Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and sodium hydrogen N-(1-oxohexadecyl)-L-glutamate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 October 2013 to 16 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of sodium hydrogen N-(1-oxooctadecyl) -L-glutamate and sodium hydrogen n-(1-oxohexadecyl) -L-glutamate

IUPAC Name:

Reaction mass of sodium hydrogen N-(1-oxooctadecyl) -L-glutamate and sodium hydrogen n-(1-oxohexadecyl) -L-glutamate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White to pale yellow powder
- Storage conditions of test material: Room temperature

Method

Target gene:

Thymidine kinase, TK+/- locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI-10 medium. The RPMI-10 medium consisted of RPMI 1640 medium with the following components added (final concentration in medium): heat inactivated horse serum 10 % v/v, antibiotic-antimycotic solution 0.01 mL/mL (containing 10 000 IU/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin–B), Pluronic-F68 0.5 mg/mL and pyruvic avid 0.2 mg/mL. L-glutamine 0.3 mg/mL was also added freshly to the media. NaHCO₃ 2 mg/mL was already included in the ready to use liquid media.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

ASSAY 1
- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL
- Three hour treatment without metabolic activation: 5, 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL

ASSAY 2
- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL
- Twenty four hour treatment without metabolic activation: 2.5, 5, 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Based on a previous chromosome aberration study performed by the same laboratory, the test material was soluble in distilled water. The highest achievable concentration using this vehicle was 250 mg/mL. As this vehicle is also compatible with the test system, it was selected to be the vehicle (solvent) for this study.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
0.2 mL of RPMI-5 medium, vehicle (solvent), test material formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or 150 mM KCl (in the case of 3 h treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatments, at least 10⁷ cells were placed in each of a series of 75 cm² sterile flasks. For the 24-hour treatment, at least 4 x 10⁶ cells were placed in each of a series of 25 cm² sterile flasks.
RPMI-5 medium was made in the same way as the RPMI-10, with the exception that it contained a reduced level of heat inactivated horse serum (5 % v/v).
Cultures were incubated at 37 ± 1 °C (approximately 5 % CO₂ in air). Gentle shaking was used during the 3-hour treatments. After the treatment period, cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in 20 mL RPMI-10. The number of viable cells in the individual samples was counted manually using a haemocytometer. Measurement of pH and osmolality was also performed after the treatment period.
Where a sufficient number of cells survived, the cell density was adjusted to a concentration of 2 x 10⁵ cells/mL. Cells were transferred to flasks for growth through the expression period (maximum 25 mL of suspension) or diluted to be plated for survival.

DURATION
- Exposure duration: Assay 1: 3 hours in the presence and absence of metabolic activation. Assay 2: 3 hours in the presence of metabolic activation, 24 hours in the absence of metabolic activation.
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): about 2 weeks

SELECTION AGENT (mutation assays): trifluorothymidine (TFT). The cultures were sub-cultured daily and the cell density adjusted to 2 x 10⁵ cells/mL.

NUMBER OF REPLICATIONS: Performed in duplicate

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency and the corresponding relative survival (%) and relative total growth
Plating for survival - Cultures of cell density 2 x 10⁵ cells/mL were further diluted to 8 cells/mL. 0.2 mL of the final concentration of each culture were placed into each well of two 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ± 0.5 °C in air containing approximately 5 % (v/v) CO₂ for about two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Plating for viability - At the end of the expression period (cultures treated with TFT), the cell density in the selected cultures was determined and adjusted to 1 x 10⁴ cells/mL with RPMI-20 (20 % heat inactivated horse serum, no addition of Pluronic F-68) for plating for a viability test. Samples from these cultures were diluted to 8 cells/mL. 0.2 mL of the final concentration of each culture was placed into each well of two 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ± 0.5 °C in air containing approximately 5 % (v/v) CO₂ for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Evaluation criteria:

The test material was considered to be mutagenic in this assay if all the following criteria were met:
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency were observed in treated cultures compared to the corresponding negative (vehicle) control values at one or more concentrations.
3. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There was a significant concentration-relationship as indicated by linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 10⁶ viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value.
Results which only partially satisfied the acceptance and evaluation criteria were evaluated on a case-by-case basis.

Statistics:

The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis in any assay with or without S9

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Due to excessive toxicity in the cultures, not all assays had eight analysable concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There were no large changes in pH
- Effects of osmolality: There were no large changes in osmolality
- Precipitation: In assay 1, minimal precipitation was observed at all concentrations in at least one replicate. In assay 2 in the presence of metabolic activation (3 hour exposure) minimal precipitation was observed at all concentrations in at least one replicate. In assay 2 in the absence of metabolic activation (24 hour exposure) minimal precipitation was observed at all concentrations in at least one replicate with the exception of the lowest dose tested in the series.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed at the following concentrations: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL for 3 hour treatments in the presence and absence of metabolic activation (toxicity tests A and B, respectively) and for 24 hours in the absence of metabolic activation (toxicity test C).
All concentrations were performed in duplicate. Due to the solubility of the test material, the test solution administration in the preliminary toxicity test was 0.4 mL. Precipitate was observed at all concentrations in all three tests in at least one replicate.

In test A, relative survival at 78.13 µg/mL was 41 % on day 0 and 92 % on day 3. All concentrations above this level demonstrated excessive toxicity (0 % relative survival).
In test B, relative survival at 78.13 µg/mL was 5 % on day 0 and 45 % on day 3. All concentrations above this level demonstrated excessive toxicity (0 % relative survival). At 36.06 µg/mL, relative survival was 61 % on day 0 and 84 % on day 3.
In test C, relative survival at 19.53 µg/mL was 21 % on day 1 and 80 % on day 3. All concentrations above this level demonstrated excessive toxicity (0 % relative survival).

COMPARISON WITH HISTORICAL CONTROL DATA:
Comparison with the historical control data confirmed that the test system was functioning correctly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Assay 1 with metabolic activation, excessive cytotoxicity of the test material was observed at 160, 140 and 120 μg/mL concentrations; cells of these samples did not survive the treatment or the expression period. Evaluation of the mutagenicity was made using data of the first surviving concentration of 100 μg/mL (relative survival value of 24 %) and the five lower concentrations (a total of six concentrations).
In Assay 1 without metabolic activation, excessive cytotoxicity of the test material was observed at the 100 μg/mL concentration; cells of this sample did not survive the treatment period. Evaluation was performed on data from the next concentration of 90 μg/mL (relative survival value of 18 %) and seven lower concentrations (a total of eight concentrations).

In Assay 2, for the 3-hour treatment with metabolic activation, cells of the 160, 140 and 120 μg/mL concentration samples did not survive the treatment or the expression period. Evaluation was performed on the data from the first surviving concentration which was 100 μg/mL (relative survival value of 46 %) and the five lower concentrations (a total of six concentrations).
In Assay 2, for the 24-hour treatment without metabolic activation, excessive cytotoxicity was observed at the 40, 35, 32.5, 30 and 27.5 μg/mL concentrations; cells of these samples died during the treatment or in the expression period. Evaluation of the data was made using the next concentration of 25 μg/mL (relative survival of 8 %) and the following six lower concentrations (a total of seven concentrations).

Any other information on results incl. tables

Table 1: Summary of Results for Assay 1

Concentration (µg/mL)	Survival Data			Viability Data	Mutation Frequency
	PE	RS (%)	RTG (%)	PE
Metabolic activation	With (+ S9-mix); 3 hour exposure
160	ND	ND	ND	ND	ND
140	0.007	0	ND	ND	ND
120	0.015	1	ND	ND	ND
100	0.374	24	1	0.860	120.7
80	0.575	51	37	0.810	116.4
60	0.748	79	59	0.706	139.1
40	0.805	89	85	0.687	109.3
20	0.787	95	108	0.727	102.2
10	0.776	97	117	0.701	123.3
Vehicle control	0.810	100	100	0.759	109.9
Vehicle control for positive control (DMSO)	0.712	94	113	0.743	99
Untreated control	0.737	98	91	0.732	111.8
Positive control CP (4 μg/mL)	0.405	45	16	0.398	1733.3*
Metabolic activation	Without (- S9-mix); 3 hour exposure
100	ND	ND	ND	ND	ND
90	0.765	18	3	0.732	100.4
80	0.732	31	13	0.754	101.6
70	0.776	41	25	0.781	104.8
60	0.712	65	54	0.712	122.6
50	0.810	89	59	0.759	108.9
40	0.707	84	78	0.754	108.6
20	0.776	98	78	0.712	106.5
10	0.816	115	108	0.816	96.5
5	0.799	105	100	0.787	110.9
Vehicle control	0.781	100	100	0.722	99.8
Vehicle control for positive control (DMSO)	0.816	108	94	0.687	103.8
Untreated control	0.754	103	99	0.793	101.3
Positive control NQO (0.15 μg/mL)	0.453	60	29	0.440	1087.9*

CP = Cyclophosphamide

RS= Relative survival values (%) corrected with the post treatment cell concentrations

ND = No data (no cells were plated for colony growing due to the observed cytotoxicity during treatment or in the expression period)

NQO = 4-Nitroquinoline-N-oxide

RTG = Relative total growth

PE = Plating efficiency

* = Statistically significant

 

Table 2: Summary of Results for Assay 2

Concentration (µg/mL)	Survival Data			Viability Data	Mutation Frequency
	PE	RS (%)	RTG (%)	PE
Metabolic activation	With (+ S9-mix); 3 hour exposure
160	ND	ND	ND	ND	ND
140	0.008	0	ND	ND	ND
120	0.008	1	ND	ND	ND
100	0.424	46	35	0.787	141.1
80	0.644	83	47	0.640	147.4
60	0.743	86	79	0.835	99.0
40	0.841	105	89	0.737	132.1
20	0.706	97	110	0.701	120.0
10	0.770	100	99	0.663	118.9
Vehicle control	0.765	100	100	0.653	94.3
Vehicle control for positive control (DMSO)	0.770	107	116	0.776	90.9
Untreated control	0.805	106	114	0.810	89.8
Positive control CP (4 μg/mL)	0.380	41	12	0.357	1935.9*
Metabolic activation	Without (- S9-mix); 24 hour exposure
40	ND	ND	ND	ND	ND
35	ND	ND	ND	ND	ND
32.5	ND	ND	ND	ND	ND
30	ND	ND	ND	ND	ND
27.5	ND	ND	ND	ND	ND
25	0.398	8	18	0.706	92.4
22.5	0.408	9	23	0.822	80.3
20	0.539	14	29	0.805	83.0
15	0.532	25	41	0.748	84.3
10	0.805	92	121	0.860	70.8
5	0.743	99	119	0.722	91.5
2.5	0.727	83	103	0.776	89.0
Vehicle control	0.754	100	100	0.717	95.2
Vehicle control for positive control (DMSO)	0.754	74	91	0.793	85.2
Untreated control	0.727	99	122	0.810	86.1
Positive control NQO (0.1 μg/mL)	0.360	17	6	0.386	1473.9*

CP = Cyclophosphamide

RS= Relative survival values (%) corrected with the post treatment cell concentrations

ND = No data (no cells were plated for colony growing due to the observed cytotoxicity during treatment or in the expression period)

NQO = 4-Nitroquinoline-N-oxide

RTG = Relative total growth

PE = Plating efficiency

* = Statistically significant

Applicant's summary and conclusion

Conclusions:

Interpretation of results: Negative with and without metabolic activation

Under the conditions of this study, the test material was found not to be mutagenic in mouse lymphoma L5178Y cells in the presence and absence of exogenous metabolic activation up to precipitating and cytotoxic concentrations.

Executive summary:

The mutagenicity of the test material was assessed in an in vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay) which was performed in accordance with the standardised guidelines OECD 476 and EU Method B.17, under GLP conditions.

The test was performed using the mouse lymphoma L5178Y cell model up to cytotoxic and precipitating concentrations in the absence and presence of an exogenous metabolic activation system (S9 -mix).

The following conditions were used in the study:

ASSAY 1

- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL

- Three hour treatment without metabolic activation: 5, 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL

ASSAY 2

- Three hour treatment with metabolic activation: 10, 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL

- Twenty four hour treatment without metabolic activation: 2.5, 5, 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL

The expression time and selection time for both assays (both in the presence and absence of metabolic activation) were 3 days and 2 weeks, respectively.

No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis was observed in any of the assays either with or without metabolic activation.

Under the conditions of this study, the test material was found not to be mutagenic in mouse lymphoma L5178Y cells in the presence and absence of exogenous metabolic activation up to precipitating and cytotoxic concentrations.