Reaction mass of L-Glutamic acid, N-coco acyl derivs., monosodium salts and sodium hydrogen N-(1-oxooctadecyl)-L-glutamate

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:

13 July 2016 to 22 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997, ninth ammendment

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:

Reaction mass of L-Glutamic acid, N-coco acyl derivs., monosodium salts and sodium hydrogen N-(1-oxooctadecyl)-L-glutamate

EC Number:

915-656-1

Molecular formula:

Not specified (UVCB substance)

IUPAC Name:

Reaction mass of L-Glutamic acid, N-coco acyl derivs., monosodium salts and sodium hydrogen N-(1-oxooctadecyl)-L-glutamate

Test material form:

solid: particulate/powder

Details on test material:

-Appearance: White to pale yellow powder

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected agent.
Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

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
- 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.
- The strains are stored at -80 ± 10 °C in the testing laboratory. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- 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) or tryptophan (Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
- The viability of each testing culture was determined by plating 0.1 mL of the 10^5, 10^6, 10^7 and 10^8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The number of viable cell of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- 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.
- The strains are stored at -80 ± 10 °C in the testing laboratory. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- 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) or tryptophan (Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
- The viability of each testing culture was determined by plating 0.1 mL of the 10^5, 10^6, 10^7 and 10^8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The number of viable cell of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

-Preliminary Concentration Range Finding Test: 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate
-Initial Mutation Test and in the Confirmatory Mutation Test: 5 000, 1 581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate in all strains with metabolic activation 1 581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate in all strains without metabolic activation.
-Complementary Confirmatory Mutation Test: 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate in S. typhimurium TA1537 strain without metabolic activation and 5 000, 1 581, 500, 158.1, 50 and 15.81 μg/plate in E. coli WP2 uvrA strain without metabolic activation.
-Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test same concentrations were used. Furthermore, a Complementary Confirmatory Mutation Test was also performed based on the results of the Confirmatory Mutation Test using a modified concentration range.

Vehicle / solvent:

- Vehicle used: Distilled water
- Justification for choice of solvent/vehicle: The appropriate vehicle and the behaviour of the test material formulations with the solution of top agar and phosphate buffer were determined in a preliminary compatibility test.
-The solubility of the test material was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-Dimethylformamide (DMF). The test material was insoluble in DMF and DMSO at 100 mg/mL concentrations. However, the formulation at 100 mg/mL concentration using Distilled water as vehicle (solvent) was a clear white solution. Therefore, Distilled water was selected as vehicle (solvent) for the study. The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

For test material and positive controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene and 4-nitro-1,2-phenylenediamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: Pre-incubation

PRELIMINARY CONCENTRATION RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
-Based on the available information and the solubility and compatibility test, 100 mg/mL stock solution was prepared in Distilled water, 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 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate of the test material. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

INITIAL MUTATION TEST AND CONFIRMATORY MUTATION TEST
-Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared in Distilled water, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5 000 μg test material/plate.
-A pre-incubation method was used in the preliminary experiment 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 a 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 (non-activated 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 was 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.

Evaluation criteria:

DATA EVALUATION
-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 (Mutation factor (MF): mean number of revertants on the test material plate / mean number of revertants on the vehicle control plate) values were calculated for each concentration level of the test material and for the controls using Microsoft Excel software.

VALIDITY 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.

CRITERIA FOR A POSITIVE RESPONSE
A test material was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than twice higher than the reversion rate of the negative (solvent) control.

CRITERIA FOR A NEGATIVE RESPONSE
The test material was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

bacteria, other: S typhimurium TA98, TA100, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S. typhimurium TA98, TA100 and TA1537 strains at 5 000 μg/plate (+S9 mix) and at 1 581, 500 and 158.1 μg/plate concentrations (-S9 mix); in S. typhimurium TA1535 strains at 5 000 μg/plate (+S9 mix) and at 1 581 and 500 μg/plate (-S9 mix)

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 CONCENTRATION RANGE FINDING TEST
-Concentrations of 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test.
In the Preliminary Concentration Range Finding Test, the numbers of revertant colonies were mostly in the normal range which is the Mutation Factor between 0.8 and 1.2 (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
-Precipitate was not detected in the Preliminary Concentration Range Finding Test.
-Inhibitory, cytotoxic effect of the test material (reduced/slightly reduced background lawn development) was observed in the Preliminary Concentration Range Finding Test in Salmonella typhimurium TA98 and TA100 strains at 5 000 and 2 500 μg/plate concentration and Salmonella typhimurium TA100 strain at 1 000 μg/plate Concentration (+S9 mix) and at 5 000, 2 500, 1 000 and 316 μg/plate concentrations (-S9 mix).

INITIAL AND CONFIRMATORY MUTATION TESTS
-In the Initial Mutation Test (using the pre incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 15.81 μg/plate concentration without metabolic activation (the observed mutation factor values were: MF: 1.36). However, there was no 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 Tests (using the pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 0.5 and 0.1581 μg/plate concentration without metabolic activation (the observed mutation factor values were: MF: 1.63). However, there was no 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 or Confirmatory Mutation Tests.
-Inhibitory, cytotoxic effect of the test item (reduced/slightly reduced background lawn development) was observed in the Initial Mutation Test and in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5 000 μg/plate concentration (+S9 mix) and at 1 581, 500 and 158.1 μg/plate concentrations (-S9 mix); in Salmonella typhimurium TA1535 strains at 5 000 μg/plate concentration (+S9 mix) and at 1 581 and 500 μg/plate concentrations (-S9 mix); in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1537 strain at 158.1 μg/plate concentration (-S9 mix).

Any other information on results incl. tables

Table 1: Summary Table of the Initial Mutation Test (Pre-Incubation Method)

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Untreated DMSO Distilled water 5000 1581 500 158.1 50 15.81 5 1.581 0.5	113.3 - 108.3 - 0.0 88.0 102.7 104.7 111.0 100.7 114.0 97.3	13.0 - 11.0 - 0.0 4.0 9.3 9.0 15.0 10.3 10.0 13.3	39.0 - 36.7 - 34.0 32.3 36.0 32.0 35.7 35.7 40.0 38.3	20.7 23.0 22.3 - 0.0 5.0 14.0 17.3 20.0 29.0 23.0 19.0	5.3 9.0 10.7 - 0.0 1.0 3.3 4.3 6.7 7.3 8.0 9.0
+	Untreated DMSO Distilled water 5000 1581 500 158.1 50 15.81 5 1.581 0.5	125.3 144.7 141.7 0.0 0.0 10.3 51.0 66.3 87.7 105.3 108.0 -	16.7 9.3 13.0 0.0 4.7 9.0 10.0 11.0 7.7 10.3 10.3 -	40.0 40.0 41.0 40.3 41.7 42.3 42.7 40.3 39.0 39.7 39.7 -	31.3 29.3 28.7 0.0 22.7 24.7 23.0 31.7 27.7 27.0 27.7 -	11.3 7.0 9.0 0.0 5.7 5.0 7.3 5.0 6.7 10.3 6.0 -
Positive Controls
-	Name	SAZ	SAZ	MMS	NPD	9AA
	Concentration (µg/plate)	2	2	2	4	50
	Mean no. colonies/plate	850.7	1234.7	989.3	410.7	424.7
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2	2	50	2	2
	Mean no. colonies/plate	2528.0	217.3	215.3	2309.3	205.3

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SAZ = Sodium azide

MMS = Methyl-methanesulfonate

NPD = 4-nitro-1,2-phenylenediamine

Table 2: Summary Table of the Confirmatory Mutation Test and the Complementary Confirmatory Mutation Test (Pre-Incubation Method)

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Untreated DMSO Distilled water 5000 1581 500 158.1 50 15.81 5 1.581 0.5 0.1581	107.7 - 100.3 - 0.0 26.7 53.3 54.3 75.3 87.7 98.7 98.3 -	13.7 - 12.7 - 0.0 6.7 12.0 13.3 10.3 15.0 11.0 14.7 -	26.7 - 30.3 28.3 34.7 29.3 25.3 28.0 28.7 - - - -	26.0 24.7 26.0 - 0.0 9.7 11.3 15.3 26.0 29.0 23.0 30.0 -	6.0 8.3 6.3 - - - 4.3 8.7 6.0 6.3 8.0 10.3 10.3
+	Untreated DMSO Distilled water 5000 1581 500 158.1 50 15.81 5 1.581 0.5 0.1581	120.7 117.0 114.7 2.3 35.0 63.3 73.0 78.7 73.0 90.0 118.7 - -	12.3 9.7 14.0 0.0 10.0 11.0 11.3 14.0 14.3 15.3 10.7 - -	42.3 46.7 45.3 45.3 51.3 52.0 51.7 50.0 59.0 49.3 52.3 - -	33.3 31.0 35.3 0.0 26.3 27.0 34.0 37.0 34.0 46.0 36.0 - -	14.0 11.3 14.3 0.0 8.3 10.3 14.7 8.7 14.7 11.0 10.0 - -
Positive Controls
-	Name	SAZ	SAZ	MMS	NPD	9AA
	Concentration (µg/plate)	2	2	2	4	50
	Mean no. colonies/plate	1042.7	1262.7	824.0	342.7	397.3
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2	2	50	2	2
	Mean no. colonies/plate	2372.0	350.0	238.7	2477.3	209.3

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SAZ = Sodium azide

MMS = Methyl-methanesulfonate

NPD = 4-nitro-1,2-phenylenediamine

Preliminary Compatability Test

-Based on the available information and the results of the solubility testing, distilled water was selected as vehicle (solvent) of the study.

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 this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100 under GLP conditions using the bacterial reverse mutation assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (S. typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (E. 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.

Based on the results of a solubility test, the test material was formulated in distilled water. Concentrations of 5 000; 2 500; 1 000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 5 000, 1 581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate in all strains with metabolic activation and 1 581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate in all strains without metabolic activation. Examined concentrations in the Complementary Confirmatory Mutation Test were 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test material/plate in Salmonella typhimurium TA1537 strain without metabolic activation and 5 000, 1 581, 500, 158.1, 50 and 15.81 μg test material/plate in Escherichia coli WP2 uvrA strain without metabolic activation.

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 material 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 or Confirmatory Mutation Tests.

Inhibitory, cytotoxic effect of the test material (reduced/slightly reduced background lawn development) was observed in the Initial Mutation Test and in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5 000 μg/plate concentration with metabolic activation and at 1 581, 500 and 158.1 μg/plate concentrations without metabolic activation; in Salmonella typhimurium TA1535 strains at 5 000 μg/plate concentration with metabolic activation and at 1 581 and 500 μg/plate concentrations without metabolic activation; in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1537 strain at 158.1 μg/plate concentration without metabolic activation.

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 analyzable 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.

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.