N-glycyl-L-tyrosine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

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:

N-glycyl-L-tyrosine

EC Number:

211-525-1

EC Name:

N-glycyl-L-tyrosine

Cas Number:

658-79-7

Molecular formula:

C11H14N2O4

IUPAC Name:

glycyl-L-tyrosine

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: 10-11 weeks old at start, 12-13 weeks old at mating
- Weight at study initiation: Males: 396-447 g, females: 235-278 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): 20.0-26.0℃ (target range: 19-25°C)
- Humidity (%): 21-75% (target range: 30-70%),
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 2022-02-08 - 2022-04-18

Administration / exposure

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:

other: 1 % methyl cellulose solution

Details on oral 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 1.15 was applied).
The test item was formulated in the selected vehicle (1 % methyl cellulose solution), 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 continuously with a magnetic stirrer until completion of treatment.
Formulations were prepared fresh every day prior to administration to animals to allow their used according to stability assessment results of the analytical method validation study

VEHICLE
- Concentration in vehicle: 20 mg/mL (low dose), 60 mg/L (mid dose), 200 mg/mL (high dose)
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): SLCH2339 / 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

equivalent to 115 mg/kg bw/day (test item dihydrate form)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

equivalent to 345 mg/kg bw/day (test item dihydrate form)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

equivalent to 1150 mg/kg bw/day (test item dihydrate 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

Observations and examinations performed and frequency:

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.

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) 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

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).

Results and discussion

Results of examinations

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 the nose was recorded for one Mid dose male (#3011) on Day 27.
Piloerection was recorded for one Control male (#1010) on Days 27-28.
Slight oedema around the right cheek was recorded for one Mid dose male (#3003) on Days 15-16.
Fur thin was observed for one Control female (#1510) and one Low dose female (#2511) from Day 35 to Day 52 and from Day 7 to Day 34, respectively.

Mortality:

no mortality observed

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 High dose males 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:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related effects observed in any of the dose groups on clinical chemistry parameters.
There were sporadic statistically significant differences in the parameters, but all of them were within the historical control range or without biological relevance or without dose response, therefore these effects were not considered as test item related effects.

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 statistically significant changes were observed in the organ weight of the dosed groups compared to controls.

Parental Females
There were no test item related changes observed in the organ weights of the test item treated females.
Statistically significant organ weight changes were observed in the adrenal in the Mid and High dose females and in the thyroid/parathyroid weights of the High dose females compared to controls.
The absolute weight of the adrenals and relative to brain weights were statically higher than control in Mid and High dose groups, but not when adjusted for body weight. However, there was no dose response relationship and no histological differences between groups, hence the statistical differences were considered to be unrelated to treatment.
The absolute weight of the thyroid/parathyroids increased by 20.0%, the relative to body weights by 16.2% and relative to brain weights by 22.1% in the High dose females compared to controls. However, in the historical control database there were control groups with comparable results, and those organ weight changes were without histopathological relevance, thus were considered as not being a test item related effect.

Gross pathological findings:

no effects observed

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted in the Irwin test, landing foot splay test and grip strength measurements. Although statistically significantly (p < 0.05) increased grip strength values were recorded on the hind paws in case of the Mid and High dose females (by 31.7 %, and 34.7 %, respectively), the differences were caused by the extreme values of one female in both groups, and no similar differences were noted in case of the forelimb values. Thus, the observed results were not considered as being a test item related effect.
All dose groups of males and females had comparable locomotor activity to the Control. In all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. Statistically significantly increased distance was observed in the Mid dose group (by 126.4 %, p < 0.01) for one period (55-60 min). This was not considered as test item related effect of the lack of differences in other periods and the overall data were not similar to Control. There was no statistical significance between the test item treated animals (males and females) and the Control when evaluating the overall total travelled distance (0-60 minutes). The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In two Control (#1501 and #1505) and in one Low dose (#2503) animals, dilatation of the uterine horns was observed. In the Mid dose female (#3508), in the thymus multifocal, moderate congestion/haemorrhage was observed. In one Control male (#1001), in the prostate multifocal, minimal inflammatory cell infiltrate was detected.
In these animals, no reason for the lack of pregnancy was identified.

Histopathological findings: neoplastic:

no effects observed

Effect levels

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 dihydrate form (test item) is 1150 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 dihydrate form (test item) is 1150 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

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The daily administration of N-Glycyl-L- tyrosine (CAS: 658-79-7; 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.