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EC number: 283-640-5 | CAS number: 84696-21-9 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Hydrocotyle asiatica, Umbelliferae.
- 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
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- 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
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- 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 mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018
- 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 guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- july 2016
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Test material
- Reference substance name:
- Hydrocotyle asiatica, ext.
- EC Number:
- 283-640-5
- EC Name:
- Hydrocotyle asiatica, ext.
- Cas Number:
- 84696-21-9
- Molecular formula:
- not applicable
- IUPAC Name:
- Hydrocotyle asiatica, ext.
- Test material form:
- solid
Constituent 1
Method
- Target gene:
- Thymidine kinase
Species / strain
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Without metabolic activation: 10, 25, 50, 100, 250, 450 and 600 microg/mL.
With metabolic activation: 100, 250, 450, 600, 650, 700 and 800 microg/mL - Vehicle / solvent:
- DMSO 1% (v/v)
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Evaluation criteria:
- Acceptability of the Assay
A mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT [8]:
- At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable
- The cloning efficiency 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 106 cells
- The cell number of the negative/solvent controls should undergo 8 - 32 fold increase during a 2 day growth period (short-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 106 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 106 cells
- The RTG must be greater than 10%. - Statistics:
- Evaluation of Results
The test item is considered mutagenic if the following criteria:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- a dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Remarks on result:
- other: entella asiatica dry ext. is considered to be mutagenic in the in vitro mammalian cell gene mutation assay
Any other information on results incl. tables
Main Experiment - Toxicity Data, without metabolic activation
Test Group |
Concen-tration [µg/mL] |
Number of Cells 4 h after Treatment |
Number of Cells 24 h after Treatment |
Number of Cells 48 h after Treatment |
SGa |
RSGb[%] |
RCEc[%] |
RTGd[%] |
C1 |
0 |
363000 |
916000 |
1520000 |
13.9 |
104.1 |
105.0 |
109.3 |
C2 |
380000 |
985000 |
1250000 |
12.3 |
92.1 |
100.0 |
92.0 |
|
S1 |
0 |
376000 |
873000 |
1370000 |
12.0 |
100.0 |
100.0 |
100.0 |
S2 |
400000 |
1020000 |
1450000 |
14.8 |
||||
2 |
10 |
392000 |
944000 |
1250000 |
11.8 |
88.2 |
105.0 |
92.6 |
3 |
25 |
364000 |
886000 |
1370000 |
12.1 |
90.8 |
89.6 |
81.3 |
4 |
50 |
385000 |
898000 |
1490000 |
13.4 |
100.0 |
116.4 |
116.5 |
5 |
100 |
369000 |
987000 |
1470000 |
14.5 |
108.5 |
93.9 |
101.8 |
6 |
250 |
336000 |
705000 |
1450000 |
10.2 |
76.4 |
93.9 |
71.7 |
7 |
450 |
336000 |
332000 |
836000 |
2.8 |
20.8 |
100.0 |
20.7 |
8 |
600 |
322000 |
276000 |
465000 |
1.4 |
10.4 |
123.0 |
12.8 |
EMS |
300 |
358000 |
925000 |
1400000 |
13.0 |
96.8 |
73.0 |
70.6 |
MMS |
10 |
362000 |
839000 |
1350000 |
11.3 |
84.7 |
70.9 |
60.0 |
Main Experiment - Mutagenicity Data, without metabolic activation
Cloning Efficiency (CE) |
Mutagenicity Data |
||||||||||
Test Group |
Concen-tration [µg/mL] |
Plate 1e |
Plate 2e |
CEf[%] |
Number of cultures / 96 wells |
MFg [mutants / 106cells] |
IMFh [mutants / 106cells] |
||||
Plate 1e |
Plate 2e |
Plate 3e |
Plate 4e |
Mean |
|||||||
C1 |
0 |
82 |
74 |
104.6 |
11 |
19 |
21 |
21 |
18.0 |
99.9 |
/ |
C2 |
76 |
77 |
99.6 |
20 |
17 |
20 |
19 |
19.0 |
110.8 |
/ |
|
S1 |
0 |
76 |
78 |
101.2 |
19 |
15 |
16 |
10 |
15.0 |
84.3 |
/ |
S2 |
72 |
80 |
98.0 |
16 |
19 |
19 |
9 |
15.8 |
92.0 |
/ |
|
2 |
10 |
80 |
76 |
104.6 |
19 |
27 |
16 |
15 |
19.3 |
107.9 |
19.7 |
3 |
25 |
74 |
72 |
89.3 |
14 |
22 |
5 |
15 |
14.0 |
89.8 |
1.6 |
4 |
50 |
84 |
78 |
116.0 |
11 |
19 |
14 |
13 |
14.3 |
69.5 |
-18.6 |
5 |
100 |
72 |
77 |
93.5 |
18 |
24 |
21 |
20 |
20.8 |
130.4 |
42.3 |
6 |
250 |
72 |
77 |
93.5 |
23 |
25 |
22 |
16 |
21.5 |
136.1 |
47.9 |
7 |
450 |
71 |
82 |
99.6 |
25 |
15 |
22 |
19 |
20.3 |
119.5 |
31.3 |
8 |
600 |
85 |
80 |
122.6 |
39 |
12 |
17 |
18 |
21.5 |
107.8 |
19.6 |
EMS |
300 |
58 |
74 |
72.7 |
67 |
68 |
72 |
60 |
66.8 |
824.7 |
736.5 |
MMS |
10 |
67 |
63 |
70.6 |
61 |
55 |
69 |
63 |
62.0 |
742.4 |
654.3 |
Main Experiment - Toxicity Data, with metabolic activation
Test Group |
Concen-tration [µg/mL] |
Number of Cells 4 h after Treatment |
Number of Cells 24 h after Treatment |
Number of Cells 48 h after Treatment |
SGa |
RSGb[%] |
RCEc[%] |
RTGd[%] |
C1 |
0 |
265000 |
965000 |
1430000 |
13.8 |
91.4 |
115.0 |
105.1 |
C2 |
327000 |
1030000 |
1450000 |
14.9 |
99.0 |
106.3 |
105.2 |
|
S1 |
0 |
330000 |
985000 |
1430000 |
14.1 |
100.0 |
100.0 |
100.0 |
S2 |
362000 |
1110000 |
1450000 |
16.1 |
||||
5 |
100 |
359000 |
1070000 |
1430000 |
15.3 |
101.4 |
107.9 |
109.4 |
6 |
250 |
403000 |
1020000 |
1410000 |
14.4 |
95.3 |
111.4 |
106.1 |
7 |
450 |
352000 |
611000 |
1430000 |
8.7 |
57.9 |
116.8 |
67.6 |
8 |
600 |
347000 |
467000 |
1200000 |
5.6 |
37.1 |
107.9 |
40.1 |
9 |
650 |
312000 |
355000 |
917000 |
3.3 |
21.6 |
127.0 |
27.4 |
10 |
700 |
333000 |
380000 |
888000 |
3.4 |
22.4 |
136.3 |
30.5 |
11 |
800 |
316000 |
294000 |
677000 |
2.0 |
13.5 |
115.0 |
15.5 |
B[a]P |
2.5 |
384000 |
843000 |
1430000 |
12.1 |
79.9 |
82.7 |
66.1 |
Main Experiment - Mutagenicity Data, with metabolic activation
Cloning Efficiency (CE) |
Mutagenicity Data |
||||||||||
Test Group |
Concen-tration [µg/mL] |
Plate 1e |
Plate 2e |
CEf[%] |
Number of cultures / 96 wells |
MFg [mutants / 106cells] |
IMFh [mutants / 106cells] |
||||
Plate 1e |
Plate 2e |
Plate 3e |
Plate 4e |
Mean |
|||||||
C1 |
0 |
77 |
76 |
99.6 |
18 |
18 |
20 |
13 |
17.3 |
99.7 |
/ |
C2 |
73 |
75 |
92.1 |
18 |
23 |
18 |
18 |
19.3 |
121.7 |
/ |
|
S1 |
0 |
67 |
78 |
88.0 |
19 |
18 |
19 |
20 |
19.0 |
125.4 |
/ |
S2 |
69 |
74 |
85.4 |
21 |
16 |
20 |
21 |
19.5 |
133.2 |
/ |
|
5 |
100 |
73 |
76 |
93.5 |
17 |
20 |
15 |
15 |
16.8 |
102.7 |
-26.6 |
6 |
250 |
76 |
75 |
96.5 |
24 |
23 |
19 |
21 |
21.8 |
133.3 |
4.0 |
7 |
450 |
77 |
77 |
101.2 |
26 |
22 |
31 |
17 |
24.0 |
143.3 |
14.0 |
8 |
600 |
78 |
71 |
93.5 |
26 |
26 |
30 |
25 |
26.8 |
174.8 |
45.5 |
9 |
650 |
76 |
83 |
110.1 |
33 |
27 |
29 |
34 |
30.8 |
175.8 |
46.5 |
10 |
700 |
80 |
83 |
118.1 |
35 |
35 |
37 |
38 |
36.3 |
200.8 |
71.5 |
11 |
800 |
77 |
76 |
99.6 |
44 |
43 |
31 |
44 |
40.5 |
277.3 |
148.0 |
B[a]P |
2.5 |
71 |
60 |
71.7 |
53 |
59 |
52 |
64 |
57.0 |
634.1 |
504.8 |
C: Negative control
S: Solvent control (1% DMSO; v/v)
e: Number of cultures with cell growth.
f: Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)
g: Mutant frequency,
MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
h: Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
Applicant's summary and conclusion
- Conclusions:
- Mutagenicity:
In the experiment without metabolic activation the mutant frequencies induced by the test item did not show any biologically relevant increase. A statistical analysis displayed that some of the mutant frequencies were significantly increased over those of the solvent controls. However, the GEF was not exceeded by the induced mutant frequency at any concentration. Therefore any differences observed in mutant frequency between the treated and concurrent control groups were concluded upon as not biologically relevant.
In the experiment with metabolic activation the mutant frequencies induced by the test item showed a distinct biologically relevant increase. The GEF of 126 was exceeded at the concentration of 800 µg/mL (148.0 mutants/106 cells). A statistical analysis displayed that the corresponding values of the mutant frequencies for the concentrations 600, 650, 700 and 800 µg/mL were significantly increased over those of the solvent controls. Moreover, a dose-response relationship was observed.
Clastogenicity:
The positive controls MMS and B[a]P induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥40%), thus proving the ability of the test system to indicate potential clastogenic effects.
In the main experiment with metabolic activation the percentage of small colonies in the negative controls and in the solvent controls, was found to be lower than 40%. Due to the increased number of small colonies and corresponding mutagenicity in the three highest dose groups, these concentrations of the test items were considered as potential clastogenic
Conclusion
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Centella asiatica dry ext. is considered to be mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
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