Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol

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:

17 Oct - 19 Dec 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

yes

Remarks:

Minor deviations in preparation of the S9 mix and dose-range finding test. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

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

Constituent 1

Reference substance name:

Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol

Molecular formula:

not applicable

IUPAC Name:

Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol

Test material form:

liquid

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+/--3.7.2C mouse lymphoma cells were obtained from American Type Culture Collection, (ATCC, Manassas, USA).
- 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.

For cell lines:
- Absence of Mycoplasma contamination: The cultures were checked for mycoplasma contamination.
- Methods for maintenance in cell culture: Stock cultures of the cells were stored in the ultra-low freezer set to maintain -150 °C. Cell density was kept below 1E6 cells/mL.
- Cell cycle length, doubling time or proliferation index: Not indicated

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Horse serum
Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum.
Exposure medium
Cells were exposed to the test material in basic medium supplemented with 5 - 10% (v/v) heat-inactivated horse serum.
Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT) (Sigma).
Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum.

Environmental conditions
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 42 - 103%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.5 - 38.0 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.

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 and ß-naphthoflavone.
- method of preparation of S9 mix: S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life Technologies). The above solution was filter (0.22 μm)-sterilized.
- concentration or volume of S9-mix and S9 in the final culture medium: To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. In the dose range finding test, another ratio of the S9-components was used, due to a technical error. The concentration of the S9-fraction in the exposure medium was 4% (v/v).
- quality controls of S9: The S9 fraction was confirmed for protein content, positive enzymatic activity, absence of contaminating microorganisms, and negative promutagen activity.

Test concentrations with justification for top dose:

Dose range finding test 1: 3.9, 7.8, 15.6, 31.3 and 62.5 μg/mL with and without S9 mix for 3 h and without S9 for 24 h
Dose range finding test 2: 125, 250, 500, 1000 and 2000 μg/mL with and without S9 mix for 3 h
Dose range finding test 3: 50, 100, 250, 500, 1000 and 2000 μg/mL without S9 mix for 24 h

Experiment 1: 10, 25, 50, 100, 125, 250, 500, 600, 700, 800, 900, 1000 μg/mL
Experiment 2: 12.5, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μg/mL

Doses were selected based on the results of the dose range finding test.

Vehicle / solvent:

The vehicle for the test material was tetrahydrofuran (THF, Hipersolv Chromanorm, VWR, Belgium).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single
- 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
- 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: 2 days
- Selection time: 11 - 12 days after treatment with 5 µg/mL of trifluorothymidine (TFT)
- Method used: For determination of the mutant frequency (MF) a total number of 9.6E5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6E5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CE day2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5 - 2 h, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Treatment with test concentrations: 1.25E5 to 1E6 cells/mL
Expression period: 4E6 cells
Plating with TFT: 9.6E5 cells/concentration
- Criteria for small (slow growing) and large (fast growing) colonies: The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8E6 cells (1E6 cells/mL for 3-hour treatment) or 6E6 cells (1.25E5 cells/mL for 24 h treatment) with a number of test material concentrations increasing by approximately half log steps. The cell cultures for the 3 h treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0 °C and 145 rpm. The cell cultures for the 24 h treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0 °C. The test material was tested in the absence and presence of S9-mix.
For the 3 h treatment, cell cultures were exposed to the test material in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium.
Initially, since the test material was poorly soluble in the exposure medium, the highest tested concentration in the dose range finding was 62.5 μg/mL exposure medium. However, since the test material was not precipitating at this dose level in the dose-range finding, the dose range finding test was repeated with higher concentrations.
For the 24 h treatment, cell cultures were exposed to the test material in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium. The cells in the final suspension were counted with the coulter particle counter.
Initially, since the test material was poorly soluble in the exposure medium, the highest tested concentration in the dose range finding was 62.5 μg/mL exposure medium, and in the second experiment up to 70 μg/mL. However, since the test material was not precipitating any more at this dose level in the second experiment, the dose-range finding test was repeated with higher concentrations.
The surviving cells of the 3 h treatment were subcultured twice to determine cytotoxicity. After 24 h of subculturing, the cells were counted and subcultured again for another 24 h, after that the cells were counted. The surviving cells of the 24 h treatment were subcultured once. After 24 h of subculturing, the cells were counted. If less than 1.25E5 cells/mL were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 h or only 24 h cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests.
Dose-range finding test:
The suspension growth (SG) for the 3-hour treatment=
SG = Suspension growth = [Day 1 cell count / 1.6E5] x [Day 2 cell count / 1.25E5]
The suspension growth (SG) for the 24-hour treatment=
SG = Suspension growth = [Day 0 cell count / 1.25E5] x [Day 1 cell count / 1.25E5]
Mutagenicity tests:
The suspension growth (SG) for the 3-hour treatment=
[Day 1 cell count / 1.6E5] x [Day 2 cell count / 1.25E5]
The suspension growth (SG) for the 24-hour treatment=
[Day 0 cell count / 1.25E5] x [Day 1 cell count / 1.25E5] x [Day 2 cell count / 1.25E5]
Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100
The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. The value obtained is the P(0), the zero term of the Poisson distribution:
P(0) = number of empty wells/total number of wells
The cloning efficiency (CE) was then calculated as follows:
CE = -ln P(0)/number of cells plated per well
The relative cloning efficiency (RCE) at the time of mutant selection =
CE (test) / CE (controls) x 100
The Relative Total Growth (RTG) was also calculated as the product of the cumulative relative suspension growth (RSG) and the relative survival for each culture:
RTG = RSG x RCE / 100

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutant frequency was expressed as the number of mutants per 1E6 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutant frequency (MF) was calculated as follows:
MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 1E6
Small and large colony mutation frequencies were calculated in an identical manner.

Rationale for test conditions:

Based on OECD test guideline 490 (2016).

Evaluation criteria:

The global evaluation factor (GEF) is the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test material is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test material is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test material is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutant frequency of MF(controls) + 126.

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:

The test material began to precipitate in the exposure medium at a concentration of 500 and 600 µg/mL in Experiment 1 and 2, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolarity: The pH and osmolarity at a concentration of 62.5 μg/mL were 7.074 and 0.337 Osm/kg respectively (compared to 7.091 and 0.331 Osm/kg in the solvent control).
- Precipitation and time of the determination: The test material began to precipitate in the exposure medium at a concentration of 500 and 600 µg/mL in Experiment 1 and 2, respectively. The test material was tested beyond the limit of the solubility to obtain adequate mutagenicity data.

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test material concentration range of 3.9 - 62.5 μg/mL in the absence of S9-mix with 3 and 24 h treatment periods and in the presence of S9-mix with a 3 h treatment period.
After 3 h, the test material did not precipitate in the exposure medium at the highest dose level of 62.5 μg/mL. Therefore, this part of the dose range finding was repeated in a second dose-range finding. In this second dose-range finding, L5178Y mouse lymphoma cells were treated with a test material concentration range of 125 - 2000 μg/mL in the absence and presence of S9-mix with a 3 h treatment period.
After 24 h, the test material precipitated in the exposure medium at the highest dose level of 62.5 μg/mL. However, during the second mutagenicity assay, precipitation was not observed at the highest concentration. Therefore, the dose-range finding for the 24 h treatment period was repeated in a third dose-range finding with a concentration range of 50 - 2000 μg/mL in the absence of S9-mix with a 24 h treatment period.
For the 3 h treatment, both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test material concentration of 2000 μg/mL compared to the solvent control. The test material precipitating dose level was 1000 μg/mL and upwards.
For the 24 h treatment, no toxicity in the relative suspension growth was observed up to test material concentrations of 2000 μg/mL compared to the solvent control. The test material precipitating dose level was 1000 μg/mL and upwards.

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. See 'Any other information on results incl. tables', Table 1.
Untreated, solvent and positive control cultures were included in each mutation experiment.
The mutant frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. In addition, the mutant frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

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)	RSG (%)	CE day2 (%)	RCE (%)	RTG (%)	Mutant frequency per 1E6 survivors
					Total	Small	Large
Experiment 1 – 3 h without S9
SC	100	98	100	100	97	31	62
SC	100	88	87	100	100	46	38
10	83	91	98	82	107	41	60
25	97	91	98	95	90	32	55
50	76	102	110	84	94	27	63
100	86	85	92	79	106	33	69
125	93	90	97	91	104	45	55
250	94	105	113	107	75	21	51
500^1	97	104	112	109	96	38	54
MMS	45	81	88	40	1187	395	519
Main assay 1 – 3 h with S9
SC	100	113	100	100	80	32	45
SC	100	108	100	100	97	29	64
10	110	123	111	122	77	33	41
25	76	116	105	80	86	41	40
50	94	115	104	98	78	29	46
100	107	107	96	103	81	25	53
125	100	107	96	96	101	34	61
250	100	94	85	85	86	20	63
500^1	121	94	85	103	85	31	51
CP	91	60	55	50	760	346	327
Main assay 2 – 24 h without S9
SC	100	91	100	100	92	28	60
SC	100	102	100	100	72	18	53
25	100	95	98	98	93	34	55
50	101	104	107	108	69	24	43
100	97	101	104	101	56	10	44
200	109	79	82	89	100	16	81
300	120	97	100	120	72	20	50
400	120	111	115	138	65	21	42
500	126	108	112	141	56	15	40
600^1	121	98	101	123	61	19	40
MMS	102	69	72	73	574	164	360
^1 The test item precipitated in the exposure medium RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth; SC = Solvent control = THF; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

 

Table 2: Historical control data of the spontaneous mutant frequencies for the mouse lymphoma assay (November 2019 and November 2022)

	Mutant frequency per 1E6 survivors
	-S9-mix		+S9-mix
	3 h	24 h	3 h
Solvent controls
Mean	101	98	99
SD	29	27	28
n	81	76	81
Lower Control Limit (95% Control Limits)	45	46	45
Upper Control Limit (95% Control Limits)	158	151	154
Positive controls
Mean	1049	808	1311
SD	380	253	653
n	77	74	78
Lower Control Limit (95% Control Limits)	305	312	31
Upper Control Limit (95% Control Limits)	1794	1305	2591
SD = Standard deviation; n = Number of observations

 

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.