Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January to 15 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Identification Dodecanoic acid, tetradecyl ester
Appearance White powder
Batch 20161228002
Purity/Composition 95%
Test item storage At room temperature
Stable under storage conditions until 27 December 2017 (expiry date)
Target gene:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Escherichia coli WP2uvrA strain: base-pair substitutions
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
Escherichia coli WP2uvrA strain
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Test concentrations with justification for top dose:
Dose range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate with heavy precipitate at 1600 and 5000 μg/plate.
Mutation assay 1: 1.7, 5.4, 17, 52, 164 and 512 μg/plate
Mutation assay 2: 51, 91, 162, 288, 512 μg/plate
Vehicle / solvent:
A solubility test was performed. The test item could not be dissolved in water or dimethyl sulfoxide. The test item was soluble in ethanol. Therefore ethanol was used as solvent in this project.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191
Remarks:
Without metabolic activation (-S9-mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
With metabolic activation (+S9-mix)
Details on test system and experimental conditions:
Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale: Recommended test system in international guidelines (e.g.OECD, EC).
Source: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2006, TA1537: 2016, TA98: 2015, TA100: 2015; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Environmental conditions: All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 35.4 – 38.3°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Rationale for test conditions:
According to International guidelines.
Evaluation criteria:
Acceptability of the assay:

a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle 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 the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up 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:
No formal hypothesis testing was done.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
Conclusions:
Based on the results of this study it is concluded that Dodecanoic acid, tetradecyl ester is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Evaluation of the mutagenic activity of Dodecanoic acid, tetradecyl ester in the Salmonella typhimurium reverse mutation assay and the Escherichia colireverse mutation assay.

Dodecanoic acid, tetradecyl ester was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains ofSalmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 20161228002 of Dodecanoic acid, tetradecyl ester was a white powder with a purity of 95%. The test item was dissolved in ethanol.

In the dose range finding test, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 164 μg/plate and upwards. Since the test item precipitated heavily on the plates at test item concentrations of 1600 and/or 5000 μg/plate, the number of revertants of these dose levels could not be determined. 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 the first mutation assay.

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 1.7 to 512 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the top dose of 512 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.

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 51 to 512 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the dose levels of 288 and 512 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.

Dodecanoic acid, tetradecyl ester did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) or in the number of revertant (Trp+) colonies in the tester strain WP2uvrA in either the absence or presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

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 Dodecanoic acid, tetradecyl ester is not mutagenic in the Salmonella typhimuriumreverse mutation assay and in the Escherichia colireverse mutation assay.

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

Additional information

Genetic toxicity endpoint

CAS 22412-97-1

Dodecanoic acid, tetradecyl ester was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch 20161228002 of Dodecanoic acid, tetradecyl ester was a white powder with a purity of 95%. The test item was dissolved in ethanol.

In the dose range finding test, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 164 μg/plate and upwards. Since the test item precipitated heavily on the plates at test item concentrations of 1600 and/or 5000 μg/plate, the number of revertants of these dose levels could not be determined. 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.

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 1.7 to 512 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the top dose of 512 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.

In a follow-up experiment, the test item was tested at a concentration range of 51 to 512 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the dose levels of 288 and 512 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.

Dodecanoic acid, tetradecyl ester did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) or in the number of revertant (Trp+) colonies in the tester strain WP2uvrA in either the absence or presence of S9-metabolic activation. 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 Dodecanoic acid, tetradecyl ester is not mutagenic in the bacterial reverse mutation assay.

CAS 26399-02-0

The cytogenetic potential of 2-ethylhexyl oleate (CAS 26399-02-0) was assessed in an in vitro mammalian chromosome aberration test in primary human lymphocytes, performed according to OECD guideline 473 (Buskens, 2010). 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 incubated with test substance concentrations of 3, 10 and 33 µg/mL in ethanol for 3 hours with and without metabolic activation. In the second experiment cells were incubated with 3, 10 and 33 µg/mL for 24 hours followed by 24 hours expression time and 48 hours following 48 hours expression time, all without metabolic activation. 33 µg/mL was chosen as maximum concentration due to limited solubility of the test substance. Evaluation of 100 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 in comparison to the negative controls. The test material demonstrated only modest cytotoxicity. The vehicle (solvent) and positive controls were shown to be valid. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations with or without metabolic activation. An in vitro mammalian cell gene mutation assay was performed with 2-ethylhexyl oleate according to OECD 476 (Verspeek-Rip, 2010). Two independent experiments (with 3 or 24 hours of exposure) were performed in mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix) with test substance concentrations up to 100 μg/mL dissolved in ethanol. Precipitation was seen at 100 µg/mL and higher. The positive and negative controls were valid and within the range of historical control data. No significant increase in mutation frequency occurred in any of the test conditions.

CAS 3234-85-3

The in-vitro genetic toxicity of tetradecanoic acid, tetradecyl ester (CAS 3234-85-3) was assessed in a bacterial reverse mutation assay (Ames test) performed similarly to OECD 471 (Marquardt, 1995). The preincubation method was applied using S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 1538 at concentrations up to 1000 µg/plate. No TA 102 or E. coli strain was used. The test substance did not induce reversions in any of the S. typhimurium strains with or without metabolic activation. All the positive controls were valid. Precipitation was observed at the highest dose level in the range-finding study with TA 98 and TA 100. More than 50% cytotoxicity was observed in the range-finding study with TA 98 from 10 µg/plate (without metabolic activation) and with TA 100 from 1000 µg/plate (without metabolic activation).

 

CAS 3687-45-4

The potential of oleyl oleate (CAS 3687-45-4) to induce chromosomal aberrations was assessed using Chinese hamster V79 cells, in a study performed according to OECD 473 (Völkner, 1994). The V79-cells were exposed to oleyl oleate at concentrations up to 100 µg/mL, with and without metabolic activation (S9-mix). One experiment with duplicate replications was performed with short-term treatment (4 h) and fixation time 18 and 28 h, without metabolic activation; and with metabolic activation using 18 h treatment time and 18 h fixation time and 28 h treatment time and 28 h fixation time, respectively. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, with or without metabolic activation. The mitotic indices of the treated cultures without metabolic activation were 83.4-119% and with metabolic activation 91-127.1%, compared with the vehicle control. Precipitation was observed at concentrations from 100 µg/mL, while no cytotoxicity was noted at any concentration. The vehicle and positive controls were valid.

 

An in vitro mammalian cell gene mutation assay was performed using oleyl oleate, according to OECD 476 (Poth, 1994). Chinese hamster lung fibroblasts (V79) were treated with oleyl oleate at concentrations of up to 100 µg/mL for 4 h both with and without metabolic activation. After an expression time of 7 days in growth medium, cells were incubated for 9 or 12 days with 6 -thioguanine as selection agent for forward mutation at the HPRT locus. Both with and without metabolic activation, no increases in mutant frequency were observed in the initial and in the confirmatory gene mutation assay. There was no evidence of excessive cytotoxicity (i.e., < 10 % relative cloning efficiency) at any of the tested concentrations either in the presence or absence of metabolic activation in any of the experiments performed.

 

Overall conclusion for genetic toxicity

The results of all the studies included in this dossier were negative. The available data on in-vitro genetic toxicity indicates that Dodecanoic acid, tetradecyl ester, as the other analogues of the LCAE category, has no genetic toxicity potential.


Short description of key information:
In none of these studies mutagenicity in bacteria could be observed.
In none of these studies clastogenic effects in mammalian cells could be observed.
In none of these studies mutagenicity in mammalian cells could be observed.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.