N-glycyl-L-tyrosine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-11-10 - 2022-04-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

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: crystalline

Method

Species / strain

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes were used as test system.

Details on mammalian cell type (if applicable):

Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Prior to the mitosis (during or after exposure of the test material) the chemical cytochalasin B was added to the cultures. Cytochalasin B arrests the formation of actin filaments. Consequently, the cell is not able to divide, but nuclear division still continues. In this way, cytochalasin B allows discrimination between cells that have undergone nuclear division (binucleated) and cells that have not (mononucleated).

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg bw) and beta-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

The highest tested concentration was the recommended dose of 2000 μg/mL. A correction factor of 1.15 based on molecular weight differences of the dihydrate form (CAS 39630-46-1; test item) and the anhydrous form (CAS 658-79-7) was applied.

Vehicle / solvent:

The vehicle for the test material was culture medium: RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Environmental conditions:
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 30 – 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 32.4 – 37.6°C).

Dose-range Finding Test:
Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 47 h and thereafter exposed to selected doses of the test material for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma; 5 μg/mL) was added to the cells simultaneously with the test material at the 24 hours exposure time. A vehicle control was included at each exposure time.
The highest tested concentration was the recommended dose of 2000 μg/mL.
After 3 hours exposure to the test material in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasine B and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.
Cytotoxicity of the test material in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 2000 μg/mL.

First Cytogenetic Assay:
Lymphocytes were cultured for 48 ± 2 hours and thereafter exposed in duplicate to selected doses of the test material for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasin B (5 μg/mL) and incubated for another 24 hours. Appropriate vehicle and positive controls were included in the first cytogenetic assay. An additional experiment was performed with a concentration of 2000 μg/mL since a lower concentration was tested in the first experiment.

Second Cytogenetic Assay:
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix. Lymphocytes were cultured for 48 ± 2 hours and thereafter exposed in duplicate to selected doses of the test material with cytochalasin B (5 μg/mL) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.

Preparation of Slides:
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately after, ethanol : acetic acid fixative (3:1 v/v) was
added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper.

Cytotoxicity Assessment:
A minimum of 500 cells (with a maximum deviation of 5%) per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
Three analyzable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. In case the test material was not cytotoxic, the highest concentration analyzed was the recommended 2000 μg/mL.

Cytogenetic Assessment/Scoring of Micronuclei:
To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with Charles River Den Bosch study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei.

Evaluation criteria:

A test material is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test material is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 8.4 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Dose-range Finding Test:
A concentration of 2000 μg/mL showed no precipitation in the culture medium and was used as the highest concentration of the test material. The pH and osmolarity of a concentration of 2000 μg/mL were 7.59 and 279 mOsm/kg
respectively (compared to 7.87 and 274 mOsm/kg in the solvent control). In the dose-range finding test blood cultures were treated with 62.5, 125, 250, 490, 962, and 1852 μg test material/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.

First Cytogenetic Assay:
Based on the results of the dose-range finding test the following dose levels were selected for the first cytogenetic assay: Without and with S9-mix : 490, 962, and 1852μg/mL culture medium (3 hours exposure time, 27 hours harvest time). All dose levels were selected for scoring of micronuclei. Due to a technical error, the highest concentration tested was lower than the recommended 2000 μg/mL. Therefore, the experiment was repeated in cytogenic assay 1A using the recommended 2000 μg/mL dose level. Both in the absence and presence of S9-mix, the test material did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei.

Second Cytogenetic Assay:
material, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay: Without S9-mix : 500, 1000, and 2000 μg/mL culture medium (24 hours exposure time, 24 hours harvest time). All dose levels were selected for the scoring of micronuclei. The test material did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei.

Any other information on results incl. tables

The positive control chemicals, mitomycin C, colchine and cyclophosphamide all produced a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Applicant's summary and conclusion

Conclusions:

In conclusion, this test is valid and N-Glycyl-L-tyrosine dihydrate is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.