N-glycyl-L-tyrosine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- An Ames test performed according to OECD guideline 471 and in compliance with GLPwas performed to investigate the potential of N-Glycyl-L-tyrosine Dihydrate to induce gene mutations according to the plate incorporation testand the pre-incubation test  using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The test item did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in several follow-up experiments.
In conclusion, based on the results of this study it is concluded that N-Glycyl-L-tyrosine dihydrate (CAS: 39630-46-1; EC: 211-525-1) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

- Gene mutation in mammalian cells (OECD 490, Mouse Lymphome Assay): negative in the mouse lymphoma L5178Y test system with and without metabolic activation.

- Cytogenicity / micronucleus assay (OECD 487, In Vitro Mammalian Cell Micronucleus Test): negative in human lymphocytes with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

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

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

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.

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.

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-11-10 - 2022-05-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type of cells: L5178Y/TK+/--3.7.2C mouse lymphoma cells.
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD).

For cell lines:
- Absence of Mycoplasma contamination: yes, cultures were checked for mycoplasma contamination
- Storage of stock cultures: in the ultra-low freezer set to maintain -150°C.
- Cell density: kept below 1 x 10E6 cells/mL.

MEDIA USED
- Horse serum: Horse serum was inactivated by incubation at 56°C for at least 30 minutes.
- Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) or RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
- Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum.
- Exposure medium (RPMI 1640): Cells will be exposed to the test material in basic medium supplemented with 5% to 10% (v/v) heat-inactivated horse serum.
- Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum and 5 μg/mL trifluorothymidine (TFT).
- Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum.

Additional strain / cell type characteristics:

other: deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) and therefore resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT).

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was 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 bw).

Test concentrations with justification for top dose:

Since the test material was poorly soluble in the exposure medium, the highest tested
concentration was 1000 μg/mL exposure medium. A correction factor of 1.15 was used to correct the water content.

Vehicle / solvent:

The vehicles for the test material were dimethyl sulfoxide DMSO (Dose range finding test) and RPMI 1640 (main experiments) exposure medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(exposure medium)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Cleansing:
Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in growth medium containing 10E-4 M hypoxanthine, 2 x 10E-7 M aminopterine and 1.6 x 10E-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on growth medium containing hypoxanthine and thymidine only. After this period cells were returned to growth medium for at least 1 day before starting the experiment.

Dose-range finding study:
In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 10E6 cells (10E6 cells/mL for 3 hour treatment) or 6 x 10E6 cells (1.25 x 10E5 cells/mL for 24 hour treatment) with a number of test material concentrations increasing by approximately half log steps. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test material was tested in the absence and presence of S9-mix.
Since the test material was poorly soluble in the exposure medium, the highest tested concentration was 1000 μg/mL exposure medium (using DMSO as solvent).
For the 3 hour treatment, cell cultures were exposed to the test material in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium.
For the 24 hour treatment, cell cultures were exposed to the test material in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium. The cells in the final suspension were counted with the coulter particle counter.
The surviving cells of the 3 hour treatment were subcultured twice to determine cytotoxicity. After 24 hour of subculturing, the cells were counted and subcultured again for another 24 hours, after that the cells were counted. The surviving cells of the 24 hour treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 10E5 cells/mL were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests.

Mutagenicity Test:
Eight doses of the test material were tested in the mutation assay. The test material was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods.
Per culture 8 x 10E6 cells (106 cells/mL for 3 hour treatment) or 6 x 10E6 cells (1.25 x 10E5 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate.
In the first experiment, cell cultures were exposed for 3 hours to the test material in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test material in exposure medium for 24 hours in the absence of S9-mix. For the 3 hour treatment, cell cultures were exposed to the test material in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium.
For the 24 hour treatment, cell cultures were exposed to the test material in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium. The cells in the final suspension were counted with the coulter particle counter.

Expression period:
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10E6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test material the cells were plated for determination of the cloning efficiency (CEday2) and the mutant frequency (MF).

Determination of the Mutant Frequency:
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a
96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in
non-selective medium.
For determination of the mutant frequency (MF) a total number of 9.6 x 10E5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10E5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection).
The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the CEday2 and MF were scored with the naked eye or with the microscope.

Determination of the Mutant Colonies:
The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.

Rationale for test conditions:

In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test material concentration range of 63 to 1000 μg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The vehicle for the test material was DMSO.
The highest concentration in the exposure medium was 1000 μg/mL. The pH and osmolarity at a concentration of 1000 μg/mL were 7.3 and 0.598 Osm/kg respectively (compared to 7.4 and 0.611 Osm/kg in the solvent control).
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test:
Without and with S9-mix: 31, 63, 125, 250, 500, 1000, 1250, 1500, 1750 and 2000 μg/mL exposure medium.

Evaluation criteria:

A test material is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test material is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test material is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutant frequency of MF(controls) + 126

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

First Mutagenicity Test
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test:
Without and with S9-mix: 31, 63, 125, 250, 500, 1000, 1250, 1500, 1750 and 2000 μg/mL exposure medium. The dose levels selected to measure mutant frequencies at the TK-locus were:
Without and with S9-mix: 31, 63, 125, 250, 500, 1000, 1500 and 2000 μg/mL exposure medium.
In the absence of S9-mix, the relative total growth of the highest test material concentration was 131 % compared to the total growth of the solvent controls.
In the presence of S9-mix, the relative total growth of the highest test material concentration was 76 % compared to the total growth of the solvent controls.
No biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test material either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second Mutagenicity Test
To obtain more information about the possible mutagenicity of the test material, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period.
Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 31, 63, 125, 250, 500, 1000, 1250, 1500, 1750 and 2000
μg/mL exposure medium.
The dose levels selected to measure mutant frequencies at the TK-locus were:
31, 63, 125, 250, 500, 1000, 1500 and 2000 μg/mL exposure medium.
The relative total growth of the highest test material was 92 % compared to the total growth of the solvent controls.
No biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test material. The numbers of small and large colonies in the test material
treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

The mean mutant frequency found in the solvent control cultures was within the range of the
acceptability criteria of this assay and within the 95 % control limits of the distribution of the
historical concurrent solvent control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. In addition, the mutant frequency 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.

Conclusions:

In conclusion, N-Glycyl-L-tyrosine dihydrate (CAS: 39630-46-1; EC: 211-525-1) is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01.11.2021 - 28.03.2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: histidine gene mutation
Escherichia coli: tryptophan gene mutation

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Salmonella typhimurium:
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions

Escherichia coli:
WP2uvrA Base-pair substitutions

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix.
Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (anhydrous N-Glycyl-L-tyrosine) were tested in triplicate.
The highest concentration of the test item used in the subsequent mutation assays was 5000 μg/plate. At least five different doses (increasing with approximately half-log steps) of
the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
The dose calculation was corrected for the correction factor. (The Correction factor takes into account the different molecular weights of the test item N-Glycyl-L-tyrosine Dihydrate and the anhydrous form.)

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 6

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
10E09 cells/mL
- Test substance added in medium: in agar (experiment I, III and V: direct plate assay); preincubation assay (experiment II, IV and VI)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period:
30 minutes
- Exposure duration/duration of treatment:
48 hours


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of revertants (below the laboratory historical control data
range)

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Mean values and standard deviations were calculated.

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

E. coli WP2

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:

In the first experiment, increase in the number of revertants were observed in tester strain
TA1535, TA1537 and TA98 in the absence of S9-mix in all dose levels tested. Increases of up
to 8.8-, 32- and 9.7- fold were observed, respectively. The observed increases were outside the
historical control data range.
In the second mutation experiment, dose related increases in the number of revertants were
observed in tester strain TA1537, TA98 and TA100. For tester strain TA1537 and TA100 in
the absence of S9-mix, the increases observed were 8.0- and 2.1-fold the concurrent solvent
control, respectively. The observed increases were outside the historical control data range. In
tester strains TA98 in the absence of S9-mix, increases up to 3.0-fold compared to the solvent
control were observed. However, this increase was within the historical control data range. In
all other tester strains, no increase in the number of revertants was observed upon treatment
with the test item under all conditions tested.
To verify the positive results obtained in the first experiment in tester strain TA1535, TA1537
and TA98 in the absence of S9-mix and in second experiment in tester strain TA100 in the
presence of S9-mix and in tester strain TA1537 and TA98 in the absence of S9-mix an
additional third (direct plate assay) and fourth experiment (preincubation assay) were
performed.
In the third mutation experiment, the test item was tested up to concentrations of
5000 μg/plate in the tester strains TA1535, TA1537, TA98, in the absence of S9-mix in the
direct plate assay. The test item did not precipitate on the plates at this dose level. The bacterial
background lawn was not reduced at any of the concentrations tested and no biologically
relevant decrease in the number of revertants was observed.
In the third experiment no increases were observed in tester strain TA1535, TA1537 and TA98
in the absence of S9-mix. The increases observed in the first experiment were therefore not
reproducible.
In the fourth mutation experiment, the test item was tested up to concentrations of 5000 μg/plate
in the tester strains TA1537, TA98, in the absence of S9-mix and in tester strain TA100 in the
presence of S9-mix in the pre-incubation assay. In addition, tester strain TA1535 in the absence
of S9-mix was also tested in this experiment. This was not necessary but occurred due to a
technical error. The test item did not precipitate on the plates at this dose level. The bacterial
background lawn was not reduced at any of the concentrations tested and no biologically
relevant decrease in the number of revertants was observed.
In the fourth experiment no increases were observed in tester strains TA98 and TA1535 in the
absence of S9-mix and TA100 in the presence S9-mix. However, 3-fold increases were
observed in TA1537 in the absence of S9-mix but were well within the historical data and
linked to a low value of the negative control. The increases observed in the second experiment
were therefore not reproducible.
All the increases that were observed showed negative or not biologically relevant increases in
their respective repeat experiment. But since an increase in the number of revertants was
observed in the initial first and second experiment it is not possible to confirm that the negative
results of the additional third and fourth experiment are the correct results. To verify this
negative result, an additional fifth (direct plate assay) and sixth experiment (preincubation
assay) were performed observing all tester strains that showed a 2- or 3-fold increase in one of
the previous experiments.
In the fifth experiment the test item was tested up to concentrations of 5000 μg/plate in the
tester strains TA1537, TA1537, and TA98 in the absence of S9-mix in the direct plate assay.
The test item did not precipitate on the plates at this dose level. The bacterial background lawn
was not reduced at any of the concentrations tested and no biologically relevant decrease in the
number of revertants was observed. All tester strains showed no increase in the number of
revertants confirming the previous obtain negative results in the third experiment.
In the sixth experiment the test item was tested up to concentrations of 5000 μg/plate in the
tester strains TA1537 and TA98 in the absence of S9-mix and in tester strain TA100 in the
presence of S9-mix in the pre-incubation assay. The test item did not precipitate on the plates
at this dose level. The bacterial background lawn was not reduced at any of the concentrations
tested and no biologically relevant decrease in the number of revertants was observed, except
for tester strain TA100 in the presence of S9-mix where toxicity, as evidenced by a reduced
background, was observed at the highest dose level tested. All tester strains showed no increase
in the number of revertants confirming the previous obtain negative results in the fourth
experiment.
Overall, all bacterial strains showed negative responses over the entire dose-range, i.e. no
biologically relevant, dose-related increase in the number of revertants.

Conclusions:

Interpretation of results: negative with and without metabolic activation.
The test substance N-Glycyl-L-tyrosine Dihydrate did not show mutagenic activity in S. typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA (with and without S9-mix).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

According to the performed QSAR prediction (Leadscope Enterprise model for the Bacterial Reverse Mutation Test (Ames test) in S. typhimurium in vitro) N-glycyl-L-tyrosine is non-mutagenic. The exact methodology as well as the respective training set and validation set data are proprietary to Leadscope but the Danish QSAR database published that the substance falls within the applicability domain.

 

Published data about the mutagenicity of the constituents of N-glycyl-L-tyrosine, glycine and tyrosine, do not exhibit mutagenic activity under the conditions of the present bacterial forward mutation assay.

Justification for classification or non-classification

The available data on genetic toxicity in vitro do not meet the criteria for classification according to Regulation No. (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

No classification for genetic toxicity is warranted according to the criteria of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.