L-Glutamine, L-alanyl-

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 and TA1537 with and without metabolic activation

Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and in E. coli strain WP2uvrA with and without metabolic activation

Cytogenicity in mammalian cells (similar to OECD 473, Chromosome aberration test): negative in Chinese hamster lung cells with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1994-02-10 to 1994-02-18

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:

26 May 1983

Deviations:

yes

Remarks:

no S. typhimurium T102 or E. coli strain tested; only 2-aminoanthracene used as positive control with S9 mix

GLP compliance:

yes (incl. QA statement)

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:

0, 62, 185, 556, 1667, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

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:
- Test substance added in agar (plate incorporation, both experiments)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 72 h at 37 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

Evaluation criteria:

A positive response is 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.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: at 5000 µg/plate slightly less dense background lawn of bacterial growth than in concomitant control plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No precipitation of the test substance was observed.

RANGE-FINDING/SCREENING STUDIES:
A preliminary test to assess the toxicity of the test substance to the bacteria was not performed as no toxicity was expected. The cytogenicity test was incorporated into the first mutagenicity assay.

 

Table 1: Detailed results of the first experiment

Dose µg/plate		TA 1535		TA 1537		TA 98		TA 100
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	Mean SD	22 25 18   22 4	18 14 17   16 2	16 13 16   15 2	20 15 12   16 4	31 30 33   31 2	47 45 52   48 4	163 197 159   173 21	177 167 181   175 7
62	Mean SD	19 19 27   22 5	13 11 23   16 6	14 12 17   14 3	17 22 12   17 5	44 40 40   41 2	52 70 46   56 12	211 175 176   187 21	174 211 190   192 19
185	Mean SD	30 22 25   26 4	24 15 19   19 5	28 24 19   24 5	19 19 13   17 3	51 36 37   41 8	62 56 47   55 8	170 172 195   179 14	175 177 170   174 4
556	Mean SD	20 23 25   23 3	23 20 26   23 3	14 12 22   16 5	23 20 19   21 2	33 38 51   41 9	53 53 58   55 3	196 174 183   184 11	178 187 174   180 7
1667	Mean SD	30 26 19   25 6	23 15 18   19 4	13 15 12   13 2	19 13 14   15 3	34 38 39   37 3	52 58 39   50 10	175 192 181   183 9	193 178 172   181 11
5000	Mean SD	12 18 23   18 6	14 12 15   14 2	11 14 16   14 3	16 22 15   18 4	40 37 38   38 2	37 59 45   47 11	107 131 126   121 13	177 208 164   183 23
Positive Control	Mean SD	540 502 502   515 22	473 487 472   477 8	1085 904 958   982 93	195 148 193   179 27	452 462 526   480 40	1367 1294 1335   1332 37	604 608 619   610 8	1663 1782 1689   1711 63

SD: Standard deviation

 

Table 2: Detailed results of the second experiment

Dose µg/plate		TA 1535		TA 1537		TA 98		TA 100
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	Mean SD	28 34 25   29 5	17 20 16   18 2	25 23 20   23 3	19 19 30   23 6	30 37 55   41 13	57 63 53   58 5	162 186 157   168 16	187 79 186   184 4
62	Mean SD	17 17 24   19 4	15 22 19   19 4	18 16 22   19 3	16 17 25   19 5	36 38 42   39 3	38 58 48   48 10	163 163 174   167 6	165 140 173   159 17
185	Mean SD	29 11 17   19 9	16 16 25   19 5	25 20 24   23 3	16 20 14   17 3	30 33 34   32 2	60 50 51   54 6	165 177 184   175 10	150 165 159   158 8
556	Mean SD	22 27 29   26 4	20 22 24   22 2	13 14 12   13 1	16 16 17   16 1	55 40 42   46 8	57 58 61   59 2	174 207 167   183 21	189 161 164   171 15
1667	Mean SD	35 40 24   33 8	27 15 22   21 6	27 23 25   25 2	16 14 22   17 4	47 48 44   46 2	59 68 40   56 14	173 188 175   179 8	173 164 189   175 13
5000	Mean SD	25 20 20   22 3	17 16 19   17 2	20 17 27   21 5	22 22 15   20 4	45 46 42   44 2	67 53 68   63 8	143 117 127   129 13	189 170 192   184 12
Positive Control	Mean SD	499 445 506   483 33	445 412 424   427 17	1408 1208 1380   1332 108	185 145 188   173 24	493 524 495   504 17	1528 1446 1539   1504 51	633 600 616   616 17	1754 1743 1687   1728 36

SD: Standard deviation

Conclusions:

The test substance shows no mutagenic activity in Salmonella typhimurium TA1535, TA 1537, TA98 or TA 100, either in the absence or in the presence of the S9-mix.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

For cell lines:
- Cell cycle length: 17 h

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: cells were grown in Eagle’s Minimal Essential Media with HEPES buffer and Earle’s Salts, supplemented with 10% foetal bovine serum and antibiotics and incubated at 37 °C with 5% CO2 in air

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: male Sprague–Dawley rats (approx. 250 g) induced with 3 consecutive daily doses of 80 mg phenobarbitone/kg bw and 100 mg beta-naphthoflavone/kg bw
- quality controls of S9: metabolic capability tested

Test concentrations with justification for top dose:

Preliminary toxicity test: 34 to 2180 µg/mL
First and second experiment: 0 to 2180 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: sterile distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION first experiment
- Exposure duration: 6 h with and without S9-mix
- Recovery phase: 18 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

DURATION second experiment
- Exposure duration: 24 h or 48 h without S9-mix
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h or 48 h

SPINDLE INHIBITOR (cytogenetic assays): 0.1 µg/ml Colcemid two hours prior to the end of the incubation
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS:
- each concentration in duplicate

NUMBER OF CELLS EVALUATED:
- the first 100 consecutive well-spread metaphases from each culture to a total of 200 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index calculated from 1000 cells counted & metaphases recorded

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Key result

Species / strain:

other: Chinese Hamster Lung (CHL) 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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no effect on pH when dosed in cell culture
- Data on osmolality: no effect on osmolality when dosed in cell culture

RANGE-FINDING/SCREENING STUDIES:
The test item at concentrations ranging from 34 to 2180 µg/mL was incubated with cell cultures for 24 and 48 h without S9-mix, and for 6 h both with and without S9-mix. The highest concentration analysed was selected based on the recommendation limit dose.

Conclusions:

Under the test conditions applied, the test substance does not induce chromosome aberrations in cultured mammalian somatic cells, in the presence and absence of a metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon for S. typhimurium strains and trp operon for E. coli strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: University of California, Berkeley, USA

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: British Industrial Biological Research Association, UK

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : male Sprague–Dawley rats (approx. 250 g) induced with 3 consecutive daily doses of 80 mg phenobarbitone/kg bw and 100 mg beta-naphthoflavone/kg bw
- quality controls of S9: metabolic capability tested

Test concentrations with justification for top dose:

Range finding assay: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Main study and repeat study: 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: sterile distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h at 37 °C

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

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 uvr A

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:
In the range finding test, the substance was tested up to 5000 µg/plate in the absence and presence of S9 mix in S. typhimurium TA100 and E. coli WP2uvrA. No reduction of the bacterial lawn and no biologically relevant decrease in the number of revertants were observed. The highest concentration analysed was selected based on the recommended limit dose in OECD guideline 471.

Conclusions:

Under the conditions of the test the test substance is considered to be not mutagenic in S. typhimurium TA1537, TA1535, TA100, and TA98 as well as E. coli WP2uvrA, in the presence and absence of a metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria

An Ames test was described in a publication (Oda_et_al_2008_Ames), which was conducted similarly to OECD guideline 471 and compliant with GLP. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA were exposed to the test substance, vehicle and positive controls in the presence and absence of metabolic activation (S9 mix). A range-finding assay with concentrations in the range of 0.15 to 5000 µg/plate was conducted prior to the main mutagenicity test, with and without S9 mix. Based on the results of this pre-test, test substance concentrations in the range of 50 to 5000 µg/plate were chosen for the mutagenicity assay. In two independent experiments with and without metabolic activation, the bacteria were exposed to the test substance or corresponding controls in agar (plate-incorporation test). After 48 h of exposure, the bacterial background lawn was inspected and the number of revertant colonies was determined.

There was no cytotoxicity up to the highest concentration tested in the presence and in the absence of S9 mix. Treatment with the test did not increase the number of revertant colonies at any dose level for any of the bacteria strains tested, with or without S9 mix. The vehicle and positive control showed the expected results, indicating that the test system was sensitive and valid.

Based on the experimental results, the test item is considered not mutagenic in bacteria, with and without metabolic activation.

The test substance was also investigated for its potential to cause gene mutation in bacteria in an Ames test which was performed according to OECD guideline 471 and in compliance with GLP (93-0104-DNT). However, compared to the current OECD guideline 471, there are deviations: only four bacterial strains were tested and only 2-aminoanthracene was used as positive control substance in the presence of a metabolic activation system.

Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to the test substance, vehicle (water) or positive controls in the presence and absence of metabolic activation (S9 mix). In two independent experiments, test substance concentrations in the range of 62 to 5000 µg/plate were added to the agar (plate-incorporation method) with and without S9 mix. After 72 h of exposure at 37 °C, the bacterial background lawn was inspected and the number of revertant colonies were counted for each strain.

There was no precipitation observed up to and including the highest concentration tested. At 5000 µg/plate in the absence of metabolic activation, slight indications of cytotoxicity were noted. Treatment with the test item did not increase the number of revertant colonies for any tester strain, neither in the presence, nor in the absence of metabolic activation. The mean number of revertant colonies was comparable to the spontaneous reversion rate of vehicle controls. The positive control induced a strong increase in the number of revertant colonies, demonstrating the functionality of the test. Under the conditions of the experimental study, there is no indication of mutagenicity in bacteria, in the presence and in the absence of metabolic activation.

In vitro cytogenicity in mammalian cells

The Genetic toxicity in vitro was tested in a chromosomal aberration assay that was published (Oda_et_al_2008_CA). The test was performed according to OECD guideline 473 and compliant with GLP. In two independent experiments, duplicate cultures of Chinese hamster lung (CHL) cells were exposed to the test substance in the presence and absence of metabolic activation (S9 mix). The cells were exposed to test substance  concentrations of 1.090, 1.635 and 2.180 µg/mL for 6 h in the presence and absence of S9 mix (first experiment, sampling 24 h after start of exposure) and for 24 or 48 h in the absence of S9 mix (second experiment, sampling at the end of exposure). A total of 200 metaphases for scored for each condition and the mitotic index (MI) indicating cytotoxicity was assessed for each condition.

There was no cytotoxicity up the highest concentration in the presence or in the absence of metabolic activation. Treatment with the test substance did not induce the mean incidence of chromosomal aberrations at any dose level, in the presence and in the absence of S9 mix. Aberrant cell frequencies induced by the positive control substances cyclophosphamide (with metabolic activation) and mitomycin C (without metabolic activation) demonstrated clastogenic activity and confirmed the validity of the test system.

Based on the results of the study the test substance does not induce chromosomal aberrations in mammalian cells.

Conclusion

In conclusion, the available experimental data on gene mutation in bacteria and chromosome aberration in mammalian cells in vitro do not indicate that the test substance is mutagenic or clastogenic in vitro, in the presence and in the absence of metabolic activation.

Justification for classification or non-classification

The available data on genetic toxicity in vitro do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP), 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.