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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA with and without metabolic activation.

Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 with and without metabolic activation.

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:

2021-11-05 - 2021-12-23

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 1.089 based on molecular weight differences of the monohydrate form (CAS 172669-64-6; test item) and the anhydrous form (CAS 13115-71-4) 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 35 – 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.8 – 37.3°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.

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 7.471 and 284 mOsm/kg respectively (compared to 7.711 and 276 mOsm/kg in the solvent control).
In the dose-range finding test blood cultures were treated with 63, 125, 250, 500, 1000 and 2000 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 : 500, 1000 and 2000 μg/mL culture medium (3 hours exposure time, 27 hours harvest time). All dose levels were selected for scoring of micronuclei.
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:
To obtain more information about the possible clastogenicity and aneugenicity of the test 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 study is valid and Glycyl-L-Glutamine monohydrate is not clastogenic or aneugenic in human lymphocytes under the experimental conditions of this study.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-11-01 - 2021-12-14

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:

The highest tested concentration was the recommended 2000 μg/mL exposure medium. A correction factor of 1.089 was used to correct for the water content.

Vehicle / solvent:

RPMI 1640 (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.
The highest tested concentration was the recommended 2000 μg/mL exposure medium.
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 125 to 2000 μg/mL in the absence of S9-mix with 3 and 24 hours treatment periods and in the presence of S9-mix with a 3 hours treatment period.
The highest concentration which did not precipitate in the exposure medium was 2000 μg/mL. The pH and osmolarity at a concentration of 2000 μg/mL were 7.41 and 0.308 Osm/kg respectively (compared to 7.52 and 0.302 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: 15.6, 31.3, 62.5, 125, 250, 500, 1000 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: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL exposure medium.
No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.
In the absence of S9-mix, the relative total growth of the highest test material concentration was 94% 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 85% 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: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL exposure medium.
No significant toxicity was observed and all dose levels were evaluated.
The relative total growth of the highest test material was 109% 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 mutant frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative 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, Glycyl-L-Glutamine monohydrate (CAS: 172669-64-6; EC: 700-144-0) is not mutagenic in the TK mutation 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:

2020-02-21 - 2020-03-09

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:

July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon, trp operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 were used as the metabolic activation system.

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate (anhydrous Glycyl-L-Glutamine)
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate (anhydrous Glycyl-L-Glutamine)
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration.
The dose calculation was corrected for the correction factor. (The Correction factor takes into account the different molecular weights of the test item Glycyl-L-Glutamine Hydrate and the anhydrous form as well as the purity of the test item.)

Vehicle / solvent:

- vehicle used: deionized water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10E08-10E09 cells/mL
- Test substance added in medium: in agar (experiment I: plate incorporation); preincubation (experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 minutes
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of revertants (below the indication factor of 0.5)

Rationale for test conditions:

To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations (3 – 5000 μg/plate) were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).
The pre-experiment is reported as main experiment I since the acceptance criteria are met.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

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:

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Glycyl-L-Glutamine Hydrate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Conclusions:

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

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 26 May, 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatstoezicht op de Volksgezondheid, Rijswijk, The Netherlands

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

Pre-test: 62, 185, 556, 1667 and 5000 μg/plate (test item glycyl-L-glutamine monohydrate), equivalent to 57, 170, 513, 1534 and 4600 μg/plate anhydrous glycyl-L-glutamine (with and without metabolic activation)
Main test (plate incorporation test): 62, 185, 556, 1667 and 5000 μg/plate (test item glycyl-L-glutamine monohydrate), equivalent to 57, 170, 513, 1534 and 4600 μg/plate anhydrous glycyl-L-glutamine (with and without metabolic activation)

Vehicle / solvent:

- Vehicle used: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2 Nitrofluorene, -S9: 2.0 µg/plate for TA98; 2-Aminoanthracene, +S9: 2.0 µg/plate for TA1535, TA98 and TA100, 5.0 µg/plate for TA1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 h at 37 °C

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn was examined microscopically and the his+ revertants were counted.

Rationale for test conditions:

A preliminary toxicity test was carried out in TA 98 in the presence and absence of metabolic activation. There was no cytotoxicity both in the absence and presence of S9 mix up to the highest tested concentration (5000 µg/plate). In view of these observations, 5000 µg/plate was chosen as the highest concentration for the mutagenicity assay, both, in the presence and absence of metabolic activation.

Evaluation criteria:

A positive response in the assay system is taken to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the vehicle, together with evidence of a dose-response.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98 and 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

Additional information on results:

RANGE-FINDING/SCREENING STUDIES
A preliminary toxicity test was performed in S. typhimurium strain TA98 using 5 concentrations of the test substance (62 – 5000 µg/plate) and the vehicle control (water), both in the presence and absence of metabolic activation. There was no toxicity and no precipitation observed up to the highest concentration tested. In view of these observations, 5000 µg/plate was chosen as the highest concentration for the mutagenicity assay, both, in the presence and absence of S9 mix.

Table 1: Results of the Ames test

Experiment 1: Plate incorporation test
Strain	TA 1535		TA 1537		TA 98		TA 100
Metabolic activation	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Vehicle control
water mean	24	20	16	24	38	58	171	160
± SD	± 4	± 4	± 2	± 2	± 6	± 6	± 11	± 7
Test item [µg/plate]
62 mean	25	17	12	19	39	59	160	164
± SD	± 2	± 3	± 3	± 8	± 5	± 1	± 7	± 2
185 mean	28	20	16	21	41	70	158	164
± SD	± 1	± 3	± 3	± 3	± 1	± 3	± 4	± 3
556 mean	30	19	14	17	38	68	164	166
± SD	± 4	± 1	± 4	± 5	± 4	± 4	± 10	± 10
1667 mean	23	18	17	14	38	61	161	171
± SD	± 3	± 4	± 1	± 3	± 7	± 9	± 6	± 6
5000 mean	19	16	17	24	35	67	140	139
± SD	± 2	± 1	± 2	± 4	± 4	± 6	± 10	± 5
Positive control
mean	359	479	1491	224	734	2044	499	2385
± SD	± 14	± 41	± 106	± 7	± 17	± 37	± 9	± 26
Experiment 2: Plate incorporation test
Strain	TA 1535		TA 1537		TA 98		TA100
Metabolic activation	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Vehicle control
water mean	25	18	20	25	41	63	177	167
± SD	± 1	± 4	± 1	± 3	± 5	± 6	± 14	± 5
Test item [µg/plate]
62 mean	21	22	18	20	40	65	172	178
± SD	± 1	± 3	± 1	± 4	± 4	± 4	± 7	± 1
185 mean	25	21	14	20	45	65	159	171
± SD	± 2	± 4	± 3	± 3	± 4	± 9	± 6	± 6
556 mean	29	20	16	17	40	72	183	175
± SD	± 2	± 3	± 3	± 3	± 5	± 6	± 6	± 6
1667 mean	27	19	21	20	40	65	168	169
± SD	± 2	± 4	± 2	± 4	± 6	± 4	± 18	± 2
5000 mean	20	19	18	22	36	67	147	144
± SD	± 3	± 3	± 2	± 2	± 6	± 2	± 11	± 6
Positive control
mean	469	360	1349	256	763	1926	556	2119
± SD	± 10	± 17	± 86	± 38	± 27	± 158	± 59	± 167

Conclusions:

Interpretation of results: negative with and without metabolic activation.
The test substance did not show mutagenic activity in Salmonella typhimurium TA1535, TA1537, TA98 or TA100 (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

Mode of Action Analysis / Human Relevance Framework

An Ames test performed according to OECD TG 471 and in compliance with GLP (2019 -5202 -DGM) was performed to investigate the potential of Glycyl-L-Glutamine Hydrate to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in two independent experiments both with and without liver microsomal activation using test item concentrations in the range of 3 to 5000 μg/plate (anhydrous glycyl-L-glutamine). The calculation of the concentration was adjusted to the purity and refer to the anhydrous form of the test item. Each concentration, including the controls, was tested in triplicate.
No precipitation of the test item occurred in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 μg/plate. In experiment II no precipitation was observed in the overlay agar on the incubated agar plates. The undissolved particles had no influence on the data recording.
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Glycyl-L-Glutamine Hydrate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

The potential of the test item to cause gene mutations in bacteria was evaluated in an Ames test performed according to OECD TG 471 and in compliance with GLP (92-0064-DNM). Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to the test item, vehicle (water) and positive controls in the presence and absence of metabolic activation (S9 mix). In a preliminary test performed with TA 98, no cytotoxicity or precipitation of the test substance in the medium was noted after and included the limit dose of 5000 µg/plate. Two independent plate-incorporation tests were performed, using test item concentrations in the range of 62 to 5000μg/plate (test item glycyl-L-glutamine monohydrate), equivalent to 57 to 4600μg/plate anhydrous glycyl-L-glutamine (with and without metabolic activation). After 72 h incubation, the background lawn of bacterial growth was assessed and the mean number of revertant colonies was determined.
There was no cytotoxicity and no precipitation was observed up to the highest dose tested, both in the presence and absence of S9 mix. Treatment with the test item did not induce a biologically relevant increase in the number of revertant colonies for any tester strain in the presence or in the absence of metabolic activation. The vehicle and positive controls demonstrated the sensitivity and validity of the test system. Based on the experimental results, the test item is not considered to be mutagenic in bacterial cells with and without metabolic activation.

 

The mutagenic potential of Glycyl-L-Glutamine monohydrate was evaluated by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions. The test was performed 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 study procedures were based on the most recent OECD TG 490.

In the first experiment, the test material was tested up to concentrations of 2000 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix.
In the second experiment, the test material was tested up to concentrations of 2000 μg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix.

In the absence of S9-mix, the test material did not induce a biologically relevant increase in the mutant frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
In the presence of S9-mix, the test material did not induce a biologically relevant increase in the mutant frequency.

In conclusion, Glycyl-L-Glutamine monohydrate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions
of this study.

 

Glycyl-L-Glutamine monohydrate was evaluated for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of the test material was tested in two independent experiments and was performed according to OECD TG 487.

In the first cytogenetic assay, the test material was tested up to 2000 μg/mL for a 3-hour exposure time with a 27-hour harvest time in the absence and presence of S9-fraction. This is the highest dose level recommended in the guideline for testing.
In the second cytogenetic assay, the test material was again tested up to 2000 μg/mL for a 24-hour exposure time with a 24-hour harvest time in the absence of S9-mix.

The number of binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C, colchicine and cyclophosphamide all produced a statistically significant increase in the number of binucleated cells with micronuclei.

The test material did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.
In conclusion, this study is valid and Glycyl-L-Glutamine monohydrate is not clastogenic or aneugenic in human lymphocytes under the experimental conditions of this study.

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.