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Genetic toxicity in vitro

Description of key information

Non-mutagenicity of [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids was predicted using a (Q)SAR model battery developed by the Danish National Food Institute at the Technical University of Denmark.  None of the constituents are predicted to have structural features which are either electrophilic or are likely to be metabolized to electrophiles.  All seven of the UVCB components present in the Danish (Q)SAR database are predicted to have a negative outcome for bacterial reverse mutation and eletrophillic reactivity with DNA. The mutagenic activity of a similar UVCB substance  was tested in the Salmonella typhimurium Reverse Mutation Assay and the Escherichia coli Reverse Mutation Assay according to OECD method 471 (1997) in 2016.  In this study, the test substance did not induce a significant dose-related increase in the number of revertant colonies in the test strains either in the absence or presence of S9-metabolic activation.  The substance was determined to be not mutagenic in the reverse mutation assay.

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Endpoint:
genetic toxicity in vitro, other
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
March, 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
See attached QPRF and QMRF documents.
Qualifier:
equivalent or similar to
Guideline:
other: US National Toxicology Program (NTP) cancer bioassay and Salmonella typhimurium mutagenicity assay data
Version / remarks:
QSAR model identifies structural moieties that are statistically correlated with mutagenicity in Salmonella and ability to induce cancer in rats and mice at several sites and in both sexes.
Deviations:
not applicable
Principles of method if other than guideline:
Use of QSAR model is consistent with ECHA "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals".
Specific details on test material used for the study:
DNA reactivity was predicted for seven individual constituents of the UVCB substance. These components, along with water, comprise ca. 98% of the quantified constituents and ca. 78% of the total composition.
Key result
Species / strain:
other: multiple strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Species / strain:
other: Model battery predicts mutagenicity (DNA reactivity) in "Rodents (rats and mice, both sexes, multiple organs)"
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
[ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids is predicted to be not reactive with DNA and therefore not mutagenic.
Executive summary:

Non-mutegenicity of UVCB substance was predicted using a battery of (Q)SAR models developed by the Danish National Food Institute at the Technical University of Denmark. The battery combines the results of three modeling systems (MultiCASE Ultra, Leadscope Enterprise, and SciMatics SciQSAR). DNA reactivity was predicted for seven (7) individual constituents which, along with water, comprise ca. 98% of the quantified constituents and ca. 78% of the total composition.

All seven of the individual constituents of [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids queried are organic fatty acids with a relatively simplistic linear structure consisting of only carbon, hydrogen, and oxygen which fall within the applicability of the Danish (Q)SAR Database model battery. All seven of the UVCB constituents present in the Danish (Q)SAR database were predicted to have a negative outcome for mutagenicity via DNA reactivity by a battery of three model systems.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
March, 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
See attached QPRF and QMRF documents.
Qualifier:
equivalent or similar to
Guideline:
other: QSAR model predicts mutagenicity consistent with OECD 471
Version / remarks:
Use of QSAR model is consistent with ECHA "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals".
Deviations:
not applicable
Principles of method if other than guideline:
Use of QSAR model is consistent with ECHA "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals".
Specific details on test material used for the study:
Mutagenicity was predicted using a QSAR model for seven individual constituents of the UVCB substance which, along with water, comprise ca. 98% of the quantified constituents and ca. 78% of the total UVCB composition.
Key result
Species / strain:
other: multiple strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
All seven of the UVCB components present in the Danish (Q)SAR database were predicted to have a negative outcome for bacterial reverse mutation by a battery of three model systems. [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids is predicted to be not mutagenic.
Executive summary:

Non-mutagenicity of [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids was predicted using a (Q)SAR model battery developed by the Danish National Food Institute at the Technical University of Denmark that combines the results of three modeling systems. Mutagenicity was predicted for seven (7) individual constituents of the substance which, along with water, comprise ca. 98% of the quantified constituents and ca. 78% of the total UVCB composition.

All of the queried constituents are within the applicability domain of the model battery. The models identify structural features and molecular descriptors which may suggest mutagenic properties. These structural features are often associated with widely accepted and clearly understood mechanisms of mutagenicity. None of the long-chain fatty acid structures which comprise the UVCB substance were flagged by any of the models as positively predictive for mutagenicity. The constituents are all simple linear structures comprised on only carbon, hydrogen, and oxygen atoms. The compounds contain no functional groups or complex structures (e.g., cyclic or branched) which would reduce confidence in the predicted results.

Given the ubiquity of long-chain fatty acids in biological systems, the absence of published literature on any mutagenic properties of long-chain saturated or mono-unsaturated fatty acids, and the negative reverse mutation assay results of a closely related and structurally similar UVCB substance, the predictive modeling results are considered appropriate for supporting the weight of evidence to classify the UVCB substance as not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
29 August 2016 - 05 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced with Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1 (direct plate assay):
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000μg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 μg/plate

Experiment 2 (pre-incubation assay):
Without and with S9-mix: 52, 164, 512, 1600 and 5000 μg/plate
Experiment 3 (pre-incubation assay):
TA1535, TA1537 and TA100, without S9-mix: 5.4, 17, 52, 164 and 512 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed. The test item was dissolved in dimethyl sulfoxide. In the dose range finding test and the third mutation experiment the test item was heated up to a maximum of 62.0°C with a maximum of 45 minutes before use.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2.5 μg/plate in DMSO for TA1537 (direct plate assay)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO; 1 μg/plate for TA98 and TA100 (direct plate assay), 2.5 μg/plate for TA1535 and TA1537, 5 μg/plate for TA100 (pre-incubation assay) and 15 μg/plate for WP2uvrA.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION: Exposure duration = 48 hours; Preincubation period = 30 minutes

NUMBER OF REPLICATIONS: Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY: Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS: The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) 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:
Statistical analysis not performed
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in the pre-incubation assay
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the pre-incubation assay without S9-mix
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the pre-incubation assay
Vehicle 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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Dose range finding test: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards in both tester strains and at 5000 μg/plate at the end of the incubation period in tester strain WP2uvrA only.
First mutation experiment: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period for all three tester strains. In addition, precipitation at the end of the incubation period was also observed at the concentration of 1600 μg/plate in tester strain TA1535 (absence of S9-mix).
Second mutation experiment: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period.
Third mutation experiment: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain.

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the tester strains TA100 and WP2uvrA in the direct plate assay. Precipitation of the test item on
the plates was only observed in tester strain WP2uvrA at the top dose level of 5000 μg/plate. 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.

HISTORICAL CONTROL DATA
The negative and strain-specific positive control values were within the testing laboratory's historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Positive historical control data:
TA1535; TA1537; TA98
S9-mix: -; +; -; +; -; +
Range: 78 - 1381; 78 - 1058; 55 - 1565; 55 – 1112; 410 - 2057; 263 - 1907
Mean: 785; 228; 653; 387; 1155; 860
SD: 167; 105; 290; 143; 370; 323
n: 1684; 1662; 1448; 1536; 1646; 1686

TA100; WP2uvrA
S9-mix: - + - +
Range: 549 – 1848 620 - 2651 127 – 1951 85 - 1390
Mean: 892 1404 1263 342
SD: 178 327 461 165
n: 1650 1677 1370 1410

Negative (solvent/vehicle) historical control data:
TA1535; TA1537; TA98; TA100; WP2uvrA
S9-mix: -; +; -; +; -; +; -; +; -; +
Range: 4 - 36; 3 - 34; 3 - 25; 3 - 28; 9 - 50; 9 - 57; 63 - 153; 60 - 156; 12 - 68; 12 - 70
Mean: 14; 13; 7; 9; 17; 25; 100; 103; 26; 32
SD: 6; 5; 3; 4; 5; 7; 16; 18; 7; 8
n: 1662; 1677; 1548; 1547; 1662; 1703; 1659; 1691; 1421; 1424

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between 31 May 2014 - 31 May 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- First mutation experiment: Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 in the absence and presence of S9-mix.
- Second mutation experiment: In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In tester strain
TA98, a slight reduction of the bacterial background lawn was observed at the top dose of 5000 μg/plate in the absence of S9-mix only and no biologically relevant decrease in the number of revertants was observed. In the tester strains TA1535, TA1537 and TA100, reductions of the bacterial background lawn and decreases in the number of revertants were observed in the absence and presence of S9-mix.
- Third mutation experiment: Cytotoxicity, as evidenced by a reduction of bacterial background lawn and/or a decrease in the number of revertants, was observed in all three tester strains.
Conclusions:
In an AMES test, performed according to OECD 471 guideline and GLP principles, (w-hydroxy) fatty acid methyl esters and (w-hydroxy) fatty acids was found not to be mutagenic with or without metabolic activation.
Executive summary:

An AMES test was performed according to OECD 471 guideline and GLP principles. The first experiment was a direct plate asaay and the second experiment a pre-incubation assay. All bacterial strains showed negative responses up to 5000 μg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. Precipitation of the test item on the plates was observed at 5000 μg/plate at the end of the incubation period. In the first experiment, cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 in the absence and presence of S9-mix. In the second experiment, toxicity was observed in all Salmonella strains except for tester strain WP2uvrA. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were 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.

Based on the results of this study it is concluded that (w-hydroxy) fatty acid methyl esters and (w-hydroxy) fatty acids is not mutagenic in the Salmonella typhimurium reverse mutation assay or in the Escherichia coli reverse mutation assay with or without metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

[ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids is a UVCB substance of biological origin. The substance is a complex mixture of long-chain fatty acids produced and excreted by an engineered Escherichia coli K-12 organism through an aqueous microbial fermentation process. The fatty acids are extracted from the fermentation by centrifugation.  The substance is predominantly comprised of linear unbranched long-chain organic fatty acids differentiated only by saturation and number of terminal carboxyl or hydroxyl groups. All of the quantified constituents are comprised of only carbon, hydrogen, and oxygen.

Many of the UVCB constituents are either naturally produced or obtained through the food chain of a broad range of organisms and have been positively identified in algae [4], plant cuticular waxes [1], seeds [3], fish [9], avian eggs [10], edible nuts [7], and berries [11].  Palmitic acid (C16:0) is synthesized endogenously and serves essential cellular functions in humans [5].  Furthermore, palmitic acid and oleic acid (C18:1) comprise more than half of the fatty acid content in human breast milk [8]. Excess dietary intake of unsaturated fatty acids including palmitic acid, which present in [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids, has been shown to affect metabolism and body weight regulation in both experimental animals and humans [2], but this is not a genotoxic or mutagenic effect.

A sufficient weight of evidence is provided by the ubiquity of long-chain fatty acids in biological systems, the absence of published literature on any genetic toxicity or mutagenic properties of these compounds, predictive modeling results, and the negative reverse mutation test results of a closely related and structurally similar UVCB substance. [ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids is considered to be not mutagenic.

References:

1.   Fernandes, A. M. Silva, Baker, E.A., Martin, J.T. Studies on plant cuticle. Annals of Applied Biology 1964, 53(1):43-58

2.   Frago LM, Canelles S, Freire-Regatillo A, et al. Estradiol Uses Different Mechanisms in Astrocytes from the Hippocampus of Male and Female Rats to Protect against Damage Induced by Palmitic Acid. Frontiers in Molecular Neuroscience. 2017;10:330

3.   Hajghanbari et al. Essential oil constituents and fatty acids in Echium amoenum grown wild in Iran. International Journal of Biosciences 2015, 6(1): 156-161

4.   Hassain, J, et al. Effects of Different Biomass Drying and Lipid Extraction Methods on Algal Lipid Yield, Fatty Acid Profile, and Biodiesel Quality. Appl Biochem Biotechnol 2015, 175: 3048

5.   Innis, Sheila. Palmitic Acid in Early Human Development. Critical Reviews in Food Science and Nutrition 2016, 56(12): 1952-1959

6.   Kakimoto, Pamela and Kowaltowski, Alicia. Effects of high fat diets on rodent liver bioenergetics and oxidative imbalance. Redox Biology 2016, 8 (2016) 216–225

7.   Maguire LS, O’Sullivan SM, et al. Fatty acid profile, tocopherol, squalene and phytosterol content of walnuts, almonds, peanuts, hazelnuts and the macadamia nut. International Journal of Food Science Nutrition 2004, 5:171-178

8.   Martin, Camilia, Ling Pei-Ra, Blackburn, George. Review of Infant Feeding: Key Features of Breast Milk and Infant Formula. Nutrients 2016, 8(5), 279

9.   Ozogul Y, Ozogul F, Cicek E, Polat A, Kuley E: Fat content and fatty acid compositions of 34 marine water fish species from the Mediterranean Sea. International Journal of Food Science Nutrition 2008, 29:1-12

10.  Toledo, A., Andersson, M.N., Wang, HL. et al. Fatty acid profiles of great tit (Parus major) eggs differ between urban and rural habitats, but not between coniferous and deciduous forests. Sci Nat 2016, 103: 55

11.  Yang B, Kallio HP. Fatty acid composition of lipids in sea buckthorn (Hippophaë rhamnoides L.) berries of different origins. J Agricultural Food Chemistry 2001, 49:1939-1947