Dl-Proline, 5-oxo-, compd. with N2-coco acyl-l-arginine Et ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 2016 to 23 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dl-Proline, 5-oxo-, compd. with N2-coco acyl-l-arginine Et ester

EC Number:

305-928-2

EC Name:

Dl-Proline, 5-oxo-, compd. with N2-coco acyl-l-arginine Et ester

Cas Number:

95370-65-3

IUPAC Name:

DL-Proline, 5-oxo-, compd. with N2-coco acyl-L-arginine Et ester

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Based on the results of the preliminary tests, 50 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in at least five steps to obtain at least six dosing formulations for lower doses. The maximum test concentration was 1000 μg test item/plate.

Method

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- Methods for maintenance in cell culture if applicable: The strains are stored at -80 ± 10 ºC in the Culture Collection of the Microbiological Laboratory of CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory Water Bath Shaker.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
The Typical Composition (g/1000 mL) of Minimal Glucose Agar:
Glucose 20.0 g, Magnesium sulfate 0.2 g, Citric acid 2.0 g, di-Potassium hydrogen phosphate 10.0 g, Sodium ammonium hydrogen phosphate 3.5 g, Agar agar 13.0 g, Distilled water q.s. ad 1000 mL.
Minimal glucose agar plates were provided by Merck and used in the Preliminary Experiment I, Preliminary Experiment II, in the Initial Mutation Test and in the Confirmatory Mutation Test.

Nutrient Broth No.2:
Nutrient Broth No.2 25.0 g + Distilled water q.s. ad 1000 mL. Sterilisation was performed at 121oC in an autoclave.

Nutrient Agar:
Nutrient Agar 20.0 g + Distilled water q.s. ad 1000 mL. Sterilisation was performed at 121oC in an autoclave.

Top Agar for Salmonella typhimurium Strains:
Agar solution: Agar Bacteriological 4.0 g, NaCl 5.0 g, Distilled water q.s. ad 1000 mL. Sterilisation was performed at 121 °C in an autoclave.
Histidine – Biotin solution (0.5 mM): D-Biotin (F.W. 244.31) 122.2 mg, L-Histidine x HCl x H2O (F.W. 209.63) 104.8 mg, Distilled water q.s. ad 1000 mL. Sterilisation was performed by filtration using a 0.22 μm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100 mL, Agar solution 900 mL.

- Properly maintained: Yes
- Periodically 'cleansed' against high spontaneous background: Yes. The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly.
Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011 to 2015 were (as guide) as follows: Salmonella typhimurium TA98: 9-46, TA100: 54-210, TA1535: 1-46, TA1537: 1-24.

Additional strain / cell type characteristics:

other: TA98/100: carries the muc+ gene and an ampicillin resistance transfer factor (R-factor)

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- Methods for maintenance in cell culture if applicable: The strains are stored at -80 ± 10 ºC in the Culture Collection of the Microbiological Laboratory of CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37oC in a Gyrotory Water Bath Shaker.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
The Typical Composition (g/1000 mL) of Minimal Glucose Agar:
Glucose 20.0 g, Magnesium sulfate 0.2 g, Citric acid 2.0 g, di-Potassium hydrogen phosphate 10.0 g, Sodium ammonium hydrogen phosphate 3.5 g, Agar agar 13.0 g, Distilled water q.s. ad2 1000 mL.
Minimal glucose agar plates were provided by Merck and used in the Preliminary Experiment I, Preliminary Experiment II, in the Initial Mutation Test and in the Confirmatory Mutation Test.

Nutrient Broth No.2:
Nutrient Broth No.2 25.0 g + Distilled water q.s. ad 1000 mL. Sterilisation was performed at 121 °C in an autoclave.

Nutrient Agar:
Nutrient Agar 20.0 g + Distilled water q.s. ad 1000 mL. Sterilisation was performed at 121 °C in an autoclave.

Top Agar for Salmonella typhimurium Strains:
Agar solution: Agar Bacteriological 4.0 g, NaCl 5.0 g, Distilled water q.s. ad 1000 mL. Sterilisation was performed at 121 °C in an autoclave.
Histidine – Biotin solution (0.5 mM): D-Biotin (F.W. 244.31) 122.2 mg, L-Histidine x HCl x H2O (F.W. 209.63) 104.8 mg, Distilled water q.s. ad 1000 mL. Sterilisation was performed by filtration using a 0.22 μm membrane filter.


Complete Top Agar for Escherichia coli strain: Nutrient Broth 50 mL, Tryptophan solution (2 mg/mL) 2.5 mL, Agar solution 947.5 mL.
- Properly maintained: Yes
- Periodically 'cleansed' against high spontaneous background: Yes. The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly.
Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to tryptophan (Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011 to 2015 were (as guide) as follows: Escherichia coli WP2 uvrA: 11-82.

Additional strain / cell type characteristics:

other: Defective in excision repair and has rfa mutation

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Escherichia coli WP2 uvrA strain with and without metabolic activation: 1000, 316, 100, 31.6, 10, 3.16, 1, 0.316 and 0.1 μg/plate
Salmonella typhimurium strains with metabolic activation: 100, 31.6, 10, 3.16, 1, 0.316 and 0.1 μg/plate
Salmonella typhimurium strains without metabolic activation were 31.6, 10, 3.16, 1, 0.316, 0.1, 0.0316 and 0.01 μg/plate.

Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test I-II (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test same concentrations were used.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO and Distilled water
- Justification for choice of solvent/vehicle: Both DMSO and Distilled water were used as vehicle controls depending on the solubility of the test material and the solubility of strain specific positive chemicals.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine 4 µg/plate in DMSO without metabolic activation for Salmonella strain TA98; 2-aminoanthracene at 2 µg/plate in DMSO with metabolic activation for all Salmonella strains and 50 µg/plate for E. coli.

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

PRELIMINARY COMPATABILITY TESTS
The solubility of the test material was examined using Distilled water, Dimethyl sulfoxide (DMSO) and Acetone. The formulations at 100 mg/mL concentration using these vehicles were suspensions. After 4 minutes ultrasonic bath no change was observed. The formulations at 50 mg/mL concentration using these vehicles were still suspensions; however after 4 minutes ultrasonic bath the formulation using DMSO as vehicle became a clear solution. The other two formulations showed no change. Therefore DMSO was selected as vehicle (solvent) for the study. The obtained stock formulation (100 or 50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

PRELIMINARY CONCENTRATION RANGE FINDING TESTV I-II (INFORMATORY TOXICITY TEST)
Based on the solubility test, in the Preliminary Concentration Range Finding Test I 50 mg/mL stock formulation was prepared in the vehicle, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test material (if possible). In the Preliminary Concentration Range Finding Test I-II the pre-incubation method was used.
In the Preliminary Concentration Range Finding Test I, excessive cytotoxicity was observed in Salmonella typhimurium TA98, TA100 strains with and without metabolic activation at several concentrations. To find the correct concentration range an additional experiment was performed (Preliminary Concentration Range Finding Test II) in both strains. The experimental conditions were the same as in the Preliminary Concentration Range Finding Test I. The following concentrations were tested in both strains without metabolic activation: 10, 3.16, 1, 0.316, 0.1 and 0.0316 μg/plate of the test material; in both strains with metabolic activation: 31.6, 10, 3.16, 1 and 0.316 μg/plate of the test material.

PROCEDURE
A pre-incubation method was used in the preliminary experiments and main tests of the study as requested by the Sponsor. In the pre-incubation procedure bacteria were exposed to the test material both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. The test material and other components were prepared freshly.
Before the overlaying, 50 μL of test material formulations or its vehicle (or positive reference controls or their solvent), 100 μL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (nonactivated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test material. These tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37 °C in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for 48 ±1 hour.

NUMBER OF REPLICATIONS: Testing was performed in triplicate

COLONY COUNTING: The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test material treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test material and for the controls using Microsoft Excel™ software. Mutation factor (MF) is the mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Evaluation criteria:

The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analysable concentrations were presented in all strains of the main tests.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY COMPATABILITY TEST
Based on the available information and the results of the solubility testing, DMSO was selected as vehicle (solvent) of the study.

PRELIMINARY CONCENTRATION RANGE FINDING TESTS
In the Preliminary Concentration Range Finding Test I-II, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
Precipitate / slight precipitate were observed in all examined Salmonella typhimurium strains without and with metabolic activation at 5000, 2500 and 1000 μg/plate concentrations in the Preliminary Concentration Range Finding Test.
Inhibitory, cytotoxic effect of the test material(pinpoint colonies, absent / reduced /slightly reduced background lawn development) was observed in Preliminary Concentration Range Finding Test I in all examined Salmonella typhimurium strains with and without metabolic activation at all concentrations, except in Salmonella typhimurium strain TA98 with metabolic activation at 10 μg/plate concentration.
Inhibitory, cytotoxic effect of the test material (reduced / slightly reduced background lawn development) was observed in Preliminary Concentration Range Finding Test II in all examined Salmonella typhimurium strains with metabolic activation at 31.6 μg/plate concentration, in Salmonella typhimurium TA98 strain without metabolic activation at 10 μg/plate concentration and in Salmonella typhimurium TA100 strain without metabolic activation at 10 and 3.16 μg/plate concentrations.

INITIAL AND CONFIRMATORY MUTATION TEST
In the Initial Mutation Test (using the pre incubation method), the highest revertant rate was observed in Salmonella typhimurium TA98 bacterial strain at 1 μg/plate concentration with metabolic activation (the observed mutation factor value was 1.39). However, there was no clear dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
In the Confirmatory Mutation Test (using the pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 3.16 μg/plate concentration with metabolic activation (the observed mutation factor values were: MF: 1.58). However, there was no clear dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were within the historical control range in all cases, thus they were considered as biological variability of the test system.
Precipitate was not detected in the Initial Mutation Test and Confirmatory Mutation Tests.
Inhibitory, cytotoxic effect of the test material (pinpoint colonies, absent / reduced /slightly reduced background lawn development) was observed in the Initial Mutation Test and in the Confirmatory Mutation Test in Salmonella typhimurium TA98 bacterial strain without metabolic activation at 31.6 and 10 μg test material/plate and with metabolic activation at 100 and 31.6 μg test material/plate; in Salmonella typhimurium TA100 bacterial strain without metabolic activation at 31.6, 10 and 3.16 μg test material/plate and with metabolic activation at 100 and 31.6 μg test material/plate; in Salmonella typhimurium TA1535 bacterial strain without metabolic activation at 31.6 and 10 μg test material/plate and with metabolic activation at 100 μg test material/plate; in Salmonella typhimurium TA1537 bacterial strain without metabolic activation at 31.6, 10 and 3.16 μg test material/plate and with metabolic activation at 100 μg test material/plate; in Escherichia coli WP2 uvrA bacterial strain without metabolic activation at 1000, 316, 100 and 31.6 μg test material/plate and with metabolic activation at 1000 and 316 μg test material/plate.

Any other information on results incl. tables

Validity of the Tests

Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the test material had no mutagenic activity in the applied bacterium tester strains.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

The study included a Preliminary Compatibility Tests, a Preliminary Concentration Range Finding Test I-II (Informatory Toxicity Test), an Initial Mutation Test (Pre- Incubation Method), a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of a solubility test, the test item was formulated in DMSO. Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test I. In the Preliminary Concentration Range Finding Test I, excessive cytotoxicity was observed inSalmonella typhimuriumTA98, TA100 strains with and without metabolic activation at several concentrations. To find the correct concentration range an additional experiment was performed (Preliminary Concentration Range Finding Test II) in both strains. The following concentrations were tested in both strains without metabolic activation: 10, 3.16, 1, 0.316, 0.1 and 0.0316 μg/plate of the test item; in both strains with metabolic activation: 31.6, 10, 3.16, 1 and 0.316 μg/plate of the test item. Based on the results of the Range Finding Tests, the test item concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 1000, 316, 100, 31.6, 10, 3.16, 1, 0.316 and 0.1 μg/plate atEscherichia coliWP2uvrAstrain with and without metabolic activation and inSalmonella typhimuriumstrains with metabolic activation were 100, 31.6, 10, 3.16, 1, 0.316 and 0.1 μg/plate and inSalmonella typhimuriumstrains without metabolic activation were 31.6, 10, 3.16, 1, 0.316, 0.1, 0.0316 and 0.01 μg/plate.

In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.

Precipitate was not detected in the Initial Mutation Test and Confirmatory Mutation Tests.

Inhibitory, cytotoxic effect of the test item (pinpoint colonies, absent / reduced / slightly reduced background lawn development) was observed in the main tests in all examined with and without metabolic activation at several concentrations.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test materialdid not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test material had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.