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Genetic toxicity in vitro

Description of key information

The Category member 3, 2 -decyltetradecanoic acid, was examined for mutagenic activity by assaying for reverse mutation to histidine prototrophy in the prokaryotic organism Salmonella typhimurium in an OECD 471 test. The five tester strains TA1535, TA1537, TA98, TA100 and TA102 were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Based on this test is concluded that 2-decyltetradecanoic acid does not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions. Additionally 2-decyltetradecanoic acid

was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. In conclusion, in the described mutagencity test according to OECD 476 under the experimental conditions reported, the Category Member 3 is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells. The Source substance 2, docosanoic acid, did not induce structural chromosomal aberrations in the absence or presence of an exogenous metabolic activation system in a test according to OECD 473.
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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-03-29 to 2002-04-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study
Qualifier:
according to
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 TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate
Test concentrations with justification for top dose:
5000, 2500, 1250, 625 and 313 µg/plate.
Vehicle / solvent:
acetone
Details on test system and experimental conditions:
SYSTEM OF TESTING
- Species/cell type: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Deficiencies/Proficiencies: histidine auxotroph
- Metabolic activation system: rat liver S9 of Phenobarbital/beta-Naphthoflavone induced male Sprague-Dawley rats
ADMINISTRATION:
- Dosing:
experiment I and II:
313, 625, 1250, 2500 and 5000 µg/plate
- Number of replicates: 3
- Application:
Experiment I (plate-incorporation method): The first experiment was perfonned using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was
then poured onto the surface of a minimal medium agar plate, and allowed to solidify prior to incubation.
The overlay mixture was composed as follows:
(i) Overlay agar (held at 45°C) 2 ml
(ii) Test or control item solution 0.1 ml
(iii) S9 mix or phosphate buffer (pH 7.4, 0.1 M) O.S ml
(iv) Bacterial suspension 0.1 ml

Experiment II and III (pre-incubation method): Since acetone is known to be toxic at 50 Ill/plate when used for the pre-incubation method, 25
Ill/plate of solvent or test item solution were used.
The components were added in turn to an empty test-tube:
(i) Bacterial suspension 0.1 ml
(ii) Test item solution or its solvent 0.025ml
Positive control solution or its solvent O.OSOml
(iii) S9 mix or phosphate buffer (PH 7.4, 0.1 M) O.S ml

The incubate was vortexed and placed at 37°C for 30 minutes. Two ml of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

Evaluation criteria:
The test item is considered as a mutagen if at least a two-fold increases in mean revertant numbers are observed at two consecutive dose-levels or at the highest practicable dose level only. In addition there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose-levels.
Statistics:
regression analysis
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of result:
negative

The test item does not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.
Executive summary:

The test item was examined for mutagenic activity by assaying for reverse mutation to histidine prototrophy in the prokaryotic organism Salmonella typhimurium. The five tester strains TA1535, TA1537, TA98, TA100 and TA102 were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions were prepared using acetone. In the toxicity test, the test item was assayed at a maximum dose-level of 5000 flg/plate and at four lower dose-levels spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 flg/plate. No signs of toxicity were observed at any doselevel tested, in any tester strain, in the absence or presence of S9 metabolic activation. Two main experiments were performed. In Main Assay I, using the plate incorporation method, the test item was assayed at a maximum dose-level of 5000 flg/plate and at four lower dose-levels, separated by two-fold dilutions: 2500,1250,625 and 313 flg/plate. As no increases in revertant numbers were observed, all treatments of Main Assay II included a pre-incubation step and used the same dose-levels as Main Assay I. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism. It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
January to March, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented study performed according to OECD Guideline and GLP
Justification for type of information:
see Category Approach
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase locus/TK+/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
Complete culture medium: RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
Treatment medium: RPMI medium supplemented with 5% heat-inactivated horse serum (in case of short term exposure) resp. 7.5% heat-inactivated horse serum (in case of long term exposure), 100 U/100 µg/mL penicillin/streptomycin, 1mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
Selective medium: RPMI 1640 medium supplemented with 20% heat-inactivated horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B, 5 µg/mL trifluorothymidine (TFT)
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
liver S9 microsomal fraction of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) induced male Wistar rats
Test concentrations with justification for top dose:
Pre-experiment for toxicity: 0.2/1.0/2.6/5.3/8.0 and 10.6 mM for experiment I with and without S9 mix, 0.05/0.1/0.2/0.5/1.0 and 1.5 mM for experiment II, long term exposure, without S9 mix
Experiment I: 0.05/0.1/0.2/0.5/0.7/0.9/1.1, and 1.3 mM without S9 mix, 0.02/0.05/0.1/0.2/0.5/1.0/1.2 and 1.4 mM with S9 mix
Experiment II: 0.002/0.005/0.01/0.02/0.05/0.1/0.2 and 0.5 mM without S9 mix, 0.15/0.3/0.7/0.9/1.1/1.2/1.3 and 1.4 mM with S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: in the absence of metabolic activation: ethyl methanesulphonate (EMS, 200/300 µg/mL) and methyl methanesulphonate (MMS, 8/10µg/mL); in the presence of metabolic activation: benzo[a]pyrene (B[a]P, 2.5 µg/ml)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4 h
Experiment 2: 24 h (without metabolic activation) and 4 h (with metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (incubation with the selection agent): 14 days (mutation selection assay)

SELECTION AGENT: trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 1 (test item and positive controls, 2 (negative control)

NUMBER OF CELLS EVALUATED: mutation selection assay: cells from each experimental group were seeded in four 96-well plates at a density of 200 cells/well in 200 µL selective medium; cloning efficiency assay: seeding a statistical number of 1.6 cells per well in two 96-well plates

DETERMINATION OF CYTOTOXICITY
- Method: measuring the colony-forming ability and the growth rate of cultures (relative suspension growth, recorded over 2 days following treatment)

OTHER EXAMINTATIONS
- Colony sizing: ratio of small to large type mutants
Evaluation criteria:
The assay is considered acceptable if it meets the following criteria:
- at least three out of four of the negative and/or solvent controls is in the range 65% - 120%
- the spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 10E6 cells
- the cell number of the negative/solvents controls should undergo 8-32 fold increase during a 2 day growth period (short-term treatment) or 32-180 fold increase during a 3 day growth period (long-term treatment)
- the clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 10E6 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 10E6 cells. The relative total growth (RTG) must be greater than 10%.

Criteria for a positive result:
- the induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10E6 cells
- a dose-dependent increase in mutant frequency is detected.
Additionally, combined with a positive effect in the mutant frequency, an increased occurence of small colonies (>= 40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations. The biological relevance is considered first for the interpretation of results. Statistical methods might be used an aid in evaluation of the test result.

Criteria for a negative result:
a test item is considered negative if the induced mutant frequency is below the GEF and the trend of the test is negative.
Statistics:
Statistical significance of mean mutant frequency was evaluated at the 5% level (p<0.05) by means of the non-parametric Mann-Whitney test.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for details see tables 1 and 2 below
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Water solubility: no data, the test substance was soluble in cell culture medium after being treated with ultrasound for 3 minutes at 37°C
- Precipitation: no precipitation was noticed
- Other confounding effects: none

COMPARISON WITH HISTORICAL CONTROL DATA: All values found in the test were within the range of the historical laboratory control data
Table 1a: Pre-experiment for Toxicity, without metabolic activation
Test group Concentration
mM
Suspension Growth Relative Suspension Growth* [%]
Negative control 0 16.5 100.0
0 16.4
1 0.2 15.5 94.2
2 1.1 7.7 46.8
3 2.6 0.3 1.9
4 5.3 no viable cells -
5 8.0 no viable cells -
6 10.6 no viable cells -
Table 1b: Pre-experiment for Toxicity, with metabolic activation
Test group Concentration
mM
Suspension Growth Relative Suspension Growth* [%]
Negative control 0 13.4 100.0
0 13.6
1 0.2 12.2 90.1
2 1.1 9.1 67.7
3 2.6 0.3 1.9
4 5.3 no viable cells -
5 8.0 no viable cells -
6 10.6 no viable cells -
Table 1c: Pre-experiment II for Toxicity, long-term exposure, without metabolic activation
Test group Concentration
mM
Suspension Growth Relative Suspension Growth* [%]
Negative control 0 35.9 100.0
0 33.9
1 0.05 32.9 94.3
2 0.1 31.5 90.2
3 0.2 23.0 65.7
4 0.5 6.0 17.2
5 1.0 0.3 0.8
6 1.5 0.1 0.4
* = (Suspension Growth/Suspension Growth of corresponding controls) x100
Table 2a: Experiment I, without metabolic activation
Test group Concentration CE RCE RTG MF IMF GEF exceeded colony sizing
mM % % % mutants/106cells mutants/106cells % small colonies
Negative control 0 101.2 100.0 100.0 80.1 / / 15.8
108.2 / / 14.8
3 0.05 104.6 99.9 97.9 71.1 -8.8 - n.d.
4 0.1 112.0 106.9 109.1 66.5 -13.6 - n.d.
5 0.2 112.0 106.9 97.6 63.8 -16.3 - n.d.
6 0.5 84.1 80.3 75.2 94.0 13.9 - n.d.
7 0.7 98.0 93.6 81.1 50.3 -29.8 - n.d.
8 0.9 95.0 90.7 61.9 89.7 9.6 - 18.3
9 1.1 114.0 108.8 58.2 79.2 -0.9 - 22.2
10 1.3 130.0 124.1 21.8 63.3 -16.8 - 24.1
EMS 300 µg/mL 93.5 89.3 68.9 690.8* 610.7* + n.d.
MMS 10 µg/mL 80.5 76.8 59.4 511.6* 431.5* + 41.9
CE: Cloning efficiency, CE = (-ln ((96 - (mean value of plates)) / 1.6) x 100
RCE: Relative Cloning Efficiency, RCE = (CE of dose group/CE of corresponding controls)x100
RTG: Relative Total Growth, RTG = (RSG x RCE)/100
MF: Mutant Frequency
GEF: Global Evaluation Factor (126): + = GEF exceeded, - = GEF not exceeded
* statistical significant increase in mutant frequency compared to negative controls
Table 2b: Experiment II, without metabolic activation (24 h treatment)
Test group Concentration CE RCE RTG MF IMF GEF exceeded colony sizing
mM % % % mutants/106cells mutants/106cells % small colonies
Negative control 0 114.0 100.0 100.0 56.7 / / 6.1
102.9 / / 22.5
1 0.002 96.5 89.0 87.1 60.3 3.5 - n.d.
2 0.005 90.7 83.6 127.2 75.8 10.9 - n.d.
3 0.01 85.4 78.7 107.5 101.4* 44.7* - n.d.
4 0.02 106.4 98.1 92.7 67.2 10.5 - n.d.
5 0.05 112.0 103.3 112.6 69.6 12.9 - n.d.
6 0.1 102.9 94.9 74.3 52.1 -4.6 - 15.4
7 0.2 102.9 94.9 72.2 62.0 5.3 - 13.0
8 0.5 101.2 93.4 20.3 67.6 10.9 - 16.3
EMS 200 µg/mL 32.0 29.5 19.4 3673.2* 3616.5* + n.d.
MMS 8 µg/mL 38.9 35.9 25.0 1191.8* 1135.1* + 51.1
CE: Cloning efficiency, CE = (-ln ((96 - (mean value of plates)) / 1.6) x 100
RCE: Relative Cloning Efficiency, RCE = (CE of dose group/CE of corresponding controls)x100
RTG: Relative Total Growth, RTG = (RSG x RCE)/100
MF: Mutant Frequency
GEF: Global Evaluation Factor (126): + = GEF exceeded, - = GEF not exceeded
* statistical significant increase in mutant frequency compared to negative controls
Table 2c: Experiment I, with metabolic activation
Test group Concentration CE RCE RTG MF IMF GEF exceeded colony sizing
mM % % % mutants/106cells mutants/106cells % small colonies
Negative control 0 90.7 100.0 100.0 81.0 / / 10.9
110.7 / / 20.8
2 0.02 80.5 80.2 90.2 109.8 28.9 - n.d.
3 0.05 101.2 100.9 110.3 89.1 8.1 - n.d.
4 0.1 86.6 86.3 99.7 96.4 15.5 - n.d.
5 0.2 75.9 75.6 77.3 100.1 19.1 - n.d.
6 0.5 90.7 90.3 92.7 92.1 11.2 - n.d.
7 1.0 93.5 93.2 73.4 95.9 15.0 - 22.2
8 1.2 93.5 93.2 46.4 83.0 2.0 - 21.8
9 1.4 93.5 93.2 10.1 121.4* 40.5* - 20.5
B[a]P 2.5 µg/mL 92.1 91.7 57.7 593.8* 512.8* + 42.9
CE: Cloning efficiency, CE = (-ln ((96 - (mean value of plates)) / 1.6) x 100
RCE: Relative Cloning Efficiency, RCE = (CE of dose group/CE of corresponding controls)x100
RTG: Relative Total Growth, RTG = (RSG x RCE)/100
MF: Mutant Frequency
GEF: Global Evaluation Factor (126): + = GEF exceeded, - = GEF not exceeded
* statistical significant increase in mutant frequency compared to negative controls
Table 2d: Experiment II, with metabolic activation
Test group Concentration CE RCE RTG MF IMF GEF exceeded colony sizing
mM % % % mutants/106cells mutants/106cells % small colonies
Negative control 0 110.1 100.0 100.0 72.2 / / 14.3
95.0 / / 18.4
5 0.15 90.7 88.4 79.9 75.6 3.5 - n.d.
6 0.3 95.0 92.6 100.1 78.5 6.3 - n.d.
7 0.7 125.0 121.9 103.3 68.1 -4.1 - n.d.
8 0.9 98.0 95.6 81.9 66.7 -5.5 - n.d.
9 1.1 82.1 89.8 56.2 79.2 7.1 - n.d.
10 1.2 120.3 117.4 57.4 63.0 -9.2 - 11.1
11 1.3 120.3 117.4 31.8 64.7 -7.5 - 12.7
12 1.4 104.6 102.0 8.7 77.2 5.1 - 17.5
B{a]P 2.5 µg/mL 85.4 83.3 59.2 477.9* 405.7* + 42.1
CE: Cloning efficiency, CE = (-ln ((96 - (mean value of plates)) / 1.6) x 100
RCE: Relative Cloning Efficiency, RCE = (CE of dose group/CE of corresponding controls)x100
RTG: Relative Total Growth, RTG = (RSG x RCE)/100
MF: Mutant Frequency
GEF: Global Evaluation Factor (126): + = GEF exceeded, - = GEF not exceeded
* statistical significant increase in mutant frequency compared to negative controls
Conclusions:
Interpretation of results:
negative without metabolic activation
negative with metabolic activation

In the described mutagencity test under the experimental conditions reported, the test item Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Executive summary:

The test item Reaction mass of 2 -butylheptanoic acid and 2 -ethylnonanoic acid and 2 -methyldecanoic acid and 2 -proyloctanoic acid was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations used in the main experiments was based on data from the pre-experiment. In experiment I 1.3 mM (without metabolic activation) and 1.4 mM (with metybolic activation) were selected as the highest concentrations. In experiment II 0.5 mM (without metabolic activation) and 1.4 mM (with metabolic activation ) were selected as the highest concentrations. Experiment I without and with metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I without metabolic activation: 0.05, 0.1, 0.2, 0.5, 0.7, 0.9, 1.1 and 1.3 mM and with metabolic activation: 0.02, 0.05, 0.1 ,0.2, 0.5, 1.0, 1.2 and 1.4 mM

Experiment II without metabolic activation: 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 mM and with metabolic activation: 0.15, 0.3, 0.7, 0.9, 1.1, 1.2, 1.3 and 1.4 mM.

No precipitation of the test item was noted in the experiments. Growth inhibition was observed in experiment I and II without and with metabolic activation.

In experiment I without metabolic activation the relative total growth (RTG) was 21.8% for the highest concentration (1.3 mM) evaluated. The highest concentration evaluated with metabolic activation was 1.4 mM with a RTG of 10.1%. In experiment II without metabolic activation the relative total growth was 20.3% for the highest concentration (0.5 mM) evaluated. The two highest concentrations evaluated with metabolic activation were 1.3 and 1.4 mM with a RTG of 31.8% and 8.7% respectively.

In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF, defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed. Aditionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental condition (with and without metabolic activation).

Ethylmethanesulfonate (EMS), methylmethanesulfonate (MMS) and benzo[a]pyrene (B[a]P) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a)P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

In conclusion, in the described mutagencity test under the experimental conditions reported, the test item Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Justification for type of information:
The carbon chain length of this acid and that of the Category member 2-decyltetradecanoic acid, is very similar. Both substances are long chain acids which have no other funcitional group. Based on the similar structure and therefore similar expected toxicokinetic it is likely that the test result of this substance is also valid for the Category member (Please see attached Category Approach.)
Reason / purpose:
other: Category Approach
Key result
Species / strain:
mammalian cell line, other: Chinese hamster lung (CHL) cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification