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EC number: 949-569-5 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
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- Flash point
- Auto flammability
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- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
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- Dissociation constant
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Genetic toxicity
- Carcinogenicity
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- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- from 2019-04-12 to 2019-06-12
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Reaction mass of 1-(3,3-dimethylcyclohexyl)ethyl acetate and 2,6,6-trimethylcycloheptanol acetate
- EC Number:
- 949-569-5
- Molecular formula:
- C12H22O2
- IUPAC Name:
- Reaction mass of 1-(3,3-dimethylcyclohexyl)ethyl acetate and 2,6,6-trimethylcycloheptanol acetate
- Test material form:
- liquid
Constituent 1
Method
Species / strainopen allclose all
- 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:
- - Type and composition of metabolic activation system:
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- method of preparation of S9 mix : according to Ames et al. (1977)
- concentration or volume of S9 mix and S9 in the final culture medium: 33.3 mg/mL - Test concentrations with justification for top dose:
- Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
Since the acceptance criterion of at least three concentrations showing no signs of toxic effects was not fulfilled in experiment II in strain TA 100 without S9 mix, this part of experiment II was repeated at the following concentrations (reported as experiment IIa):
Experiment IIa: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate - Vehicle / solvent:
- - Solvent used: DMSO (purity > 99 %)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine, 4-NOPD - without S9 mix: TA 1537, TA 98; 2-aminoanthracene, 2-AA - with S9 mix: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 3
METHOD OF TREATMENT/ EXPOSURE:
A pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I since the acceptance criteria are met.
Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension,
2000 μL Overlay agar
Experiment II and IIa (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37 °C for 60 minutes.
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension.
After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube.
The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
In parallel to each test a sterile control of the test item was performed. Therefore, 100 μL of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates. - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- 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
- Remarks:
- at 2500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- 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:
- at 1000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- 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:
- at 1000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- No precipitation of the test item occurred in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I and II at 5000 μg/plate in the absence of S9 mix and from 2500 to 5000 μg/plate in the presence of S9 mix and in experiment IIa at 5000 μg/plate in the absence of S9 mix.
The plates incubated with the test item showed reduced background growth in strains TA 1537, TA 98, and TA 100 in experiments I, II, and IIa, and additionally in strain TA 1535 with S9 mix in experiment II.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 100 in experiments I and IIa and in strains TA 98 and TA 100 in experiment II.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Any other information on results incl. tables
The plates incubated with the test item showed reduced background growth at the following concentrations (μg/plate):
Strain |
Experiment I |
Experiment II |
Experiment IIa |
||
|
without S9 mix |
with S9 mix |
without S9 mix |
with S9 mix |
without S9 mix |
TA 1535 |
/ |
/ |
/ |
5000 |
n.p. |
TA 1537 |
5000 |
2500 – 5000 |
2500 – 5000 |
2500 – 5000 |
n.p. |
TA 98 |
2500 – 5000 |
1000 – 5000 |
2500 – 5000 |
1000 – 5000 |
n.p. |
TA 100 |
2500 – 5000 |
1000 – 5000 |
1000 – 5000 |
1000 – 5000 |
1000 – 5000 |
WP2 uvrA |
/ |
/ |
/ |
/ |
n.p. |
/
= normal background growth
n.p. = not performed
Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (μg/plate):
Strain |
Experiment I |
Experiment II |
Experiment IIa |
||
|
without S9 mix |
with S9 mix |
without S9 mix |
with S9 mix |
without S9 mix |
TA 1535 |
/ |
/ |
/ |
/ |
n.p. |
TA 1537 |
/ |
/ |
/ |
/ |
n.p. |
TA 98 |
/ |
/ |
/ |
2500 – 5000 |
n.p. |
TA 100 |
/ |
5000 |
333 – 5000 |
1000 – 5000 |
1000 – 5000 |
WP2 uvrA |
/ |
/ |
/ |
/ |
n.p. |
/
= no toxic effects (induction factor ≥ 0.5)
n.p. = not performed
Summary results from Experiment I:
Metabolic Activation |
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ± SD) |
||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|||
Without activation |
DMSO (Solvent control) |
|
18 ± 3 |
14 ± 4 |
24 ± 6 |
115 ± 10 |
51± 5 |
Untreated control |
|
15 ± 1 |
9 ± 2 |
29 ± 2 |
121 ± 15 |
52 ± 6 |
|
Test Item |
3 µg |
15 ± 1 |
13 ± 4 |
25 ± 7 |
105 ± 2 |
47 ± 5 |
|
10 µg |
16 ± 5 |
15 ± 1 |
27 ± 6 |
112 ± 9 |
58 ± 5 |
||
33 µg |
17 ± 3 |
11 ± 7 |
33 ± 4 |
120 ± 11 |
57 ± 5 |
||
100 µg |
16 ± 1 |
12 ± 3 |
30 ± 9 |
113 ± 16 |
54 ± 6 |
||
333 µg |
13 ± 3 |
14 ± 2 |
22 ± 1 |
110 ± 11 |
51 ± 3 |
||
1000 µg |
15 ± 1 |
11 ± 5 |
19 ± 5 |
89 ± 3 |
57 ± 10 |
||
2500 µg |
14 ± 5 |
13 ± 2 |
18 ± 4R |
102 ± 7R |
50 ± 5 |
||
5000 µg |
11 ± 1P |
8 ± 3R,P |
17 ± 5P,R |
78 ± 4P,M,R |
58 ± 14P |
||
NaN3 |
10 µg |
1308 ± 71 |
|
|
2083 ± 13 |
|
|
4-NOPD |
10 µg |
|
|
368 ± 9 |
|
|
|
4-NOPD |
50 µg |
|
94 ± 7 |
|
|
|
|
MMS |
2.0 µL |
|
|
|
|
1137± 33 |
|
With activation |
DMSO (Solvent control) |
|
13 ± 4 |
10 ± 2 |
32 ± 5 |
124 ± 15 |
62± 11 |
Untreated control |
|
17 ± 4 |
11 ± 5 |
32 ± 4 |
137 ± 10 |
57 ± 11 |
|
Test Item |
3 µg |
12 ± 3 |
11 ± 0 |
34 ± 6 |
134 ± 16 |
52 ± 9 |
|
10 µg |
14 ± 4 |
10 ± 3 |
31 ± 6 |
151 ± 9 |
66 ± 12 |
||
33 µg |
13 ± 2 |
15 ± 1 |
35 ± 6 |
145 ± 25 |
55 ± 8 |
||
100 µg |
14 ± 3 |
16 ± 2 |
30 ± 9 |
141 ± 5 |
63 ± 12 |
||
333 µg |
12 ± 3 |
10 ± 3 |
26 ± 1 |
115 ± 9 |
60 ± 8 |
||
1000 µg |
11 ± 3 |
9 ± 3 |
27 ± 6R |
91 ± 6R |
55 ± 7 |
||
2500 µg |
8 ± 2P |
7 ± 2R,P |
21 ± 7P,R |
73 ± 6P,M,R |
53 ± 6P |
||
5000 µg |
8 ± 1P |
7 ± 3R,P |
16 ± 2P,R,M |
42 ± 7P,M,R |
41 ± 6P |
||
2-AA |
2.5 µg |
440 ± 27 |
298 ± 36 |
3576 ± 236 |
4288 ± 385 |
|
|
2-AA |
10.0 µg |
|
|
|
|
282 ± 13 |
Positive Controls: NaN3 – sodium azide, 2-AA – 2-aminoanthracene, 4-NOPD – 4-nitro-o-phenylene-diamine, MMS – methyl methane sulfonate.
Plate Postfix Codes: R – Reduced background growth, P – Precipitate, M – Manual count.
Summary results from Experiment II:
Metabolic Activation |
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ± SD) |
||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|||
Without activation |
DMSO (Solvent control) |
|
11 ± 2 |
13 ± 3 |
33 ± 8 |
214 ± 7 |
47± 8 |
Untreated control |
|
11 ± 1 |
10 ± 4 |
40 ± 8 |
217 ± 19 |
40 ± 3 |
|
Test Item |
33 µg |
11 ± 4 |
15 ± 3 |
28 ± 9 |
197 ± 26 |
41 ± 13 |
|
100 µg |
12 ± 6 |
11 ± 3 |
23 ± 3 |
104 ± 13 |
38 ± 4 |
||
333 µg |
9 ± 3 |
9 ± 5 |
24 ± 3 |
82 ± 7 |
36 ± 6 |
||
1000 µg |
12 ± 2 |
11 ± 4 |
23 ± 2 |
22 ± 4M,R |
32 ± 2 |
||
2500 µg |
7 ± 3 |
8 ± 3R |
24 ± 5R |
18 ± 2M,R |
44 ± 7 |
||
5000 µg |
7 ± 2P |
8 ± 3P,R |
26 ± 1P,R |
18 ± 2P,M,R |
34 ± 8P |
||
NaN3 |
10 µg |
1211 ± 65 |
|
|
1257 ± 44 |
|
|
4-NOPD |
10 µg |
|
|
401 ± 21 |
|
|
|
4-NOPD |
50 µg |
|
98 ± 11 |
|
|
|
|
MMS |
2.0 µL |
|
|
|
|
573± 21 |
|
With activation |
DMSO (Solvent control) |
|
13 ± 5 |
19 ± 2 |
45 ± 8 |
207 ± 12 |
46± 15 |
Untreated control |
|
14 ± 4 |
15 ± 1 |
37 ± 4 |
209 ± 11 |
56 ± 5 |
|
Test Item |
33 µg |
13 ± 1 |
21 ± 5 |
49 ± 9 |
214 ± 22 |
53 ± 10 |
|
|
100 µg |
12 ± 4 |
14 ± 3 |
40 ± 9 |
141 ± 12 |
48 ± 6 |
|
|
333 µg |
13 ± 3 |
14 ± 3 |
39 ± 7 |
105 ± 13 |
54 ± 3 |
|
|
1000 µg |
12 ± 4 |
15 ± 5 |
42 ± 3R |
22 ± 4M,R |
41 ± 6 |
|
|
2500 µg |
11 ± 1P |
16 ± 7P,M,R |
14 ± 4P,M,R |
9 ± 2P,M,R |
37 ± 6P |
|
|
5000 µg |
10 ± 5P,R |
9 ± 2P,M,R |
9 ± 1P,R,M |
5 ± 2P,M,R |
34 ± 6P |
|
2-AA |
2.5 µg |
417 ± 2 |
136 ± 22 |
3787 ± 652 |
2401 ± 153 |
|
|
2-AA |
10.0 µg |
|
|
|
|
489 ± 3 |
Positive Controls: NaN3 – sodium azide, 2-AA – 2-aminoanthracene, 4-NOPD – 4-nitro-o-phenylene-diamine, MMS – methyl methane sulfonate.
Plate Postfix Codes: R – Reduced background growth, P – Precipitate, M – Manual count.
Summary results from Experiment IIa:
Metabolic Activation |
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ± SD) |
TA 100 |
|||
Without activation |
DMSO (Solvent control) |
|
102 ± 8 |
Untreated control |
|
120 ± 10 |
|
Test Item |
3 µg |
103 ± 13 |
|
10 µg |
99 ± 12 |
||
33 µg |
91 ± 10 |
||
100 µg |
90 ± 8 |
||
333 µg |
62 ± 8 |
||
1000 µg |
39 ± 2M,R |
||
2500 µg |
16 ± 4M,R |
||
5000 µg |
1 ± 1P,M,R |
||
NaN3 |
10 µg |
1995 ± 86 |
Positive Controls: NaN3 – sodium azide.
Plate Postfix Codes: R – Reduced background growth, P – Precipitate, M – Manual count.
Applicant's summary and conclusion
- Conclusions:
- In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
- Executive summary:
A study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The test was conducted according to OECD 471 (1997) and EU Method B.13/14 (30 May 2008). The assay was performed in three independent experiments. Experiment I and II were performed with and without liver microsomal activation, experiment II without liver microsomal activation, only. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
Since the acceptance criterion of at least three concentrations showing no signs of toxic effects was not fulfilled in experiment II in strain TA 100 without S9 mix, this part of experiment II was repeated at the following concentrations (reported as experiment IIa):
Experiment IIa: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
No precipitation of the test item occurred in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I and II at 5000 μg/plate in the absence of S9 mix and from 2500 to 5000 μg/plate in the presence of S9 mix and in experiment IIa at 5000 μg/plate in the absence of S9 mix. The undissolved particles had no influence on the data recording.
The plates incubated with the test item showed reduced background growth in strains TA 1537, TA 98, and TA 100 in experiments I, II, and IIa, and additionally in strain TA 1535 with S9 mix in experiment II. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 100 in experiments I and IIa and in strains TA 98 and TA 100 in experiment II.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
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