at necropsy.

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

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

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

Key result

Critical effects observed:

no

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

This study followed the procedures indicated by the following internationally accepted guideline and recommendations:
• OECD Guideline No. 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (2016)
• OECD No. 43 Guidance Document on Mammalian Reproductive Toxicity Testing and Assessment (2008)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Considering that no effects were observed in the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (OECD TG 422), the substance Glycyl-L-Glutamine is not classified according to Regulation (EC) No 1272/2008.