1,2,4-Benzenetricarboxylic acid, tri-C9-11-alkyl esters

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:

01 Apr - 02 Oct 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

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)

Version / remarks:

adopted in 2016

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

Test material information

1

Reference substance name:

1,2,4-Benzenetricarboxylic acid, tri-C9-11-alkyl esters

EC Number:

304-780-6

EC Name:

1,2,4-Benzenetricarboxylic acid, tri-C9-11-alkyl esters

Cas Number:

94279-36-4

Molecular formula:

C36H60O6 to C42H72O6

IUPAC Name:

tris(C9-11-alkyl) benzene-1,2,4-tricarboxylate

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y TK+/− (Clone 3.7.2C) mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518).
- Suitability of cells: The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated and is accepted by many regulatory authorities.
- Normal cell cycle time (negative control): The generation time and mutation rates (spontaneous and induced) have been checked at ERBC.

For cell lines:
- Absence of Mycoplasma contamination: The cells are checked at regular intervals for the absence of mycoplasmal contamination.
- Number of passages if applicable: Not indicated
- Methods for maintenance in cell culture: Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Cell cycle length, doubling time or proliferation index: Not indicated
- Modal number of chromosomes: Not indicated
- Periodically checked for karyotype stability: Not indicated
- Periodically ‘cleansed’ of spontaneous mutants: Prior to use, cells were cleansed of pre-existing mutants.


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Cytotoxicity test
Minimal medium A: 521 mL RPMI 1640 with L-glutamine (1X), 6.0 mL sodium pyruvate (100 mM), 5.4 mL non-essential amino acids (100X), 1.1 mL streptomycin sulphate 50000 IU/mL + Penicillin G 50000 IU/mL, 6.0 mL F 68 Pluronic, 1.1 mL penicillin G 50000 IU/mL.
Complete medium A (5%): 475 mL minimal medium A, 25 mL horse serum (heat-activated)
Complete medium A (10%): 450 mL minimal medium A, 50 mL horse serum (heat-activated)
Complete medium A (20%): 400 mL minimal medium A, 100 mL horse serum (heat-activated)
Complete medium B (20%): 391 mL RPMI 1640 with L-glutamine (1X), 4.45 mL sodium pyruvate (100 mM), 4.00 mL non-essential amino acids (100X), 0.818 mL streptomycin sulphate 50000 IU/mL + Penicillin G 50000 IU/mL, 100 mL horse serum (heat-inactived)

Main assays
Minimal medium A: 516.1 mL RPMI 1640 (1X), 5.4 mL L-glutamine (200 mM), 6.0 mL sodium pyruvate (100 mM), 5.4 mL non-essential amino acids (100X), 1.1 mL streptomycin sulphate 50000 IU/mL + Penicillin G 50000 IU/mL, 6.0 mL F 68 Pluronic
Minimal medium B: 522.1 mL RPMI 1640 (1X), 5.4 mL L-glutamine (200 mM), 6.0 mL sodium pyruvate (100 mM), 5.4 mL non-essential amino acids (100X), 1.1 mL streptomycin sulphate 50000 IU/mL + Penicillin G 50000 IU/mL
Complete medium (5%): 950 mL minimal medium A, 50 mL horse serum (heat-activated)
Complete medium (10%): 900 mL minimal medium A, 100 mL horse serum (heat-activated)
Complete medium A (20%): 800 mL minimal medium A, 200 mL horse serum (heat-activated)
Complete medium B (20%): 800 mL minimal medium B, 200 mL horse serum (heat-activated)

The plates were incubated at 37 °C in a 5% CO2 atmosphere (100% nominal relative humidity).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH; prepared from Sprague Dawley rats that had been induced with Phenobarbital – 5,6-Benzoflavone.
- method of preparation of S9 mix: The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 mL): 0.408 mL S9 tissue fraction; 0.204 mL NADP (100 mM); 0.204 mL G-6-P (100 mM); 0.204 mL KCL (330 mM); 8.98 mL Complete medium (5%).
- concentration or volume of S9 mix and S9 in the final culture medium: 9.8 mL S9 mix containing 0.4 mL S9 fraction.
- quality controls of S9: The S9 fraction was confirmed for protein content (35 mg/mL), positive enzymatic activity, absence of contaminating microorganisms, and negative promutagen activity.

Test concentrations with justification for top dose:

Migrated Data from field(s)
Field "Justification for deviation from the high dose level" (Path: ENDPOINT_STUDY_RECORD.GeneticToxicityVitro.MaterialsAndMethods.Method.JustificationForDeviationFromTheHighDoseLevel): Preliminary cytotoxicity study: 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/mL
Experiment 1 and 2: 16.0, 40.0, 100, 250, 625 μg/mL

Doses were selected based on the results of the preliminary cytotoxicity study

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone (batch nos.: 17A304006 and 21A154014 obtained from VWR)

- Justification for choice of solvent/vehicle: The test item was found to be soluble up to 500 mg/mL in acetone.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2 (Experiment 1 with and without metabolic activation and Experiment 2 without metabolic activation)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E6 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 h (Experiment 1; with and without metabolic activation); 24 h (Experiment 2; without metabolic activation)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 d
- Selection time: 14 days after treatment with 3 µg/mL of 5-trifluorothymidine (TFT)
- Method used: 96-well plates were incubated at 37 °C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored..
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Treatment with test concentrations: 1E6 cells / mL
Expression period: 2E5 cells / mL
Plating with TFT: 2E3 cells / mL
Plating for viability: 1.6 cells / well
- Criteria for small (slow growing) and large (fast growing) colonies:
Small colonies: Colonies having a diameter less than 25% of the diameter of the well. A small colony should also have a compact morphology.
Large colonies: Colonies having a diameter greater than 25% of the diameter of the well. Morphology is diffused, totally or in periphery.
For the unteated and positive controls, the small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and presence of S9 metabolic activation for
3 hrs and for 24 hrs only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells wereresuspended in 20 mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2 mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominalrelative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.
In order to aid toxicity data interpretation, the relative total growth (RTG), expressed as a percentage of the concurrent negative control, was also calculated. This is a product of the relative suspension growth (RSG) and the Day 2 relative cloning efficiency (RCE), expressed as a percentage of the concurrent negative control, for each culture, as follows:
RTG = RSG x RCE (Day 2)
RSG = (Total suspension growth (TSG)test/TSGcontrol) x 100
TSG = (post-treatment cell count/pre-treatment cell count) x (Day 1 cell count/2E5) x (Day 2 cell count/2E5)
The main experiment and the preliminary toxicity test used the same methodology to determine cytotoxitiy.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutant frequency (MF) = ((-ln (ym/nm))/fm) / (-ln (ys/ns))/fs)) x 10^6
ym = number of empty wells (mutant plates); nm = total number of wells (mutant plates); fm = number of cells plated per well (2E3 cells/well); ys = number of empty wells (viability plates); ns = total number of wells (viability plates); fs = number of cells plated per well (1.6 cells/well)
Induced mutant frequency (IMF) = MFtest - MFcontrol

Rationale for test conditions:

Based on OECD test guideline 490 (2016).

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126E−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.

Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines.

Results and discussion

Test results

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

Remarks:

Upon addition of the test item to the cultures, precipitation was observed at the highest dose level, while dose related opacity of treatment mixtures was noticed in all treatment series from 156 μg/mL onwards.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.
- Precipitation and time of the determination: Upon addition of the test item to the cultures, precipitation was observed at the highest dose level, while dose related opacity of treatment mixtures was noticed in all treatment series from 156 μg/mL onwards. By the end of the treatment, dose related opacity was seen in all treatment series starting from 156 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and presence of S9 metabolic activation for 3 h and for 24 h only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20 mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2mL was plated into 96 microtiter wells. The plates were incubated at 37 °C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.
Both in the absence and presence of S9 metabolic activation, the test item was assayed at dose levels of 19.5, 39.1, 78.1, 156, 313, 625, 1250, and 5000 μg/mL. Slight or no relevant toxicity was noted at all concentrations tested, in each treatment series.

STUDY RESULTS.
- Genotoxicity results: No statistically significant or biologically relevant increase in mutant frequency values was observed in any experiment, at any concentration tested, in the absence or presence of S9 metabolism, using the short or long treatment time. The IMF was not higher than the GEF and there was not a significant dose-relationship. See 'Any other information on results incl. tables', Table 1.
Untreated, solvent and positive control cultures were included in each mutation experiment. The mutant frequencies in the untreated control cultures fell within the normal range (50 − 170E−6 viable cells).
Both in Experiment 1 and 2, the positive control item induced an increase in the small colony mutation frequency higher than 150E-6 above that seen in the concurrent solvent control, demonstrating that one criterion of acceptance for the small colony mutant frequency was achieved. The study was accepted as valid.

HISTORICAL CONTROL DATA
See 'Any other information on results incl. tables', Table 2.

Any other information on results incl. tables

Table 1: L5178Y TK^+/- mouse lymphoma cell mutation assay

Dose (µg/mL)	RTG	MF §	P-value	IMF §	Proportion small colony mutants	Precipitation
Main assay 1 – 3 h without S9
Untreated	100%	55.1	-	-	0.32	-
0.00	100%	57.2	-	-	0.26	-
16.0	83%	51.0	NS	-	-	-
40.0	81%	52.4	NS	-	-	-
100	105%	46.4	NS	-	-	-
250	69%	48.5	NS	-	-	+
625	89%	45.0	NS	-	-	+
Main assay 1 – 3 h with S9
Untreated	100%	53.5	-	-	0.31	-
0.00	100%	67.0	-	-	0.27	-
16.0	103%	47.8	NS	-	-	-
40.0	103%	43.7	NS	-	-	-
100	88%	66.1	NS	-	-	-
250	106%	53.7	NS	-	-	+
625	94%	42.0	NS	-	-	+
B(a)P 2.00	26%	1136.6	-	1083.1@	0.37	-
Main assay 2 – 24 h without S9
Untreated	100%	55.8	-	-	0.29	-
0.00	100%	75.5	-	-	0.28	-
16.0	94%	56.6	NS	-	-	-
40.0	93%	79.9	NS	4.39	-	-
100	82%	69.8	NS	-	-	-
250	50%	101.6	NS	26.12	-	+
625	74%	66.7	NS	-	-	+
MMS 5.00	49%	573.3	-	517.5@	0.40	-
§ = per 10^6 viable cells B(a)P = benzo(a)pyrene IMF = induced mutation frequency MF = mutation frequency MMS = methylmethansulfonate NS = not statistically significant RTG = relative total growth + = Opacity of treatment medium @ = IMF > global evaluation factor (GEF = 126 x 10^-6)

Table 2: Historical data of negative / solvent and positive controls (Mutation frequencies per million surviving cells)

	Without S9; 3 h		With S9; 3h		Without S9; 24 h
	Negative control	Positive control	Negative control	Positive control	Negative control	Positive control
Mean Value	81.0	447	83.4	759	81.1	635
Standard Deviation	24.2	140	26.0	266	25.1	285
Upper Confidence Limit (p < 5%)	128	NC	135	NC	130	NC
Min	41.9	209	44.9	187	50.4	221
Max	234	1103	234	1507	217	2325
n	172	136	262	262	118	118
NC = Not calculated n = number of experiments

 

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported the test item did not induce gene mutations at the TK locus in L5178Y mouse lymphoma cells in the absence or presence of S9 metabolic activation. Therefore, the test item is considered to be non-mutagenic in the TK assay.