Linseed oil, ester with pentaerythritol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

In vitro mamammlian cell gene mutation tests using the thymidine kinase gene" following OECD 490

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 11, 2017 to July 03, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

OECD Guideline for the testing of chemicals, part 490, adopted July 26, 2016,”In vitro mammalian Cell gene mutation tests using the Thymidine Kinase Gene“

Deviations:

no

Remarks:

As all acceptability criteria of the assay were met or the deviations were considered as uncritical.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EU-Method B.17 of the commission regulation (EC) no. 440/2008, adopted May 30, 2008,”In vitro mammalian Cell gene mutation test”

Deviations:

no

Remarks:

As all acceptability criteria of the assay were met or the deviations were considered as uncritical.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mamammlian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Linseed oil, ester with pentaerythritol

EC Number:

271-985-4

EC Name:

Linseed oil, ester with pentaerythritol

Cas Number:

68648-28-2

Molecular formula:

UVCB

IUPAC Name:

2,3-dihydroxypropyl (7E,10E,13E)-hexadeca-7,10,13-trienoate; bis(3-[(7E,10E,13E)-hexadeca-7,10,13-trienoyloxy]-2-hydroxypropyl (9E)-octadec-9-enoate); 3-hydroxy-2-(hydroxymethyl)-2-[(octadec-9-enoyloxy)methyl]propyl (9E,12E,15E)-octadeca-9,12,15-trienoate; 3-hydroxy-2-{[(9E)-octadec-9-enoyloxy]methyl}-2-{[(9E,12E)-octadeca-9,12-dienoyloxy]methyl}propyl (9E,12E,15E)-octadeca-9,12,15-trienoate

Test material form:

liquid

Specific details on test material used for the study:

Name: Linseed oil, ester with pentaerythritol
Batch no.: 16.343.096
Appearance: Yellow liquid
Composition: 100% UVCB
Purity: 100% as the substance is an UVCB
Homogeneity: Homogeneous
Expiry date: October 05, 2017
Storage Room Temperature:(20 ± 5°C), After opening keep under inert gas

Method

Target gene:

Thymidine kinase gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y is a murine T-cell lymphoma cell line, which grows as single or aggregated
round cells in suspension. This cell line is characterized by a high sensitivity to chemical
mutagens, by a high proliferation rate (doubling time 10-12 h in stock cultures), a high
cloning efficiency (CE) and a stable spontaneous mutant frequency. The L5178Y consists
of a stable karyotype and shows a diploid chromosome number (40 ± 2). The cells were
purchased by ATCC (Wesel, Germany) and were sold under the name L5178Y TK+/-
clone (3.7.2C) [TK+/- (clone 3.7.2C)] (ATCC® CRL-9518™).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver enzyme S9 fraction/liver S9 mix from male rats, treated with Aroclor 1254

Test concentrations with justification for top dose:

Nominal concentrations in pre-test [μL/mL]: 5 2.5 1.25 0.63 0.31 0.16 0.08.
According to the OECD guideline 490 the highest concentration should be 0.01 M or 2 mg/mL or 2 μL/mL (whichever is lowest), unless limited by the solubility or toxicity of the test substance. RCE values below 20 % are considered toxic. In case of toxic effects, the highest test substance concentration of the main experiment should reduce the RSG value to 10 -20 %. In reference to the results of the pretest, 7-8 concentrations were chosen for experiment I and II:
Nominal concentrations in experiment I [μL/mL]: 2.5; 1.25; 0.63; 0.31; 0.16; 0.08; 0.04; 0.02
Nominal concentrations in experiment II [μL/mL]: 2.5; 0.63; 0.31; 0.16; 0.08; 0.04; 0.02

Vehicle / solvent:

In a non-GLP pre-test, the solubility of the test substance was determined. The test substance was sufficiently soluble in dimethyl sulfoxide (DMSO). Therefore, DMSO was used as solvent for the test substance.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640 medium without supplements was used as solvent control for the positive control methyl methanesulfonate (MMS). 0.9% NaCl was used as solvent control for the positive control cyclophosphamide (CPA). DMSO was used as solvent for the test item.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

(1) Methyl methanesulfonate (MMS) (-S9) 19.5 μg/mL (Experiment I) and 12.5 μg/mL (Experiment II). (2) Cyclophosphamide (CPA) monohydrate (+S9) 4.5 μg/mL

Details on test system and experimental conditions:

Method of application: Test substance in DMSO
Incubation period: 4 h (Experiment I) and 24 h (Experiment II)
Number of replications: Two replicates per culture: Experiment I with 8 concentrations + Experiment II with 7 concentrations
Determination of genotoxicity method: Relative total growth (RTG) % and mutant per 106 cells

The TK is able to catalyse the conversion of the purine analogue Trifluorothymidine (TFT) to its cytostatic and cytotoxic derivative trifluorothymidine-monophosphate. Therefore, cells deficient in TK are resistant to TFT. These cells are able to proliferate in the presence of the chemical substance whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 48 ± 4 h. The mutant frequency is determined by cloning a known number of cells in 96-well plates containing the selective agent to detect mutant cells, and in 96-well plates without selective agent to determine the number of surviving cells (cloning efficiency). After a suitable period of time the colonies are counted. Mutation frequencies are calculated from the number of mutant colonies (growth in selective medium) divided by the number of surviving cells (non-selective medium). Furthermore the size of the colonies is evaluated. Despite the exact genetic reason for the different colony forms is not yet clear, it was postulated many times, that the induction of large mutant colonies is generally associated with chemicals inducing single point mutations and the induction of small-colony mutants with chemicals inducing massive chromosomal aberrations (Schisler et al., 2013). In the pre-test the cytotoxicity of the cells is given by the relative cloning efficiency, RCE of the cells. In the case of a clear reduction of the RCE value in comparison to the solvent control a readjustment of the tested concentrations should be made for the main experiments. In the main experiments the cytotoxicity was determined by the more exactly but also more complex relative total growth (RTG). Generally, RTG and RCE values below 20 % were defined as cytotoxic (Moore et al., 2002). In this case a determination of the mutation frequency is not valid. Reference mutagens are tested in parallel to the test substance in order to demonstrate the sensitivity of the test system.

Rationale for test conditions:

Reasons for the Choice of the Cell Line L5178Y
The L5178Y is a murine T-cell lymphoma cell line, which grows as single or aggregated round cells in suspension. This cell line is characterized by a high sensitivity to chemical mutagens, by a high proliferation rate (doubling time 10-12 h in stock cultures), a high cloning efficiency (CE) and a stable spontaneous mutant frequency. The L5178Y consists of a stable karyotype and shows a diploid chromosome number (40 ± 2).

Evaluation criteria:

Cell Numbers for Determination of RSG
- Cell numbers in suspension were determined with a cell counter. Afterwards, the total suspension growth (TSG) and the relative suspension growth (RSG) was calculated

Viability
- Number of empty wells per microwell plate was counted manually. All generated data of each experiment (test item, solvent controls and positive control) were recorded in the raw data. Afterwards the relative cloning efficiency (RCE) and the relative total growth (RTG) were calculated

Mutant Colonies
Colonies were counted manually under a binocular magnifying glass. In accordance with their size, the colonies were classified into two groups:
- Less than 25 % of the well’s diameter = small colony
- More than 25 % of the well’s diameter = large colony

All generated data of each experiment (test item, solvent controls and positive control) were recorded in the raw data. Afterwards the mutant frequency (MF) was calculated

Statistics:

A linear regression (least squares) of the test substance concentrations was performed to assess a possible dose dependent increase of mutant frequencies. With the assessment of this regression, it can be evaluated whether mutations increase with increasing dose of the test substance. A p-value of 0.05 or lower (significance level 95%) is considered as critical. No significant correlation between the test substance concentrations and the mutant frequency was detected. The positive controls were tested at one concentration only. Therefore, no dose depend- ency could be evaluated, although the positive controls showed considerable increases in mutants. In the following table, the statistical significance values are presented. The chi- square test was used. Statistically significant increase in mutants is considered as given if p is below 0.01.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicty was observed for 0.63 and 2.5 μL/mL test substance concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Deviations from the study plan and guideline
In experiment I -S9, the TSG value in replicate B of the solvent control DMSO was 6.84% and therefore below 8%. The deviation is classified as uncritical, since it can be excluded that the end result is affected due to the slightly reduced growth rate in experiment I. In addition the mean of the two replicates was above 8. The deviation was assessed and signed by the study director on July 06, 2017.

In all experiments the RTG values of the cytotoxic test substance concentrations were not exactly between 10% and 20%. This deviation was considered as uncritical, since more important than a RTG value between 10-20% is, that a cytotoxic concentration occurs. In most cases, there is a point of turnover from complete cytotoxicity to low/moderate cytotoxicity. Therefore, a setting of such an exact RTG value is sometimes technically not possible. The deviation was assessed and signed by the study director on July 18, 2017.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test substance is considered to be nonmutagenic under the conditions of the mouse lymphoma assay.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substance, Linseed oil, ester with pentaerythritol, with the in vitro mammalian cell gene mutation tests using the thymidine kinase gene according to OECD Guideline 490, in compliance with GLP. This study was performed to investigate the potential of the test substance to induce mutations at the thymidine kinase locus (Tk1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178Y Tk+/- cells. The assay was performed in a pre-test and three independent experiments whereby the first experiment I was declared invalid and was repeated with different concentrations. The results of the invalid experiment are not included in this final report. The pre-test was performed to detect a potential cytotoxic effect of the test substance. Based on the results of this test the concentrations for the two main experiments were determined. Experiment I was performed with and without metabolic activation (liver enzyme S9 fraction / “liver S9 mix from male rats, treated with Aroclor 1254”) and a treatment period of 4 h. Experiment II was performed with a treatment period of 24 h in the absence of metabolic activation. The highest nominal tested concentration was 2.5 μL/mL. Turbidity of the test substance was visible in all experimental parts at the test substance concentrations 2.5 μL/mL, 1.25 μL/mL, 0.63 μL/mL and 0.31 μL/mL. For that reason, in accordance to the OECD 490, the highest analyzable test substance concentration for mutagenicity is 0.31 μL/mL. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test substance. Under the study conditions, the test substance did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test substance is considered to be nonmutagenic under the conditions of the mouse lymphoma assay (Fruhmesser, 2017).