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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Genetic toxicity in bacteria (OECD 471, GLP): negative with and without metabolic activation in S. typhimurium TA 1535, 1537, 98 and 100, and E. coli WP2 uvr A

- Micronucleus test (OECD 487): negative in primary human peripheral lymphocytes with and without metabolic activation

- Gene mutation in mammalian cells (OECD 490): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

TK locus

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).

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.

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.

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

 

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 May - 28 Jun 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted in 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human primary culture

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human peripheral blood lymphocytes
- Suitability of cells: selected according to OECD guideline 487

For lymphocytes:
- Sex, age and number of blood donors: donor 1: male, 28 yrs; donor 2: female, 27 yrs
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: Blood from different donors was not pooled.
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel Europe Ltd., Dartford, United Kingdom) was added.
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 47 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.3 - 37.4 °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. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Cytokinesis block (if used):

5 µg/mL Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital - 5,6-Benzoflavone induced rat liver S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany.
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. 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. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was 1.8% (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: 0, 0.5, 1, 2, 3.9, 7.8, 15.6 µg/mL (without metabolic activation, 24 h exposure)
Experiment I: 0, 3.9, 7.8, 15.6 µg/mL (without metabolic activation, 3 h exposure); 0, 3.9, 7.8, 15.6 µg/mL (with metabolic activation, 3 h exposure)
Experiment II: 0, 2.0, 3.9, 7.8 µg/mL (without metabolic activation, 24 h exposure)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF, Sigma, Zwijndrecht, The Netherlands)

- Justification for choice of solvent/vehicle: In order to obtain a suitable solvent, a solubility test for this batch of test material was performed within the laboratory, which was based on visual assessment.

- Justification for percentage of solvent in the final culture medium: The final concentration of the solvent in the culture medium was 0.25% (v/v). According to OECD technical guideline 487 organic solvents should not exceed 1%.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: in medium

TREATMENT AND HARVEST SCHEDULE:
- Pretreatment period: Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test material for 3 h and 24 h in the absence of S9-mix or for 3 h in the presence of S9-mix. Cytochalasine B (Sigma) was added to the cells simultaneously with the test material at the 24 h exposure time. A vehicle control was included at each exposure time.
- Exposure duration/duration of treatment: Short treatment: 3 h with and without metabolic activation. Continuous treatment: 24 h without metabolic activation.
- Harvest time after the end of treatment (sampling/recovery times): After 3 h exposure to the test material in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasine B (5 μg/mL) and incubated for another 24 h (1.5 times normal cell cycle). The cells that were exposed for 24 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 1000 binucleate cells per culture were scored, and in total 2000 cells per concentration.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index (CBPI); 500 cells per culture were used. A CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

Rationale for test conditions:

The test was performed according to the specifications of the OECD guideline 487 (2016).

Evaluation criteria:

The following criteria for scoring of binucleated cells were used:
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.

The following cells were not scored:
- Mononucleated, trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).

The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

Statistics:

The Fisher’s exact test showed that there are statistically significant differences between one or more of the test material groups and the vehicle control group. Therefore a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Species / strain:

lymphocytes: human primary culture

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation at concentrations of 7.8 μg/mL and upwards in Experiment 1

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
- The pH and osmolarity of a concentration of 125 μg/mL were 7.54 and 332 mOsm/kg respectively (compared to 7.52 and 335 mOsm/kg in the solvent control).
- At a concentration of 7.8 μg/mL the test material precipitated in the culture medium.

RANGE-FINDING/SCREENING STUDIES (if applicable):
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose-range finding test. The highest tested concentration was determined by the solubility of the test material in the culture medium. No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the slides were scored for micronuclei. The pilot study (short term exposure period) was used as the first cytogenetic assay. See 'Any other information on results incl. tables', Table 1.

STUDY RESULTS
Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: No cytotoxicity was observed up to and including the highest concentration.
- Genotoxicity results: In Experiment 1, in absence of S9-mix, a significant increase in micronuclei (p = 0.0107) was observed at the medium dose (7.8 μg/mL). In presence of S9-mix, in the lowest dose (3.9 μg/mL) a significant increase was observed in micronuclei (p = 0.0194). The increases in both conditions were considered to be not biologically relevant, since the increases were within the solvent control historical data, confined to one dose level, and no significant trend was observed (trend test: p = 0.0634 and p = 0.6729, respectively in the absence and presence of S9-mix). In Experiment 2, the test material did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei. See 'Any other information on results incl. tables', Tables 2 - 7.
The positive controls were shown to be valid. The solvent control was shown to be valid.

HISTORICAL CONTROL DATA
- See 'Any other information on results incl. tables', Tables 8 and 9.

Table 1: Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures in the Dose-range Finding Test

Without metabolic activation (-S9-mix) 24 h exposure time, 24 h harvest time
Concentration (μg/mL)	Number of cells with nuclei			CBPI	% cytostasis
	1	2	3 or more
0	145	271	84	1.88	0
0.5	130	277	93	1.93	-5
1	114	296	90	1.95	-8
2	134	289	77	1.89	-1
3.9	143	297	60	1.83	5
7.8^1	115	306	79	1.93	-6
15.6^1	147	275	78	1.86	2
Note: All calculations were performed without rounding off. ^1 The test material precipitated in the culture medium

 

Table 2: Cytokinesis-Block Proliferation Index of Human Lymphocytes Cultures Treated in the First Cytogenetic Assay

Without metabolic activation (-S9-mix) 3 h exposure time, 27 h harvest time
Concentration (μg/mL)	CBPI	Mean CBPI	% cytostasis
0	2.06 - 2.10	2.08	0
3.9	2.04 - 2.14	2.09	-1
7.8^1	2.08 - 2.12	2.10	-2
15.6^1	2.06 - 2.07	2.06	2
0.2 MMC-C	1.76 - 1.79	1.78	28
0.25 MMC-C	1.75 - 1.79	1.77	29
0.05 Colch	1.94 - 1.98	1.96	11
0.1 Colch	1.23 - 1.26	1.24	78
With metabolic activation (+S9-mix) 3 h exposure time, 27 h harvest time
Concentration (μg/mL)	CBPI	Mean CBPI	% cytostasis
0	2.05 - 2.07	2.06	0
3.9	1.98 - 2.02	2.00	5
7.8^1	1.95 - 1.96	1.96	10
15.6^1	1.95 - 1.99	1.97	8
7.5 CP	1.69 - 1.71	1.70	34
10 CP	1.02 - 1.56	1.29	73
Note: All calculations were performed without rounding off. ^1 The test material precipitated in the culture medium

 

Table 3: Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated in the First Cytogenetic Assay

Without metabolic activation (-S9-mix) 3 h exposure time, 27 h harvest time
Concentration (μg/mL)	% cytostasis	Number of binucleated cells with micronuclei^1
		1000	1000	2000
		A	B	A+B
0	0	1	0	1
3.9	-1	3	2	5
7.8	-2	2	7	9*
15.6	2	1	5	6
0.20 MMC-C	28	12	18	30****
0.05 Colch	11	11	2	13***
0.1 Colch	78	36	36	72****
With metabolic activation (+S9-mix) 3 h exposure time, 27 h harvest time
0	0	1	0	1
3.9	5	2	6	8*
7.8	10	3	3	6
15.6	8	1	2	3
7.5 CP	34	12	6	18****
10 CP	73	19^2	19	38****
* Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001. ^1 1000 binucleated cells were scored for the presence of micronuclei. Duplicate cultures are indicated by A and B. ^2 57 binucleated cells were scored for the presence of micronuclei

 

Table 4: Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix) 24 h exposure time, 24 h harvest time
Concentration (μg/mL)	CBPI	Mean CBPI	% cytostasis
0	1.90 - 1.92	1.91	0
2.0	1.94 - 1.95	1.94	-4
3.9	1.97 - 2.01	1.99	-9
7.8^1	1.90 - 1.95	1.92	-2
0.125 MMC-C	1.79 - 1.80	1.80	12
0.15 MMC-C	1.66 - 1.82	1.74	19
0.01 Colch	1.80 - 1.84	1.82	10
0.05 Colch	1.01 - 1.02	1.02	98
Note: All calculations were performed without rounding off. ^1 The test material precipitated in the culture medium

 

Table 5: Number Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix) 24 h exposure time, 24 h harvest time
Concentration (μg/mL)	% cytostasis	Number of binucleated cells with micronuclei^1
		1000	1000	2000
		A	B	A+B
0	0	1	2	3
2.0	-4	1	1	2
3.9	-9	4	2	6
7.8	-2	2	0	2
0.125 MMC-C	12	21	20	41****
0.01 Colch	10	4	4	8
0.05 Colch	98	24^2	18^2	42****
* Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001. ^11000 binucleated cells were scored for the presence of micronuclei. Duplicate cultures are indicated by A and B. ^2 722 and 674 binucleated cells were scored for the presence of micronuclei, respectively.

 

Table 6: Scoring of Cells with One, Two or More Nuclei of Human Lymphocyte Cultures Treated in the First Cytogenetic Assay

Without metabolic activation (-S9-mix) 3 h exposure time, 27 h harvest time
Concentration (μg/mL)	Culture	Number of cells with nuclei			CBPI
		1	2	3 or more
0	A	85	280	135	2.10
	B	111	247	142	2.06
3.9	A	84	264	152	2.14
	B	115	250	135	2.04
7.8	A	108	244	148	2.08
	B	102	235	163	2.12
15.6	A	109	249	142	2.07
	B	107	258	135	2.06
0.2 MMC-C	A	133	337	30	1.79
	B	159	303	38	1.76
0.25 MMC-C	A	146	315	39	1.79
	B	155	315	30	1.75
0.05 Colch	A	124	260	116	1.98
	B	132	267	101	1.94
0.1 Colch	A	386	98	16	1.26
	B	400	87	13	1.23
With metabolic activation (+S9-mix) 3 h exposure time, 27 h harvest time
0	A	110	246	144	2.07
	B	112	250	138	2.05
3.9	A	129	250	121	1.98
	B	120	250	130	2.02
7.8	A	143	238	119	1.95
	B	131	258	111	1.96
15.6	A	113	278	109	1.99
	B	133	260	107	1.95
7.5 CP	A	171	305	24	1.71
	B	181	294	25	1.69
10 CP	A	491	9	0	1.02
	B	236	248	16	1.56

 

Table 7: Scoring of Cells with One, Two or More Nuclei of Human Lymphocyte Cultures Treated in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix) 24 h exposure time, 24 h harvest time
Concentration (μg/mL)	Culture	Number of cells with nuclei			CBPI
		1	2	3 or more
0
	B	134	284	82	1.90
2	A	100	332	68	1.94
	B	112	300	88	1.95
3.9	A	89	315	96	2.01
	B	109	296	95	1.97
7.8	A	108	310	82	1.95
	B	130	290	80	1.90
0.125 MMC-C	A	128	349	23	1.79
	B	126	346	28	1.80
0.15 MMC-C	A	192	288	20	1.66
	B	117	355	28	1.82
0.01 Colch	A	183	232	85	1.80
	B	176	230	94	1.84
0.05 Colch	A	492	8	0	1.02
	B	493	7	0	1.0

 

Table 8: Historical Control Data for in vitro Micronucleus Studies of the Solvent Control

	Binucleated
	-S9 mix		+S9 mix
	3 h exposure	24 h exposure	3 h exposure
Mean number of micronucleated cells (per 2000 cells)	4.0	4.9	4.7
SD	2.9	3.6	3.3
n	101	100	101
Lower Control Limit (95% Control Limits)	-2	-2	-2
Upper Control Limit (95% Control Limits)	10	12	11
SD = Standard deviation n = Number of observations Distribution historical negative control data from experiments performed between May 2019 and May 2022.

 

Table 9: Historical Control Data for in vitro Micronucleus Studies of the Positive Control Substances

	Binucleated			Binucleated
	-S9 mix (MMC-C)		+S9 mic (CP)	-S9 mix (Colch)
	3 h exposure	24 h exposure	3 h exposure	3 h exposure	24 h exposure
Mean number of micronucleated cells (per 2000 cells)	48.9	43.4	38.8	57.9	54.3
SD	25.1	20.8	18.0	143.0	97.9
n	106	105	108	103	99
Lower Control Limit (95% Control Limits)	0	3	4	-222	-138
Upper Control Limit (95% Control Limits)	98	84	74	338	246
SD = Standard deviation n = Number of observations Distribution historical positive control data from experiments performed between May 2019 and May 2022.

Conclusions:

The study was performed according to OECD guideline 487 and under GLP conditions. Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol was found not to induce micronuclei in cultured human peripheral blood lymphocytes neither in the presence nor absence of a metabolic activation system. Therefore, the test substance is considered to be neither clastogenic nor aneugenic under the test conditions used.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 - 25 Apr 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997; corrected in 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains)
trp operon (for the E.coli strain)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: rfa (deep rough (defective lipopolysaccharide cellcoat)), gal (mutation in galactose metabolism); chl (mutation in nitrate reductase); bio (defective biotin synthesis); uvrB (loss of excision repair system (deletion of ultraviolet-repair B gene))

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH (Giessen, Germany); prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg bw)
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck, Darmstadt, Germany); 1 mL 0.33 M KCl solution (Merck, Darmstadt, Germany). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix (corresponds to 0.025 mL S9)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Test concentrations with justification for top dose:

Dose-range finding test / Experiment 1 (plate incorporation):
- S. typhimurium TA 100 / E. coli WP2 uvr A: 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate with and without metabolic activation,
- S. typhimurium TA 1535 / 1537 / 98: 17, 52, 164, 512, 1600, 5000 µg/plate with and without metabolic activation

Experiment 2 (preincubation):
- all strains: 17, 52, 164, 512, 1600, 5000 µg/plate with and without metabolic activation

5000 µg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances according to OECD 471.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone (Merck, Darmstadt, Germany)
- Justification for choice of solvent/vehicle: The test material proved insoluble in Milli-Q water, DMSO and ethanol. The test material formed a clear colourless solution in acetone, tetrahydrafuran and hexane. Acetone was chosen to be the solvent of the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other:

Remarks:

3) 2-aminoanthracene (2AA; in DMSO): +S9 / TA 1535 / 2.5 µg/plate (Exp 1 and 2), TA 1537 / 2.5 µg/plate (Exp 1 and 2), TA 98 / 1 µg/plate (Exp 1 and 2), TA 100 / 1 µg/plate (Exp 1), TA 100 / 5 µg/plate (Exp 2), WP2uvrA / 15 µg/plate (Exp 1 and 2)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: three replicate plates / dose
- Number of independent experiments: 2 (direct plate and pre-incubation assay)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation; dose- range finding experiment and Experiment 1); preincubation (Experiment 2)
- Cell density at seeding (if applicable): 10^9 cells/mL

TREATMENT AND HARVEST SCHEDULE:
Direct plate assay
- Exposure duration/duration of treatment: 48 ± 4 h at 37.0 ± 1.0 °C
Pre-incubation assay
- Preincubation period: 30 ± 2 min at 37.0 ± 1.0 °C
- Exposure duration/duration of treatment: 48 ± 4 h at 37.0 ± 1.0 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction of the bacterial lawn and decrease in the number of revertant colonies per plate

OTHER
- The Salmonella typhimurium strains were checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
- The Escherichia coli WP2uvrA strain was checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year.

Evaluation criteria:

Rationale for test conditions according to OECD 471
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA 1535, TA 1537 or TA 98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA 1535, TA 1537 or TA 98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow-up experiment.

Statistics:

Mean values and standard deviations were calculated.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation at concentrations of 1600 μg/plate and upwards in Experiment 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation at concentrations of 1600 μg/plate and upwards in Experiment 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation at concentrations of 1600 μg/plate and upwards in Experiment 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation at concentrations of 1600 μg/plate and upwards in Experiment 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation at concentrations of 1600 μg/plate and upwards in Experiment 1 and 2

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
- Water solubility: The test material proved insoluble in Milli-Q water.
- Precipitation: Precipitation of the test material was observed on the plates at concentrations of 1600 μg/plate and upwards for both experiments.

STUDY RESULTS:
In the Direct Plate Assay (Dose-Range finding study / Experiment 1), there was no increase in the number of revertants observed upon treatment with the test material under all conditions tested (refer to table 1 and 2).
The positive controls induced a two-fold or greater increase in the number of revertant colonies compared to the solvent control for each strain, showing the study was properly executed. There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants observed upon treatment with the test material under all conditions tested.

In the Pre-Incubation Assay (Experiment 2), there was no increase in the number of revertants observed upon treatment with the test material at all concentrations, in the absence and presence of S9-mix in the tester strains TA 98, TA 100, and WP2uvrA10. In tester strain TA1537 in the absence of S9 mix, the test material induced up to 4.0-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was within the historical data range and related to a relative low solvent control. Therefore, the 4.0-fold increase observed is considered to be not biologically relevant (refer to table 3).
In tester strain TA1535 in the absence of S9-mix, the test material induced up to 3.7-fold increase in the number of revertant colonies compared to the solvent control at 512 μg/plate. There is an absence of any observed dose effect and a possibility of an outlier in the three measurements at this dose level. Therefore, the 3.7-fold increase observed is considered to be related to a specific outlier and to be not biologically relevant (refer to table 3).
The positive controls induced two-fold or greater increases in the number of revertant colonies compared to the negative control for each strain, showing the study was properly executed. There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants observed upon treatment with the test material under all conditions tested.

HISTORICAL CONTROL DATA
See 'Any other information on results incl. tables', table 4 and 5.

Table 1: Dose-Range Finding Test: Mutagenic Response in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay (Direct Plate Assay / Part of Experiment 1)

Dose (µg / plate)	Mean number of revertant colonies / 3 replicate plates (± SD) with one Salmonella typhimurium and one Escherichia coli strain
	TA 100		WP2uvrA
	Mean	± SD	Mean	± SD
Without S9-mix
Positive control	692	75	945	54
Solvent control	108	12	21	5
1.7	91	6	24	8
5.4	100	11	19	3
17	89	5	21	2
52	94	8	23	8
164	94	6	21	7
512	90	25^NP	14	3^NP
1600	92	2^SP	25	4^SP
5000	85	18^n SP	24	9^n SP
With S9-mix
Positive control	1108	237	289	113
Solvent control	92	6	24	4
1.7	76	10	26	6
5.4	75	21	25	3
17	75	27	24	5
52	76	10	28	8
164	84	10	25	12
512	71	7^NP	27	3^NP
1600	72	5^SP	19	6^SP
5000	88	6^n SP	22	4^n SP
NP = No precipitate SP = Slight precipitate n = Normal bacterial background lawn

 

Table 2: Experiment 1: Mutagenic Response in the Salmonella typhimurium Reverse Mutation Assay (Direct Plate Assay)

Dose (µg / plate)	Mean number of revertant colonies / 3 replicate plates (± SD) with different Salmonella typhimurium strains
	TA 1535		TA 1537		TA 98
	Mean	± SD	Mean	± SD	Mean	± SD
Without S9-mix
Positive control	1082	38	917	48	1342	74
Solvent control	10	3	7	2	16	3
17	9	4	7	4	13	5
52	9	1	2	1	14	6
164	12	6	9	3	15	3
512	10	2^NP	3	2^NP	17	5^NP
1600	10	3^SP	4	1^SP	17	2^SP
5000	15	5^n SP	8	6^n SP	20	4^n SP
With S9-mix
Positive control	353	20	390	44	1135	114
Solvent control	9	3	5	2	26	4
17	10	5	4	2	21	3
52	6	2	3	0	14	2
164	10	2	3	2	17	6
512	14	5^NP	6	1^NP	18	4^NP
1600	12	5^SP	6	4^SP	20	6^SP
5000	8	3^n SP	3	1^n SP	20	3^n SP
NP = No precipitate SP = Slight precipitate n = Normal bacterial background lawn

 

Table 3: Experiment 2: Mutagenic Response in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay (Pre-incubation Assay)

Dose (µg / plate)	Mean number of revertant colonies / 3 replicate plates (± SD) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
	Mean	± SD	Mean	± SD	Mean	± SD	Mean	± SD	Mean	± SD
Without S9-mix
Positive control	976	64	179	12	1535	175	740	66	1454	66
Solvent control	9	2	2	1	29	15	117	15	15	4
17	22	15	8	6	19	3	113	23	19	8
52	15	10	6	3	15	1	139	0^i	22	9
164	10	3^i	8	1	12	8	122	16	14	3
512	33	21^NP	4	1^NP	14	8^NP	107	20^NP	21	5^NP
1600	21	12^SP	2	2^SP	16	3^SP	123	11^SP	19	1^SP
5000	8	2^n SP	7	6^n SP	16	2^n SP	106	29^n SP	25	17^n SP
With S9-mix
Positive control	272	17	339	20	1233	43	199	41	416	99
Solvent control	15	4	8	6	31	5	89	20^i	24	5
17	15	4	7	4	24	2	88	7	24	4
52	8	6	4	2	29	4	100	10	25	3
164	11	4	4	1	22	4	99	8	20	6
512	12	6^NP	3	1^NP	43	24^NP	91	7^NP	15	1^NP
1600	17	6^SP	6	3^SP	28	6^SP	90	4^SP	22	9^SP
5000	14	7^n SP	11	1^n SP	40	8^n SP	90	4^n SP	27	4^n SP
NP = No precipitate SP = Slight precipitate i = Plate infected; mean of two plates n = Normal bacterial background lawn

 

Table 4: Historical Control Data of the Solvent Control

	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	2 - 26	3 - 23	1 - 24	1 - 20	3 - 43	5 - 62	58 - 188	39 - 169	9 - 61	9 - 68
Mean	9	10	5	5	12	17	100	90	21	25
SD	3	3	2	2	4	5	16	21	8	10
Total number of plates	2110	2080	2135	2118	2200	2221	2226	2156	2038	2068
95% upper limit	14	15	9	10	20	27	131	131	37	44
95% lower limit	4	5	0.7	0.5	4	7	69	49	5	6
SD = Standard deviation Historical control data from experiments performed between Dec 2018 and Dec 2021.

 

Table 5: Historical Control Data of the Positive Control Materials

	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	95 - 1373	78 - 1195	71 - 1587	48 - 1989	379 - 2141	251 - 3369	173 - 1852	166 - 2666	89 - 2027	109 - 1942
Mean	897	272	855	265	1371	951	800	1339	1312	424
SD	169	110	350	167	304	405	192	428	369	189
Total number of plates	1924	1911	1520	1955	2064	2001	2007	1984	1910	1879
95% upper limit	1229	488	1541	593	1966	1745	1177	2177	2035	795
95% lower limit	565	56	169	-63	776	157	423	501	589	53
SD = Standard deviation Historical control data from experiments performed between Dec 2018 and Dec 2021.

Conclusions:

Under the present test conditions, Esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol showed negative mutagenic responses in all bacterial strains over the entire dose-range.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation in bacteria

The ability of the registered substance to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA, either in the presence or absence of a metabolic activation system (S9-mix) was examined according to OECD  471 and GLP principles (Charles River, 2022f). The test was performed in two independent experiments: a plate incorporation assay (experiment 1, including results from the dose-range finder) and a pre-incubation assay (experiment 2). The vehicle used was acetone. In the dose-range finding study, the test item was initially tested at concentrations of 1.7 - 5000 µg/plate in the strains S. typhimurium TA100 and E. coli WP2uvrA in a plate incorporation assay with and without metabolic activation. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of experiment 1. In the first mutation experiment, the test item was tested at concentrations of 17 - 5000 µg/plate in the strains S. typhimurium TA1535, TA1537 and TA98 with and without metabolic activation. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. No toxicity was observed at any of the dose levels tested: the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second mutation experiment, the test item was tested at concentrations of 17 - 5000 µg/plate in the tester strains S. typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2uvrA in a pre-incubation assay with and without metabolic activation. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. No toxicity was observed at any of the dose levels tested: the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a biologically relevant, dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (S. typhimurium TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain E. coli WP2uvrA both in the absence and presence of S9-metabolic activation.

Based on the results of this study it was concluded that esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol was not mutagenic in the bacterial reverse mutation assay.

 

In vitro micronucleus study in mammalian cells

The ability of the registered substance to induce micronuclei in human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix), was examined according to OECD 487 and GLP principles (Charles River, 2022g). Three treatment conditions were tested: A short-term treatment (Experiment 1), where the cells were treated for 3 h, in the absence and presence of S9 metabolism with a harvest time of approximately 27 h, and a long-term (continuous) treatment (Experiment 2) only in the absence of S9 metabolism, until harvest at 24 h. The vehicle used was tetrahydrofuran. Based on solubility features, the maximum dose level of 15.6 μg/mL was selected as top dose for Experiment 1 and 7.8 μg/mL for Experiment 2. Two replicate cell cultures were prepared at each test concentration In the short-term treatment, the test item was tested at concentrations of 3.9 – 15.6 µg/mL with and without metabolic activation, along with a solvent control, and a positive control (cyclophosphamide for with metabolic activation and colchicine and mitomycin C for without metabolic activation). The test item precipitated at the 7.8 μg/mL dose level. No toxicity was observed at any of the dose levels tested and no significant increase in micronucleated cells was observed. In the long-term treatment, the test item was tested at concentrations of 2.0 – 7.8 µg/mL without metabolic activation, along with a solvent control, and a positive control (colchicine and mitomycin C). The test item precipitated at the 7.8 μg/mL dose level. No toxicity was observed at any of the dose levels tested and no significant increase in micronucleated cells was observed. The negative and positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate, and that the metabolic activation system functioned properly. Based on the results of this study it was concluded that esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol substance was not clastogenic in the in vitro micronucleus test.

 

In vitro: gene mutation in mammalian cells

The ability of the registered substance to induce 5-trifluorothymidine resistant mutants in mouse lymphoma TK+/− L5178Y cells, either in the presence or absence of a metabolic activation system (S9-mix), was examined according to OECD 490 and GLP principles (Charles River, 2023). The vehicle used was tetrahydrofuran. Multiple preliminary toxicity tests were performed indicating that the maximum practicable concentration of the test item was 1000 µg/mL. Based on the results obtained in the preliminary toxicity test, two independent assays for mutation at the TK locus were performed: a short-term treatment (Experiment 1), where the cells were treated for 3 h, in the absence and presence of S9 metabolism and a long-term (24 h) treatment (Experiment 2) only in the absence of S9 metabolism. In the short-term treatment, the test item was tested at concentrations of 10 - 1000 µg/mL with and without metabolic activation. The test item precipitated at 500 μg/mL and upwards. No toxicity was observed at any of the dose levels tested and no significant increase in mutant frequency was observed. In the long-term treatment, the test item was tested at concentrations of 12.5 - 1000 µg/mL without metabolic activation. The test item precipitated at 600 μg/mL and upwards. No toxicity was observed at any of the dose levels tested and no significant increase in mutant frequency was observed. The negative and positive control values were within the normal ranges indicating that the test conditions were adequate, and that the metabolic activation system functioned properly. Based on the results of this study it was concluded that esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritolsubstance was not mutagenic in mouse lymphoma TK+/− L5178Y cells.

 

Conclusion for genetic toxicity

The results of the available in vitro studies on mutagenicity in bacterial cells, cytogenicity in mammalian cells, and gene mutation in mammalian cells were consistently negative. Based on the available data performed with the registered substance, no mutagenic or clastogenic potential is expected for esterification products of fatty acids, C18 (unsaturated) alkyl and adipic acid with pentaerythritol.

 

 

 

Justification for classification or non-classification

According to Regulation (EC) 1272/2008 or Directive 67/548/EEC, the data on genotoxicity are conclusive but not sufficient for classification.