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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 03 June 2019 to 25 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
427-430-5
EC Name:
-
Cas Number:
54301-26-7
Molecular formula:
C13H23NO3
IUPAC Name:
(undec-10-enoylamino)acetic acid
Test material form:
solid
Details on test material:
Identification: Undecylenoyl Glycine
Chemical Name: Undecylenoyl Glycine
CAS Number: 54301-26-7
Physical State / Appearance: Off white solid
Batch: SS 655
Molecular Weight: 241
Purity: 95.6%
Expiry Date: 01 May 2020
Storage Conditions: Room temperature, in the dark
Specific details on test material used for the study:
Identification: Undecylenoyl Glycine
Chemical Name:Undecylenoyl Glycine
CAS Number: 54301-26-7
Physical State / Appearance: Off white solid
Batch: SS 655
Molecular Weight: 241
Purity: 95.6%
Expiry Date: 01 May 2020
Storage Conditions: Room temperature, in the dark

Method

Target gene:
Thymidine kinase (tk) locus of L5178Y mouse lymphoma tk ( +/- ) cells
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Type and identity of media:
The stocks of cells were stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium.

- Properly maintained:
yes

- Periodically checked for Mycoplasma contamination:
yes

- Periodically checked for karyotype stability:
no

- Periodically "cleansed" against high spontaneous background:
yes








Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Lot No. PB/βNF S9 mix used in this study, was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test and a certificate of S9 efficacy is available.

- method of preparation of S9 mix :S 9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM)

- concentration or volume of S9 mix and S9 in the final culture medium : 20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test.
Test concentrations with justification for top dose:
- Preliminary Cytotoxicity Test Range finding study: 0; 7.81; 15.63; 31.25; 62.5; 125; 250; 500; 1000; 2000 (μg/mL)
- Main study: 0; 62.5;125; 250; 300; 350; 400; 450; 500 (μg/mL)
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity).The molecular weight of the test item was 241 therefore the maximum proposed dose level in the solubility test was set at 2000 μg/mL, the maximum recommended dose level, and a correction for the purity of the test item of 95.6% was applied to the dose formulations.The dose range used in the preliminary toxicity test was 7.81 to 2000 μg/mL for all three of the exposure groups. In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival.Based on the %RSG values observed, the maximum dose level in the Mutagenicity Test was limited by test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Solvent DMSO was used.
The test item formed a solution in DMSO at 200 mg/mL considered acceptable for dosing. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (DMSO) exposure groups were used as the vehicle controls.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Ethylmethanesulphonate (EMS) , was used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (DMSO) exposure groups were used as the vehicle controls.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Cyclophosphamide was used as the positive control in the presence of metabolic activation.
Details on test system and experimental conditions:
Cell Line:

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture:

The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing:

The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to 10% donor horse serum medium.

Test Item Preparation:

The molecular weight of the test item was 241 therefore the maximum proposed dose level in the solubility test was set at 2000 μg/mL, the maximum recommended dose level, and a correction for the purity of the test item of 95.6% was applied to the dose formulations. The test item was found to be insoluble in RPMI 1640 medium at 20 mg/mL. The test item formed a solution in DMSO at 200 mg/mL considered acceptable for dosing. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991). The pH and osmolality readings are in the following table (see more details on Any other information on materials and methods incl. tables).

Control Preparation:

Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO) exposure groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) (Sigma batch BCBW8635, purity treated as 100%, expiry 10/01/24) at 400 μg/mL and 150 μg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation. Cyclophosphamide (Acros Organics batch A0389646, purity 99.9%, Expiry 01/10/22) at 1.5 μg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO.

EXPOSURE:

Preliminary Toxicity Test:

A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 7.81 to 2000 μg/mL for all three of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL.
The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments.

Mutagenicity Test:

Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (62.5 to 500 μg/mL for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 12.5 to 300 μg/mL for the 24-hour exposure group in the absence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).
The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Plate Scoring:

Microtitre plates were scored using a magnifying mirror box after ten days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutant plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutant plates. The plates were incubated for two hours.


Evaluation criteria:
Cytotoxicity was measured by relative survival growth percentage (relative cloning efficiency) or RTG. The highest concentration for the main study was chosen on the basis that it exhibited a 10 - 20% RTG. Scoring of large and small colonies was done visually and microscopically in the test and control plates to understand the mechanistic action of the test substance. Small colonies were defined as less than a quarter of the diameter of the well.
Statistics:
The experimental data was analyzed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear-trend events.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Preliminary Cytotoxicity Test:


The dose range of the test item used in the preliminary toxicity test was 7.81 to 2000 μg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:







































































Dose
(μg/mL)
% RSG (-S9)
4-Hour Exposure
% RSG (+S9)
4-Hour Exposure
% RSG (-S9)
24-Hour Exposure
0100100100
7.818810796
15.638911788
31.259911176
62.5938363
125949932
25074645
500110
1000100
2000200

There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all three of the exposure groups, when compared to the concurrent vehicle control groups. Precipitate of the test item was observed at 2000 μg/mL at the end of the exposure period in the 4-hour exposure groups in both the absence and presence of metabolic activation. Based on the %RSG values observed, the maximum dose level in the Mutagenicity Test was limited by test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline.


Main Experiment results:
































































































































Concentration (μg/mL)4-Hours -S9Concentration
(μg/mL)
4-Hours+S9
% Relative Suspension GrowthRelative Total Growth5-TFT resistant mutants/106 viable cells 2 days after exposure % Relative Suspension GrowthRelative Total Growth5-TFT resistant mutants/106 viable cells 2 days after exposure
01001.00155.1301001.00140.86
62.5870.99152.2062.5950.92150.12
125910.97160.68125870.91125.96
250600.68148.69250580.55152.51
300410.48134.95     300   Ø170.12153.71
350100.08149.57     350   Ø2  
     400   Ø1       400   Ø0  
     450   Ø0      450   Ø1  
    500   Ø0      500    Ø1  
Mutant Frequency threshold for a positive response = 281.13Mutant Frequency threshold for a positive response = 266.86
Positive controlPositive control
EMS

400

 


72



 


0.59



 


1534.07



CP


1.5



 


74



 


0.51



 


1235.67



 

















































































Concentration
(μg/mL)
24-Hours-S9
 %RSGRTGMF§
01001.00141.88
12.5950.96140.40
25700.88139.04
50791.01121.28
100540.77120.11
150320.52141.03
200120.20191.03
       250      Ø4  
        300       Ø1  
MF threshold for a positive response = 267.88
Positive control

Ethylmethanesulphonate



150



 


52



 


0.46



 


1589.23



Ø = Not plated due to excessive toxicity


 

Applicant's summary and conclusion

Conclusions:
The maximum dose level in the Mutagenicity Test was limited by test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, using a dose range that achieved optimum levels of toxicity in all three of the exposure groups as recommended by the OECD 490 guideline.

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.
Executive summary:

Introduction. 


The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.


Methods :


One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (dimethyl sulfoxide (DMSO)), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.
The dose range of test item used in the main test was selected following the results of a preliminary toxicity test.


Results:  


The maximum dose level in the Mutagenicity Test was limited by test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline. The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, using a dose range that achieved optimum levels of toxicity in all three of the exposure groups.


Conclusion:


The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.