Fatty acids, tallow, compds. with triethanolamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 January 2016 to 25 January 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 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

``Kanpoan No. 287 - - Environment Protection Agency``
``Eisei No. 127 - - Ministry of Health and Welfare``
``Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry``

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Test material

Test material information

1

Reference substance name:

Fatty acids, tallow, compds. with triethanolamine

EC Number:

263-158-1

EC Name:

Fatty acids, tallow, compds. with triethanolamine

Cas Number:

61790-67-8

IUPAC Name:

Fatty acids, tallow, esters of triethanolamine

Test material form:

liquid

Details on test material:

Appearance: Brown liquid
Storage: At ambient temperature, protected from light

Method

Target gene:

Thymidine kinase (TK) locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Methods for maintenance in cell culture if applicable: 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.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 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.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes. Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically 'cleansed' against high spontaneous background: 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 R10 medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Preliminary Cytotoxicity Test:
0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, and 2500 µg/mL for 4-hour treatment without S9, 4-hour treatment with S9 (2 %) and 24-hour treatment without S9.
Main Mutagenicity Test:
2.5, 5, 10, 20, 40 and 80 µg/mL for 4-hour treatment without S9, 4-hour treatment with S9 (2 %) and 24-hour treatment without S9.
The maximum dose level in the main Mutagenicity Test was limited by the onset of precipitate in the Preliminary Cytotoxicity Test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Following solubility checks performed on the test material, the test material was accurately weighed and formulated in acetone prior to serial dilutions being prepared.
Acetone is toxic to L5178Y cells at dose volumes greater than 0.5 % of the total culture volume. Therefore, the test item was formulated at 500 mg/mL and dosed at 0.5 % to give a maximum achievable dose level of 2500 µg/mL. There was no marked change in pH when the test item was dosed into media in the solubility test and the osmolality did not increase by more than 50 mOsm. No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate (EMS) at 400 µg/mL and 150 µg/mL in the 4- and 24-h exposure groups without metabolic activation. Cyclophosphamide (CP) at 1.5 µg/mL was used in the presence of metabolic activation. Both were formulated in DMSO.

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium
- Cell density at seeding: 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 10^6 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 10^6 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.

DURATION
- Pre-incubation period:
- Exposure duration: 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.
- Expression time: At the end of the exposure periods, the cells were washed twice using R10 medium then re-suspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured every 24 hours for the expression period of two days, by counting and diluting to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
- Selection time: On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

NUMBER OF REPLICATIONS: Performed in duplicate

- PLATE SCORING:
Microtitre plates were scored using a magnifying mirror box after ten to twelve 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 mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item. 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 mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

- CALCULATION OF PERCENTAGE RELATIVE SUSPENSION GROWTH (%RSG)
The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

- CALCULATION OF DAY 2 VIABILITY (%V)
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.

P(0) = number of negative wells / total wells plated

%V = (-1n P(0) x 100) / (number of cells/well)

- CALCULATION OF RELATIVE TOTAL GROWTH (RTG)
For each culture, the relative cloning efficiency, RCE, was calculated:

RCE = 5V / Mean Solvent control %V

Finally, for each culture RTG is calculated:

RTG = (RCE x RSG)/100 %

- CALCULATION OF MUTATION FREQUENCY (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.

- OTHER: PRELIMINARY TOXICITY TEST
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 9.77 to 2500 µg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, re-suspended in R20 medium, counted and then serially diluted to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
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 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. 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.

Rationale for test conditions:

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations was 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration was 5 mg/mL.
ii) Precipitating dose levels are not tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) 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. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Evaluation criteria:

See below.

Statistics:

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS. The statistical package used indicates the presence of statistically significant increases and linear-trend eve

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared; acetone is toxic to L5178Y cells at dose volumes greater than 0.5 % of the total culture volume. Therefore, the test item was formulated at 500 mg/mL and dosed at 0.5 % to give a maximum achievable dose level of 2500 µg/mL.
- Precipitation: The precipitate of the test item was observed at and above 39.06 µg/mL at the end of the preliminary cytotoxicity test exposure period. Therefore, the maximum dose level in the subsequent Mutagenicity Test was limited by the onset of precipitate.
In the Main Study, precipitate of the test item was observed at 40 and 80 µg/mL at the end of the exposure period.

RANGE-FINDING/SCREENING STUDIES: The dose range of the test material used in the preliminary toxicity test was 9.77 to 2500 µg/mL. There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls in all three of the exposure groups. However, the most marked reductions were only observed at dose levels at and / or above the onset of test material precipitate. The precipitate of the test material was observed at and above 39.06 µg/mL at the end of the exposure period. Therefore, the maximum dose level in the subsequent Mutagenicity Test was limited by the onset of precipitate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: There was no evidence of any marked toxicity following exposure to the test item in any of the three exposure groups, as indicated by the RTG values. There was also no evidence of any reductions in viability (%V) in any of the three exposure groups, indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with the positive control substances.

- OTHER OBSERVATIONS: The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels, in any of the three exposure groups.
The test material did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic in this assay.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material is not mutagenic in the presence or absence of metabolic activation.

Executive summary:

A study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The study was conducted in accordance with the standardised guidelines OECD 490, EU Method B.17 and EPA OPPTS 870.5300 under GLP conditions.

One main mutagenicity test was performed. In the test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels in duplicate, together with vehicle (acetone), 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 material used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were 2.5, 5, 10, 20, 30, 40 and 80 µg/mL for 4-hour treatment without S9, 4-hour treatment with S9 (2 %) and 24-hour treatment without S9. The maximum dose level used in the Mutagenicity Test was limited by the presence of precipitate of the test material. Precipitate of the test material was observed at 40 and 80 µg/mL in the 4-hour exposure groups, and at 40 µg/mL in the 24-hour exposure group, at the end of the exposure period in the Mutagenicity Test. The vehicle control cultures had mutant frequency values that were 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 material did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. Under the conditions of this study the test material is not mutagenic in the presence and absence of metabolic activation.

Under the conditions of this study, the test material is not mutagenic in the presence and absence of metabolic activation.