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EC number: 218-039-9 | CAS number: 2040-64-4
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20 Sep - 15 Oct 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: USA, EPA OCSPP harmonized guideline 870.5100 – Bacterial Reverse Mutation Test
- Version / remarks:
- adopted in 1998
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
- Version / remarks:
- adopted in 2000
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- The Departement Of Health Of The Government of the United Kingdom
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Dodecyl myristate
- EC Number:
- 218-039-9
- EC Name:
- Dodecyl myristate
- Cas Number:
- 2040-64-4
- Molecular formula:
- C26H52O2
- IUPAC Name:
- dodecyl tetradecanoate
Constituent 1
Method
- Target gene:
- his operon, trp operon
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats, induced with Phenobarbital/ ß-Naphta flavone (80/100 mg/kg bw)
- Test concentrations with justification for top dose:
- Following concentrations were used in the main experiments:
First experiment (plate incorporation, all strains): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate with and without metabolic activation (tested up to the limit concentration)
Second experiment (preincubation, all strains): 15, 50, 150, 500, 1500, 5000 μg/plate with and without metabolic activation (tested up to the limit concentration) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Acetone was selected because of a good solubility of the test substance in this vehicle during the solubility checks.
Controls
- Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- benzo(a)pyrene
- other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), 2-aminoanthracene (2AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation) (first experiment); preincubation (second experiment)
DURATION
- Preincubation period: 20 min
- Exposure duration: approx. 48 h
NUMBER OF REPLICATIONS: triplicates in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: microscopical inspection of bacterial background lawn - Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal. - Statistics:
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989). Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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:
- 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:
- no cytotoxicity, 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 100
- 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:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- 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:
- 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, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed in all strains starting at a concentration of 1500 μg/plate in experiment 1 and starting at 500 μg/plate in experiment 2. Thus for this concentration and above manual counts were performed (instead of the automated colony counting system). Nevertheless this observation did not prevent the scoring of revertant colonies.
- Other confounding effects: Minor statistical values were noted in Experiment 1 (TA 100 at 5, 50 and 500 μg/plate in the absence of S9-mix and at 50 μg/plate in the presence of S9-mix), however these responses were within the in-house historical vehicle/untreated control values for the strain and were, therefore considered of no biological relevance.
HISTORICAL CONTROL DATA
Refer to Table 3 in "Any other information on results incl. tables"
Any other information on results incl. tables
Table 1: Test results (experiment 1, plate incorporation)
With or without S9-Mix |
Test substance concentration (μL/plate) |
Mean number of revertant colonies per plate |
||||
Frameshift type |
Base-pair substitution type |
|||||
TA98 |
TA1537 |
TA100 |
TA1535 |
WP2 uvr A |
||
– |
Negative control (untreated) |
46 |
19 |
106 |
35 |
30 |
Solvent control (acetone) |
45 ± 2.1 |
17 ± 2.5 |
95 ± 5.0 |
35 ± 2.1 |
34 ± 2.9 |
|
1.5 |
45 ± 1.2 |
17 ± 2.0 |
109 ± 7.0 |
43 ± 14.6 |
26 ± 4.9 |
|
5 |
48 ± 3.1 |
18 ± 0.0 |
116 ± 13.7 |
40 ± 1.7 |
27 ± 8.5 |
|
15 |
47 ± 3.6 |
18 ± 3.2 |
104 ± 17.0 |
37 ± 3.5 |
31 ± 3.1 |
|
50 |
44 ± 3.6 |
20 ± 1.5 |
115 ± 7.5 |
35 ± 5.5 |
23 ± 3.5 |
|
150 |
50 ± 1.2 |
18 ± 6.1 |
112 ± 2.5 |
36 ± 0.0 |
25 ± 2.5 |
|
500 |
49 ± 3.1 |
15 ± 2.1 |
115 ± 2.1 |
33 ± 8.0 |
39 ± 2.0 |
|
1500 P |
45 ± 1.5 |
18 ± 3.1 |
111 ± 10.0 |
40 ± 2.6 |
33 ± 6.6 |
|
5000 P |
45 ± 6.7 |
18 ± 4.0 |
103 ± 7.6 |
40 ± 1.5 |
34 ± 2.1 |
|
Positive controls (µg/plate) |
4NQO |
9AA |
ENNG |
ENNG |
ENNG |
|
Mean No. of colonies/plate (average of 3 plates) |
193 ± 7.1 |
263 ± 22.0 |
672 ± 89.2 |
833 ± 12.5 |
824 ± 49.4 |
|
+ |
Solvent control (distilled water) |
52 ± 3.5 |
21 ± 2.5 |
101 ± 1.2 |
35 ± 5.0 |
39 ± 9.6 |
1.5 |
62 ± 18.1 |
21 ± 4.0 |
99 ± 10.4 |
38 ± 1.7 |
37 ± 13.1 |
|
5 |
50 ± 4.0 |
24 ± 1.0 |
112 ± 16.0 |
39 ± 2.1 |
37 ± 3.8 |
|
15 |
50 ± 9.3 |
19 ± 2.0 |
118 ± 6.0 |
40 ± 1.7 |
34 ± 5.7 |
|
50 |
45 ± 3.2 |
22 ± 1.7 |
124 ± 3.5 |
38 ± 1.7 |
37 ± 10.7 |
|
150 |
53 ± 3.0 |
23 ± 2.5 |
99 ± 8.9 |
40 ± 2.1 |
29 ± 9.3 |
|
500 |
49 ± 4.5 |
20 ± 2.1 |
109 ± 3.8 |
36 ± 0.0 |
30 ± 2.9 |
|
1500 P |
51 ± 9.5 |
17 ± 2.0 |
104 ± 6.7 |
38 ± 7.4 |
35 ± 4.7 |
|
5000 P |
53 ± 4.0 |
17 ± 1.2 |
100 ± 5.1 |
37 ± 4.4 |
37 ± 2.1 |
|
Positive controls (µg/plate) |
B(a)P |
2AA |
2AA |
2AA |
2AA |
|
Mean No. of colonies/plate (average of 3 plates) |
277 ± 19.4 |
392 ± 24.7 |
2058 ± 82.7 |
324 ± 25.3 |
514 ± 29.1 |
|
2AA = 2-aminoanthracene
4NQO = 4-nitroquinoline-N-oxide
9AA = 9-aminoacridine
B(a)P = benzo(a)pyrene
ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine
P = precipitations
Table 2: Test results (experiment 2, preincubation)
With or without S9-Mix |
Test substance concentration (μL/plate) |
Mean number of revertant colonies per plate |
||||
Frameshift type |
Base-pair substitution type |
|||||
TA98 |
TA1537 |
TA100 |
TA1535 |
WP2 uvr A |
||
– |
Negative control (untreated) |
18 |
9 |
102 |
12 |
28 |
Solvent control (acetone) |
23 ± 4.0 |
14 ± 5.7 |
106 ± 6.4 |
15 ± 1.2 |
31 ± 4.4 |
|
15 |
18 ± 6.4 |
15 ± 7.1 |
114 ± 17.6 |
9 ± 2.6 |
24 ± 4.6 |
|
50 |
28 ± 7.5 |
15 ± 3.2 |
106 ± 10.6 |
17 ± 1.0 |
29 ± 2.1 |
|
150 |
22 ± 5.0 |
12 ± 4.0 |
117 ± 11.0 |
15 ± 1.5 |
32 ± 3.1 |
|
500 P |
21 ± 3.5 |
16 ± 1.0 |
109 ± 9.3 |
14 ± 2.5 |
28 ± 2.6 |
|
1500 P |
23 ± 4.0 |
14 ± 4.0 |
112 ± 8.3 |
13 ± 2.1 |
29 ± 1.7 |
|
5000 P |
26 ± 0.6 |
12 ± 3.5 |
107 ± 12.5 |
15 ± 1.2 |
30 ± 6.0 |
|
Positive controls (µg/plate) |
4NQO |
9AA |
ENNG |
ENNG |
ENNG |
|
Mean No. of colonies/plate (average of 3 plates) |
257 ± 22.2 |
202 ± 14.3 |
1147 ± 108.1 |
994 ± 257.2 |
865 ± 50.3 |
|
+ |
Solvent control (distilled water) |
30 ± 1.2 |
10 ± 2.3 |
109 ± 6.8 |
16 ± 2.5 |
39 ± 4.7 |
15 |
26 ± 4.0 |
11 ± 2.1 |
115 ± 6.7 |
12 ± 3.2 |
40 ± 11.5 |
|
50 |
27 ± 4.6 |
13 ± 1.5 |
108 ± 22.8 |
13 ± 3.8 |
46 ± 2.6 |
|
150 |
26 ± 6.2 |
11 ± 4.4 |
114 ± 8.7 |
12 ± 3.2 |
31 ± 1.5 |
|
500 P |
31 ± 2.0 |
10 ± 1.5 |
107 ± 3.1 |
13 ± 5.1 |
38 ± 2.6 |
|
1500 P |
27 ± 2.5 |
10 ± 2.1 |
111 ± 3.5 |
10 ± 2.1 |
41 ± 3.6 |
|
5000 P |
29 ± 4.4 |
11 ± 3.8 |
112 ± 3.5 |
14 ± 2.0 |
42 ± 2.6 |
|
Positive controls (µg/plate) |
B(a)P |
2AA |
2AA |
2AA |
2AA |
|
Mean No. of colonies/plate (average of 3 plates) |
210 ± 7.5 |
457 ± 53.1 |
2342 ± 148.4 |
304 ± 2.1 |
401 ± 4.7 |
|
2AA = 2-aminoanthracene
4NQO = 4-nitroquinoline-N-oxide
9AA = 9-aminoacridine
B(a)P = benzo(a)pyrene
ENNG =N-ethyl-N'-nitro-N-nitrosoguanidine
P = precipitations
Table 3: Historical control values (2016)
|
S9- Mix |
Mean number of revertant colonies per plate [mean ± SD] |
||||
TA 100 |
TA 1535 |
WP2 uvr A |
TA 98 |
TA 1537 |
||
Negative/ vehicle controls |
- |
90 ± 14.5 |
15 ± 4.5 |
22 ± 5.8 |
21 ± 4.8 |
12 ± 3.5 |
+ |
93 ± 14.3 |
15 ± 5.2 |
27 ± 6.3 |
25 ± 5.7 |
13 ± 3.5 |
|
Positive controls |
- |
724 ± 320.4 |
854 ± 664.9 |
718 ± 338.6 |
186 ± 49.8 |
406 ± 227.0 |
+ |
1264 ± 562.6 |
240 ± 62.1 |
240 ± 98.2 |
188 ± 230.8 |
290 ± 92.7 |
|
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results: negative
Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells. The data provide not sufficient evidence that would imply any classification and labelling with respect to mutagenicity/genotoxicity. However, as no information regarding gene mutation in mammalian cells and cytogenicity or chromosome aberration in mammalian cells are available, the overall conclusion for classification regarding mutagenicity/genotoxicity is ‘data lacking’.
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