Linseed oil, ester with pentaerythritol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced sprague dawley rat liver preparations (S9 mixture)

Test concentrations with justification for top dose:

1, 10, 100, 1000, 10,000 and 100,000 µg/plate of the test substance (with and without S9 mix), a density of 1 gm/mL was cited for the test substance in the report. Test substance was tested neat and at 10, 100, 1000, 10000 and 100000 fold dilution in DMSO.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Key result

Species / strain:

other: Salmonella typhimurium TA98, TAlOO, TA1535, TA1537 and Escherichia coli I WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No mutagenic activity was observed

Conclusions:

Under the study conditions, the test substance was non-mutagenic with or without metabolic activation in the bacterial reverse mutation assay.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, in compliance with GLP. The strains used were Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2. 1, 10, 100, 1000, 10,000 and 100,000 µg/plate of the test substance (with and without S9 mix), a density of 1 gm/mL was cited for the test substance in the report. Test material was tested neat and at 10, 100, 1000, 10000 and 100000 fold dilution in DMSO.  The test substance was negative for mutagenic activity in the four Salmonella tester strains and in the E. coli strain, with or without metabolic activation. No mutagenic activity was observed at concentrations ranging from 1 µg/plate to the highest concentration of 100,000 µg/plate (neat). The bacterial strains tested included Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2. The negative (vehicle) control and positive controls gave the appropriate responses as expected. Under the study conditions, the test substance was non-mutagenic with or without metabolic activation in the bacterial reverse mutation assay (US EPA HPV, 2004).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

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:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsomal enzyme homogenate

Test concentrations with justification for top dose:

10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

for TA 1535

Positive control substance:

sodium azide

Remarks:

5 µg/plate dissolved in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

for TA 98

Positive control substance:

2-nitrofluorene

Remarks:

20 µg/plate dissolved in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

for TA 100

Positive control substance:

methylmethanesulfonate

Remarks:

650 µg/plate dissolved in DMSO

Untreated negative controls:

yes

Remarks:

ethanol

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

for WP2 uvrA

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

10 µg/plate dissolved in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

For the bacterial test with metabolic activation: TA1535, TA98 and TA100

Positive control substance:

other: 1 µg/plate 2-aminoanthracene dissolved in DMSO

Remarks:

none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

For the bacterial test with metabolic activation: TA1537 and TA100

Positive control substance:

other: 2.5 µg/plate 2-aminoanthracene dissolved in DMSO

Remarks:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

For the bacterial test with metabolic activation: TA1537

Positive control substance:

other: 5 µg/plate 2-aminoanthracene dissolved in DMSO

Remarks:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

For the bacterial test with metabolic activation: WP2uvrA

Positive control substance:

other: 10 µg/plate 2-aminoanthracene dissolved in DMSO

Remarks:

None

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

Evaluation criteria:

The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain. The positive control chemicals, sodium azide for TA1535; 9-aminoacridine for TA1537, 2-nitrofluorene for TA98, methylmethanesulphonate for TA100 and 4-nitroquinoline N-oxide for WP2uvrA should produce positive responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group means.
A test substance was considered negative (not mutagenic) in the test if (a) The total number of revertants in tester strain TA100 was not greater than
two times the concurrent control, and the total number of revertants in the tester strains TA1535, TA1537, TA98 or WP2uvrA was not greater than three times the concurrent control, (b) The negative response was reproducible in at least one independently repeated
experiment.

Statistics:

Not applicable

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Under the conditions of the study, the test substance was considered to be non-mutagenic.

Executive summary:

A study was conducted to determine the potential mutagenicity of glycerides, C16-18 and C18-unsatd. (in the fomr of pine nut oil), according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test substance using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 3 to 5000 μg/plate. The experiment was repeated on using the same dose range as the range-finding test. The test substance caused a precipitation in the two highest concentration tested. The test substance was, therefore, tested up to the third dose level of 1000 μg/plate. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation. Under the conditions of the study, the test substance was considered to be non-mutagenic (Speijers, 2009).

Reference 3

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

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.

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

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

Target gene:

Thymidine kinase gene

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.

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.

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.

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).

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254~induced Sprague-Dawly rat liver S9 mixture

Test concentrations with justification for top dose:

0.05, 0.5 and 5 µL/mL (approx. 50, 500 and 5000 µg/ml based on a density of 1 g/mL)

Untreated negative controls:

yes

Remarks:

HHam’s F- 12 complete medium

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Study was carried out to assess the ability of test substance to induce chromosomal aberrations in CHO cells cultured in vitro. Negative and positive control cultures were also prepared. One hour before the end of the incubation period, cell division was arrested with Colemid, the cells harvested and slides prepared so that the metaphase cells could be examined for chromosomal damage.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Remarks:

Ham’s F- 12 complete medium

Positive controls validity:

valid

Remarks:

mitomycin-C (-S9), cyclophosphamide (+S9)

Additional information on results:

The positive and negative controls gave the expected responses to fulfill the requirements of a valid test.

Negative control (Ham’s F- 12 complete medium) gave 1% aberrations with and without metabolic activation.

Positive control (cyclophosphamide) produced 18% aberrations in the metabolically activated CHO cell assay. Positive control (mitomycin C) produced 14% aberrations in the non-activated CHO cell assay.

Test results:

Exposure (h)	Metabolic activation	Doses tested (µL/mL)	Aberrations (%) at doses respectively	Test results
2	With	0.05, 0.5 and 5	0.5, 1.0 and 3.0	Negative
18	Without	0.05, 0.5 and 5	0.0, 2.0 and 3.5	Negative

Conclusions:

Under the study conditions, the test substance was not clastogenic in the CHO cell culture test system

Executive summary:

A study was conducted to assess the clastogenic properties of the test substance according to OECD Guideline 473, in compliance GLP. The test was carried out using Chinese hamster ovary (CHO) cells. The positive and negative controls gave the expected responses to fulfil the requirements of a valid test. Negative control (Ham’s F- 12 complete medium) gave 1% aberrations with and without metabolic activation. Positive control (cyclophosphamide) produced 18% aberrations in the metabolically activated CHO cell assay. Positive control (mitomycin C) produced 14% aberrations in the non-activated CHO cell assay. Regardless of dose level (from 0.05µL/mL to as high as 5µL/mL) and dosing regimen, the test substance was concluded to be negative for structural and numerical chromosome aberrations, with or without S-9. Under the study conditions, the test substance was not clastogenic in the CHO cell culture test system (US EPA HPV, 2004).

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From July 16, 1991 to July 27, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Strain S. typhimurium TA102 or E.coli WP2 were not tested.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Strain S. typhimurium TA102 or E.coli WP2 not tested.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa-, uvR-, Strains 98 and 100 also R+

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: rfa-, uvR-

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix (Wistar rats, male, Aroclor 1254 induced)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

for strains TA 100 and TA 1535 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

for strains TA 98 and TA 1538 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for strain TA 1537 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

all strains with metabolic activation

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Table 1: Mutagenicity of glycerol triacetate on bacteria - experiment I 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA1538	TA98	TA1537
-	Buffer	116	9	10	22	7
	Vehicle	112	11	12	34	10
-	8	114	10	7	29	10
-	40	111	10	8	34	7
-	200	114	8	9	29	8
-	1000	120	12	7	25	6
-	5000	132	6	9	29	6
Positive controls - S9	Name	SA	SA	4NP	4NP	9AA
	Concentrations (μg/plate)	2.0	2.0	40	40	80
	Number of colonies/plate	818	621	1792	1571	1025
+	Buffer	116	11	15	38	6
+	Vehicle	103	12	15	35	10
+	8	115	12	16	34	8
+	40	120	9	11	39	8
+	200	113	12	13	38	10
+	1000	126	11	14	39	7
+	5000	130	13	12	3934	7
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	5.0	2.5	5.0	5.0	2.5
	Number of colonies/plate	1594	210	1769	1572	66

 

4NP= 4-nitro-o-phenylendiamine

SA = Sodium azide

9AA = 9-Aminoacridine

2AA = 2-Aminoanthracene

 

Table 2: Mutagenicity of glycerol triacetate on bacteria - experiment II 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA1538	TA98	TA1537
-	Buffer	122	11	13	34	6
	Vehicle	127	13	9	39	13
-	8	116	12	11	30	10
-	40	110	14	12	31	8
-	200	121	13	14	39	5
-	1000	111	17	18	29	7
-	5000	122	16	13	32	10
Positive controls - S9	Name	SA	SA	4NP	4NP	9AA
	Concentrations (μg/plate)	2.0	2.0	40	40	80
	Number of colonies/plate	1018	824	1929	1643	1017
+	Buffer	127	15	19	40	9
+	Vehicle	116	21	20	43	10
+	8	128	11	19	46	10
+	40	126	17	19	49	6
+	200	115	13	19	45	9
+	1000	121	19	16	49	10
+	5000	127	21	14	40	7
Positive controls + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	5.0	2.5	5.0	5.0	2.5
	Number of colonies/plate	1535	327	1384	1515	64

 

4NP= 4-nitro-o-phenylendiamine

SA = Sodium azide

9AA = 9-Aminoacridine

2AA = 2-Aminoanthracene

Under the tested experimental conditions the test substance did not induce gene mutations in S. typhimurium strains up to the maximum of solubility. Therefore it is not considered to be mutagenic in this bacterial mutagenicity test in vitro.

Conclusions:

Under the study conditions, the test substance was not considered to be mutagenic in the Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100 and TA 1538) reverse mutation assays.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance Fatty acids, C16 -18 (even numbered), esters with pentaerythritol, was tested in a reverse mutation assay comparable to OECD guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 1538 were used. Tester strains were incubated with test material dissolved in Tween 80 at concentrations of 8, 40, 200, 1000 and 5000 µg/plate (no toxicity but tested up to precipitating concentrations) with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9-mix). Vehicle, negative and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent controls was observed in all strains treated with the test substance, neither in the presence nor in the absence of metabolic activation. Under the study conditions, the test substance was not considered to be mutagenic in the Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100 and TA 1538) reverse mutation assays (BASF, 1991).

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 06, 2004 to February 16, 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP - Guideline study. (Purity of test substance not given, evaluation criteria not given.)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

purity of test substance is not given (responsibility of the sponsor)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

-Type and identity of media: Eagles essential medium with HEPES buffer (MEM), supplemented with:
L-glutamine, penicillin/streptomycin, amphotericin B, 15% foetal calf serum
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats pretreated with phenobarbitone (80 mg/kg) and ß-naphtoflavone (100 mg/kg)

Test concentrations with justification for top dose:

Experiment I:
- 40, 80, 160, 240*, 320*, 400* µg/mL (with and without metabolic activation)
Experiment II:
- 40, 80, 160, 240*, 320*, 400* µg/mL (with and without metabolic activation)
* Dose levels (plus control dose) selected for metaphase analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9), 24 h (without)
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 20 h; 24 h treatment: 0 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL (demecolcine)
STAIN (for cytogenetic assays): 5% Gurrs Giemsa for 5 minutes

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

No data

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's exact test.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: cloudy precipitates were observed at and above 40 and 80 µg/mL in the 24-hour continuous and 4-hour pulse treatment groups, respectively

RANGE-FINDING/SCREENING STUDIES:
The dose range tested was 10-320 µg/mL. The test material produced some weak toxicity in the 4-hour treatment group but not the 24-hour treatment group. Toxicity could not be reproduced in the main experiment (scorable metaphases at every dose level).

COMPARISON WITH HISTORICAL CONTROL DATA:
The results are in range with historical control data.

Test results of experiment I.

Test item	Concentration	Mitotic Index	Aberrant cells in %
Experiment I	in µg/mL	in %	with gaps	without gaps
Exposure period 4h, fixation time 20h, without S9 mix
control	0	100	1	0
MMC	0.4	35	53	37
Test substance	240	98P	0.5	0
	320	110P	0	0
	400	91P	0.5	0.5
Exposure period 4h, fixation time 20h, with S9 mix
control	0	100	1	0.5
CP	10	20	35.5	28.5
Test substance	240	89P	1.5	0
	320	89P	0.5	0
	400	108P	3.5	1

Test item	Concentration	Mitotic Index	Aberrant cells in %
Experiment II	in µg/mL	in %	with gaps	without gaps
Exposure period + fixation time 24h, without S9 mix
control	0	100	1.5	0
MMC	0.4	41	70	67
Test substance	240	61	0.5	0.5
	320	53	0.5	0
	400	83	0	0
Exposure period 4h, fixation time 20h, with S9 mix
control	0	100	0.5	0
CP	10	30	67	56
Test substance	240	103	1	0
	320	76	1	0
	400	110	1.5	0.5

Mean Frequency of Polyploid Cells (%)

Experiment I dose level µg/mL	harvest time 24 hours
	4 hours without S9	4 hours with S9
0	0.0	0.0
240	0.0	0.0
320	0.0	0.0
400	0.0	0.0
MMC 0.4	0.0	NA
CP 10	NA	0.0

dose level µg/mL	harvest time 24 hours
	24 hours without S9	4 hours with S9
0	0.5	0.0
240	0.0	0.0
320	0.0	0.0
400	0.0	0.5
MMC 0.4	0.0	NA
CP 10	NA	0.0

CP: Cyclophosphamide

MMC: Mitomycin C

Conclusions:

Under the study conditions, the test substance did not induce structural chromosome aberrations in the human lymphocytes with and without metabolic activation.

Executive summary:

A study was conducted to determine in vitro cytogenicity of fatty acids, C16-18 and C18-unsatd., branched and linear ester with trimethylolpropane in cultured peripheral human lymphocyte study comparable to OECD Guideline 473, in compliance with GLP. Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). In the first experiment cells were exposed for 4 h to the test substance dissolved in acetone at concentrations of 240, 320, 400 µg/mL with and without metabolic activation. In the second experiment cells were exposed for 4 h to 240, 320, 400 µg/mL with metabolic activation and for 24 h to 240, 320, 400 µg/mL followed by 24 h expression time without metabolic activation. The test substance did not induce cytotoxicity but a cloudy precipitate was already visible at 40 µg/mL. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. Mitomycin C and cyclophosphamide were used as positive control substances inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 200 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level tested in comparison to the negative controls. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro. Under the study conditions, the test substance did not induce structural chromosome aberrations in the human lymphocytes with and without metabolic activation (Arizona, 2004).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity was assessed based on toxicity studies for the test substance itself, but also on substances representative of the main constituents, which can be categorised as pentaerythritol esters (PE) and glycerol esters (GE). The results are presented below:

 

In vitro gene mutation study in bacteria (Ames test)

Linseed oil, ester with pentaerythritol

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, in compliance with GLP. The strains used were Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2. 1, 10, 100, 1000, 10,000 and 100,000 µg/plate of the test substance (with and without S9 mix), a density of 1 gm/mL was cited for the test substance in the report. Test material was tested neat and at 10, 100, 1000, 10000 and 100000 fold dilution in DMSO.  The test substance was negative for mutagenic activity in the four Salmonella tester strains and in the E. coli strain, with or without metabolic activation. No mutagenic activity was observed at concentrations ranging from 1 µg/plate to the highest concentration of 100,000 µg/plate (neat). The bacterial strains tested included Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2. The negative (vehicle) control and positive controls gave the appropriate responses as expected. Under the study conditions, the test substance was non-mutagenic with or without metabolic activation in the bacterial reverse mutation assay (US EPA HPV, 2004).

Pentaerythritol esters (PE)

A study was conducted to determine the mutagenic potential of fatty acids, C16-18 (even numbered), esters with pentaerythritol in a reverse mutation assay comparable to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 1538 were used. Tester strains were incubated with test material dissolved in Tween 80 at concentrations of 8, 40, 200, 1000 and 5000 µg/plate (no toxicity but tested up to precipitating concentrations) with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9-mix). Vehicle, negative and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent controls was observed in all strains treated with the test substance, neither in the presence nor in the absence of metabolic activation. Under the study conditions, the test substance was not considered to be mutagenic in the Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100 and TA 1538) reverse mutation assay (BASF, 1991).

Glycerol esters (GE)

A study was conducted to determine the potential mutagenicity of glycerides, C16-18 and C18-unsatd. (in the fomr of pine nut oil), according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test substance using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 3 to 5000 μg/plate. The experiment was repeated on using the same dose range as the range-finding test. The test substance caused a precipitation in the two highest concentration tested. The test substance was, therefore, tested up to the third dose level of 1000 μg/plate. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation. Under the conditions of the study, the test substance was considered to be non-mutagenic (Speijers, 2009).

 

In vitro chromosomal aberration test

Linseed oil, ester with pentaerythritol

A study was conducted to assess the clastogenic properties of the test substance according to OECD Guideline 473, in compliance GLP. The test was carried out using Chinese hamster ovary (CHO) cells. The positive and negative controls gave the expected responses to fulfil the requirements of a valid test. Negative control (Ham’s F- 12 complete medium) gave 1% aberrations with and without metabolic activation. Positive control (cyclophosphamide) produced 18% aberrations in the metabolically activated CHO cell assay. Positive control (mitomycin C) produced 14% aberrations in the non-activated CHO cell assay. Regardless of dose level (from 0.05µL/mL to as high as 5µL/mL) and dosing regimen, the test substance was concluded to be negative for structural and numerical chromosome aberrations, with or without S-9. Under the study conditions, the test substance was not clastogenic in the CHO cell culture test system (US EPA HPV, 2004).

 

Pentaerythritol esters (PE)

A study was conducted to determine in vitro cytogenicity of fatty acids, C16-18 and C18-unsatd., branched and linear ester with trimethylolpropane in cultured peripheral human lymphocyte study comparable to OECD Guideline 473, in compliance with GLP. Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). In the first experiment cells were exposed for 4 h to the test substance dissolved in acetone at concentrations of 240, 320, 400 µg/mL with and without metabolic activation. In the second experiment cells were exposed for 4 h to 240, 320, 400 µg/mL with metabolic activation and for 24 h to 240, 320, 400 µg/mL followed by 24 h expression time without metabolic activation. The test substance did not induce cytotoxicity but a cloudy precipitate was already visible at 40 µg/mL. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. Mitomycin C and cyclophosphamide were used as positive control substances inducing statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 200 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level tested in comparison to the negative controls. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro. Under the study conditions, the test substance did not induce structural chromosome aberrations in the human lymphocytes with and without metabolic activation (Arizona, 2004).

 

In vitro mammalian mutation test

Linseed oil, ester with pentaerythritol

A study was conducted to determine the genotoxic potential of the test substance in an 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 non-mutagenic under the conditions of the mouse lymphoma assay (Fruhmesser, 2017).

 

In vivo genotoxicity

Glycerol esters (GE)

A study was conducted to evaluate possible clastogenic and cytotoxic activity of glycerides, C16-18 and C18-unsatd. (in the form of red palm oil) in mouse bone marrow cells in vivo. Three groups of Balb/C female mice, 10 per group, were dosed with test substance supernatant, test substance sediment or a mixture of the two at a dose level of 4500 mg/kg bw/d by gavage for 5 consecutive days. A negative group was dosed (by gavage) with corn oil and the positive control group was injected intraperitoneally with cyclophosphamide. 24 h after the last treatment each animal were killed by cervical dislocation. The femurs from each animal were dissected and stripped clean of muscles, for cytological preparations from bone marrow. For each animal 100 bone marrow metaphase cells were analysed for evaluation of chromosome aberration and 1000 cells for determination of mitotic index. No statistically significant differences were observed in the frequency of chromosomal aberrations and the mitotic index in bone marrow samples in any of the three groups. Under the study conditions, test substance did not induce chromosomal aberrations in mouse bone marrow cells, in vivo, after daily expositions of 4500 mg/kg bw/d. Furthermore, this dose did not promote any alteration in the mitotic index, suggesting that test substance had no cytotoxic effects (Oliveria, 1994).

Justification for classification or non-classification

Based on the information available for the substance itself and/or its main constituents, the test substance is not considered to meet the requirements for genetic toxicity classification according to the EU CLP (Regulation 1272/2008/EC) criteria.