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EC number: 213-796-1 | CAS number: 1013-75-8
- 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
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- Nanomaterial pour density
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- 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 bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- December 8, 2016 - January 5, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Ethyl ethyl(phenyl)carbamate
- EC Number:
- 213-796-1
- EC Name:
- Ethyl ethyl(phenyl)carbamate
- Cas Number:
- 1013-75-8
- Molecular formula:
- C11H15NO2
- IUPAC Name:
- ethyl ethyl(phenyl)carbamate
- Test material form:
- liquid
- Details on test material:
- Colorless
Constituent 1
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes
Method
- Target gene:
- Salmonella typhimurium - histidine
Escherichia coli - tryptophane
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: delta uvr B and rfa
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Additional strain / cell type characteristics:
- other: delta uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction prerpared from rat liver homogenate
- Test concentrations with justification for top dose:
- ASSAY 1: 5 000, 4000, 1 500, 500, 150 and 50 μg/plate - the highest concetration showed one toxicity consistent with the maximum tolerated 75%. A supplementary dose at 4000 μg/plate was tested to be sure this hight toxicity no hindering the scoring of the revertants.
ASSAY 2: 5 000, 1 500, 500, 150 and 50 μg/plate. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no react with test item and is compatible with the survival of the bacteria and S9 activity.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- NaCL 0.15 M, Acetone, DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2-Anthramine, cis-Platinum (II) Diammine Dichloride
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- plate incorporation method: ASSAY 1- Performed with and without metabolic activation. Test item tested at dose level (5 000, 4000, 1 500, 500, 150 and 50 μg/plate). The test item diluted in DMSO or controls (0.1 mL) and other components (overnight culture of test strain(0.1mL), phosphate buffer or S9 mix (0.5mL)) were prepared freshly and added to 2 mL of overlay agar, maintained supercooled at 45° C, containing 10 % (v/v) of a L-Histidine-D-Biotine solution (0.5 mM) in case of salmonella strain or containing 5% (v/v) of nutrient broth n° 2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL in case of escherichia coli strain.
This solution was mixed and poured on the surface of minimal agar plates. For the assay with metabolic activation 500 μL of S9-mix fraction were quickly added, before pouring the mixture onto the plates. After preparation, the plates were incubated at 37°C for 48 hours.
- pre-incubation: ASSAY 2- Performed with and without metabolic activation with dose level (5 000, 1 500, 500, 150 and 50 μg/plate.) Solution of the test item with the test strain were preincubated with shaking for 30 min., at 37° C prior to mixing with the overlay agar and pouring onto the minimal agar plate. When metabolic activation is used, 500 μL of S9-mix fraction are added before the pre-incubation.
- Cell density at seeding (if applicable):1-9 x109 bacteria/mL
If the first assay is positive, the second one is performed in the same manner.
If the first assay, in presence of test item, is negative, the pre-incubation test is performed for the second assay.
DURATION
- Preincubation period:30 min at 37ºC (Assay 2)
- Exposure duration:48-72 hours at 37°C
SELECTION AGENT (mutation assays): absence histidine or tryptophane,the lack of amino-acid in the medium.
Mutants can grow only if they can produce essential amino acid.
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY (Strain TA100)
- Method: relative total growth
A preliminary cytotoxicity test was performed with the strain TA100 to determine any sign of cytotoxicity. Test substance was tested at dose level 5 000, 4000, 1 500, 500, 150 and 50 μg/plate. Suplementary dose 4000 μg/plate tested on ASSAY 1 because of sign of toxicity at the highest dose level.
In a test tube 0.1 mL of the bacterial suspension (1-9 x 103 bacteria/mL) and 0.1 mL of the stock solution and dilutions, are successively added to 2 mL of top agar at 45°C, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube is poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). The three plates per concentration are incubated for 48-72 hours at 37°C, and the colonies counted. A negative control containing the blank alone is run in parallel. In case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less.
OTHER :
Sterility tests:
Test item and the corresponding dilutions are added to 2 mL of top agar maintained at 45°C, and poured after homogenization on the bottom agar (20 ml) onto a Petri plate (90 mm in diameter) (n = 3). Plates are incubated for 48 - 72 hours at 37°C and then examined. There should be no bacterial growth on any plate. S9-mix sterility is checked using the same protocol. See table No.3 - Evaluation criteria:
- The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains without and with metabolic activation.
The result of the test is considered positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.
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:
- no cytotoxicity
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- 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:
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation observed
RANGE-FINDING/SCREENING STUDIES: The preliminary cytotoxicity testing on strain TA100 showed in presence of 5 000 μg/plate one toxicity consistent with the maximum tolerated 75%. A supplementary dose at 4 000 μg/plate (80 % of the maximal dose) was tested on the first mutagenic assay to be sure this hight toxicity no hindering the scoring of the revertants.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
without metabolic activation:
TA 1535 Sodium Azide, N=975, Mean=702.3+-203.4, (min/max=190.0/1481.0)
TA 1537 9-Aminoacridine, N=975, Mean=851.2+-428.1 (min/max=219.0/1967.0)
TA 98 2-Nitrofluorene N=975, Mean=512.6+-219.5 (min/max=187.0/1667.0)
TA100 Sodium Azide, N=975, Mean=934.4+-325.2 (min/max=381.0/1690.0)
E.coli WP2 CIS-PLATINUM (II) DIAMINUM DICHLORIDE N=717, Mean=502.8+-168.1 (min/max=248.0/1089.0)
with metabolic activation, without pre-incubation
TA 1535 2-Anthramine, N=525, Mean=12.3+-4.0, (min/max=3.0/23.0)
TA 1537 2-Anthramine, N=525, Mean=55.8+-23.4 (min/max=24.0+-170.0)
TA 98 2-Anthramine, N=525, Mean=574.5+-209.5 (min/max=219.0/1499.0)
TA 100 2-Anthramine, N=522, Mean=862.1+-359.1 (min/max=361.0/2163.0)
E.coli WP2 dimethyl benzoanthracane N=372, Mean=707.3+-248.0 (min/max=378.0/1680.0)
with metabolic activation, with pre-incubation
TA 1535 2-Anthramine, N=519, Mean=73.2+-34.9, (min/max=25.0/185.0)
TA 1537 2-Anthramine, N=519, Mean=49.5+-22.5 (min/max=21.0/182.0)
TA 98 2-Anthramine, N=519 Mean=474.6+-196.3, (min/max=174.0/1370.0)
TA 100 2-Anthramine, N=519, Mean=682.4+-290.0 (min/max=309.0/1839.0)
E.coli WP2 dimethyl benzoanthracane N=372, Mean=701.8+-229.7 (min/max=397.0/1680.0)
- Negative (solvent/vehicle) historical control data (with ranges, means and standard deviation and confidence interval (e.g. 95%):
without metabolic activation
TA1535 N=975, Mean= 10.9+-3.6 (min/max=40/230)
TA1537 N=975, Mean= 6.0+-2.4 (min/max=10/140)
TA 98,N=975, Mean= 16.0+-3.8 (min/max=60/290)
TA 100 N=975, Mean= 62.2+-14.3 (min/max=410/126.0)
E.coli WP2 N=717, Mean= 75.0+-29.2 (min/max=410/188.0)
with metabolic activation, without pre-incubation
TA1535 N=525, Mean= 12.3+-4.0 (min/max=30/23.0)
TA1537 N=525, Mean= 8.0+-3.1 (min/max=1.0/24.0)
TA 98,N=525, Mean= 23.2+-4.8 (min/max=12.0/38.0)
TA 100 N=522 Mean= 101.7+-22.9 (min/max=58.0/189.0)
E.coli WP2 N=372, Mean= 154.8+-33.6 (min/max=80.0/264.0)
with metabolic activation, with pre-incubation
TA1535 N=519, Mean= 12.7+-4.2 (min/max=5.0/25.0)
TA1537 N=519, Mean= 8.3+-3.2 (min/max=1.0/19.0)
TA 98,N=519, Mean= 23.3+-5.2 (min/max=11.0/36.0)
TA 100 N=519, Mean= 101.3+-24.8(min/max=51.0/189.0)
E.coli WP2 N=372, Mean= 157.5+-35.4 (min/max=69.0/250.0),
Any other information on results incl. tables
Table No.3: Sterility control
Serie |
Doses |
Colony number/plate |
||
Control nº1 |
|
1 |
2 |
3 |
Solution of test item Lemi code : GGA191216-S2
|
5000ug/plate |
0 |
0 |
0 |
4000ug/plate |
0 |
0 |
0 |
|
1500ug/plate |
0 |
0 |
0 |
|
500ug/plate |
0 |
0 |
0 |
|
150ug/plate |
0 |
0 |
0 |
|
|
50ug/plate |
|
|
|
S9-mix |
500ul/plate |
0 |
0 |
0 |
Control nº2 |
|
1 |
2 |
3 |
Solution of test item Lemi code : GGA020117-S2
|
5000ug/plate |
0 |
0 |
0 |
1500ug/plate |
0 |
0 |
0 |
|
500ug/plate |
0 |
0 |
0 |
|
150ug/plate |
0 |
0 |
0 |
|
50ug/plate |
0 |
0 |
0 |
|
S9-mix |
500ul/plate |
0 |
0 |
0 |
Table No. 4: Bacteriostatic activity
|
|
0 Negative control |
DMSO |
50ug |
150ug |
500ug |
1500ug |
2500ug |
5000ug |
||
Solution of test item Lemi code : GGA081216-S2
|
N1 |
259 |
311 |
279 |
304 |
298 |
261 |
279 |
68 |
||
N2 |
288 |
307 |
311 |
309 |
297 |
272 |
278 |
74 |
|||
N3 |
304 |
255 |
301 |
287 |
284 |
304 |
302 |
76 |
|||
N4 |
284+-23 |
291+-31 |
297+-16 |
300+-12 |
293+-8 |
279+-22 |
286+-14 |
73+-4 |
|||
% |
- |
103 |
105 |
106 |
103 |
98 |
101 |
26 |
|||
|
|
0 Negative control |
DMSO |
2500ug |
3000ug |
4000ug |
5000ug |
|
|||
Solution of test item Lemi code : GGA121216-S1
|
N1 |
388 |
428 |
341 |
358 |
205 |
89 |
|
|||
N2 |
330 |
329 |
334 |
301 |
229 |
88 |
|
||||
N3 |
394 |
401 |
370 |
326 |
158 |
95 |
|
||||
N4 |
371+-35 |
386+-51 |
348+-19 |
328+-29 |
197+-36 |
91+-4 |
|
||||
% |
- |
104 |
94 |
89 |
53 |
24 |
|||||
|
|
0 Negative control |
DMSO |
50ug |
150ug |
500ug |
1500ug |
4000ug |
5000ug |
||
Solution of test item Lemi code : GGA191216-S2
|
N1 |
221 |
220 |
263 |
232 |
248 |
178 |
150 |
59 |
||
N2 |
241 |
241 |
246 |
234 |
245 |
218 |
148 |
64 |
|||
N3 |
223 |
248 |
238 |
198 |
231 |
168 |
139 |
66 |
|||
N4 |
228+-11 |
236+-15 |
249+-13 |
221+-20 |
241+-9 |
188+-26 |
146+-6 |
63+-4 |
|||
% |
- |
104 |
109 |
97 |
106 |
82 |
64 |
28 |
N1 Number of colonies in plate 1
N2 Number of colonies in plate 2
N3 Number of colonies in plate 3
N Mean per plate
% Percent of survival compared to negative control
Table No.5 : TA 1535, ASSAY 1 without metabolic activation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
7 |
6 |
7 |
6.67 |
0.58 |
- |
Positive control solvent |
5uL |
6 |
8 |
6 |
6.67 |
1.15 |
- |
Positive control Sodium Azide |
5ug In 5uL |
513 |
616 |
583 |
570.67 |
52.60 |
85.6 |
Vehicle |
50uL |
8 |
10 |
5 |
7.67 |
2.52 |
- |
Solution of test item Lemi code GGA191216-S2 |
5000ug |
2 |
2 |
2 |
2.00 |
0.00 |
0.26 |
4000ug |
10 |
18 |
5 |
11.00 |
6.56 |
1.43 |
|
1500ug |
9 |
8 |
6 |
7.67 |
1.53 |
1.00 |
|
500ug |
4 |
11 |
6 |
7.00 |
3.61 |
0.91 |
|
150ug |
14 |
8 |
8 |
10.00 |
3.46 |
1.30 |
|
50ug |
6 |
14 |
10 |
10.00 |
4.00 |
1.30 |
Table No.6: TA 1535 Assay 1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
14 |
8 |
5 |
9.00 |
4.58 |
- |
Positive control solvent |
20uL |
7 |
6 |
5 |
6.00 |
1.00 |
- |
Positive control 2-Anthramine
|
2ug In 20uL |
90 |
71 |
61 |
74.00 |
14.73 |
12.33 |
Vehicle |
50uL |
15 |
8 |
11 |
11.33 |
3.51 |
- |
Solution of test item Lemi code GGA191216-S2 |
5000ug |
5 |
5 |
5 |
5.00 |
0.00 |
0.44 |
4000ug |
5 |
6 |
5 |
5.33 |
0.58 |
0.47 |
|
1500ug/ |
5 |
13 |
9 |
9.00 |
4.00 |
0.79 |
|
500ug |
10 |
9 |
10 |
9.67 |
0.58 |
0.85 |
|
150ug |
15 |
21 |
16 |
17.33 |
3.21 |
1.53 |
|
50ug |
17 |
13 |
6 |
12.00 |
5.57 |
1.06 |
Table No.7: TA 1535 Assay 2 – without metabolic activation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
14 |
16 |
7 |
12.33 |
4.73 |
- |
Positive control solvent |
5uL |
11 |
12 |
12 |
11.67 |
0.58 |
- |
Positive control Sodium azide |
5 ug In 5uL |
735 |
642 |
692 |
689.67 |
46.54 |
59.11 |
Vehicle |
50uL |
14 |
14 |
16 |
14.67 |
1.15 |
- |
Solution of test item Lemi code :GGA020117-S2
|
5000ug |
7 |
12 |
15 |
11.33 |
4.04 |
0.77 |
1500ug |
15 |
10 |
11 |
12.00 |
2.65 |
0.82 |
|
500ug |
8 |
7 |
13 |
9.33 |
3.21 |
0.64 |
|
150ug |
14 |
13 |
12 |
13.00 |
1.00 |
0.89 |
|
50ug |
17 |
8 |
12 |
12.33 |
4.51 |
0.84 |
Table No.8: TA 1535 Assay 2 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
13 |
17 |
11 |
13.67 |
3.06 |
- |
Positive control solvent |
10uL |
12 |
12 |
10 |
11.33 |
1.15 |
- |
Positive control 2-Anthranine |
1 ug In 10uL |
64 |
49 |
67 |
60.00 |
9.64 |
5.29 |
Vehicle |
50uL |
14 |
16 |
14 |
14.67 |
1.15 |
- |
Solution of test item Lemi code :GGA020117-S2
|
5000ug* |
6 |
10 |
1 |
5.67 |
4.51 |
0.39 |
1500ug* |
5 |
3 |
3 |
3.67 |
1.15 |
0.25 |
|
500ug |
10 |
15 |
20 |
15.00 |
5.00 |
1.02 |
|
150ug |
23 |
14 |
14 |
17.00 |
5.20 |
1.16 |
|
50ug |
9 |
10 |
10 |
9.67 |
0.58 |
0.66 |
* Moderate thinning of the background bacterial lawn.
Table No.9: TA 1537 Assay 1 – without metabolic activation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
5 |
5 |
7 |
5.67 |
1.15 |
- |
Positive control solvent |
20uL |
4 |
3 |
6 |
4.33 |
1.53 |
- |
Positive control 9-aminoacridine
|
50ug In 20uL |
1055 |
1110 |
933 |
1032.67 |
90.59 |
238.31 |
Vehicle |
50uL |
4 |
5 |
2 |
3.67 |
1.53 |
- |
Solution of test item Lemi code GGA191216-S2 |
5000ug |
2 |
2 |
1 |
1.67 |
0.58 |
0.45 |
4000ug |
2 |
5 |
5 |
4.00 |
1.73 |
1.09 |
|
1500ug |
4 |
3 |
4 |
3.67 |
0.58 |
1.00 |
|
500ug |
5 |
5 |
5 |
5.00 |
0.00 |
1.36 |
|
150ug |
6 |
6 |
3 |
5.00 |
1.73 |
1.36 |
|
50ug |
6 |
3 |
2 |
3.67 |
2.08 |
1.00 |
Table No.10: TA 1537, Assay 1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
7 |
4 |
6 |
5.67 |
1.53 |
- |
Positive control solvent |
20uL |
12 |
8 |
10 |
10.00 |
2.00 |
- |
Positive control 2-Anthramine
|
2ug In 20uL |
25 |
39 |
30 |
31.33 |
7.09 |
3.13 |
Vehicle |
50uL |
5 |
6 |
6 |
5.67 |
0.58 |
- |
Solution of test item Lemi code GGA191216-S2 |
5000ug |
2 |
3 |
1 |
2.00 |
1.00 |
0.35 |
4000ug |
1 |
1 |
2 |
1.33 |
0.58 |
0.24 |
|
1500ug |
3 |
5 |
3 |
3.67 |
1.15 |
0.65 |
|
500ug |
3 |
6 |
9 |
6.00 |
3.00 |
1.06 |
|
150ug |
9 |
10 |
8 |
9.00 |
1.00 |
1.59 |
|
50ug |
13 |
9 |
10 |
10.67 |
2.08 |
1.88 |
Table No.11, TA 1537, Assay 2 – without metabolic activation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
7 |
4 |
4 |
5.00 |
1.73 |
- |
Positive control solvent |
20uL |
3 |
2 |
4 |
3.00 |
1.00 |
- |
Positive control 9-aminoacridine |
50 ug In 20 uL |
1716 |
1860 |
1415 |
1663.67 |
227.07 |
554.56 |
Vehicle |
50uL |
3 |
2 |
2 |
2.33 |
0.58 |
- |
Solution of test item Lemi code :GGA020117-S2
|
5000ug |
5 |
4 |
3 |
4.00 |
1.00 |
1.71 |
1500ug |
5 |
5 |
3 |
4.33 |
1.15 |
1.86 |
|
500ug |
3 |
2 |
1 |
2.00 |
1.00 |
0.86 |
|
150ug |
2 |
2 |
2 |
2.00 |
0.00 |
0.86 |
|
50ug |
3 |
2 |
4 |
3.00 |
1.00 |
1.29 |
Table No.12: TA 1537, Assay 2 – Assay 2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
8 |
5 |
9 |
7.33 |
2.08 |
- |
Positive control solvent |
10uL |
2 |
3 |
5 |
3.33 |
1.53 |
- |
Positive control 2-Anthramine
|
1 ug In 10 uL |
28 |
25 |
22 |
25.00 |
3.00 |
7.50 |
Vehicle |
50uL |
5 |
4 |
3 |
4.00 |
1.00 |
- |
Solution of test item Lemi code :GGA020117-S2
|
5000ug |
5 |
5 |
4 |
4.67 |
0.58 |
1.17 |
1500ug |
7 |
5 |
6 |
6.00 |
1.00 |
1.5 |
|
500ug |
7 |
4 |
5 |
5.33 |
1.53 |
1.33 |
|
150ug |
7 |
6 |
6 |
6.33 |
0.58 |
1.58 |
|
50ug |
4 |
5 |
7 |
5.33 |
1.53 |
1.33 |
Table No.13: TA 98 Assay 1 – without metabolic activation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
22 |
11 |
22 |
18.33 |
6.35 |
- |
Positive control solvent |
20uL |
22 |
25 |
18 |
21.67 |
3.51 |
- |
Positive control 2-Nitrofluorene
|
2ug In 20uL |
191 |
229 |
217 |
212.33 |
19.43 |
9.80 |
Vehicle |
50uL |
35 |
24 |
16 |
25.00 |
9.54 |
- |
Solution of test item Lemi code GGA191216-S2 |
5000ug* |
27 |
23 |
24 |
24.67 |
2.08 |
0.99 |
4000ug* |
15 |
25 |
19 |
19.67 |
5.03 |
0.79 |
|
1500ug |
22 |
26 |
24 |
24.00 |
2.00 |
0.96 |
|
500ug |
11 |
24 |
32 |
22.33 |
10.60 |
0.89 |
|
150ug |
26 |
19 |
28 |
24.33 |
4.73 |
0.97 |
|
50ug |
18 |
18 |
21 |
19.00 |
1.73 |
0.76 |
Table No.14: TA 98 Assay 1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
18 |
22 |
23 |
21.00 |
2.65 |
- |
Positive control solvent |
20uL |
30 |
23 |
34 |
29.00 |
5.57 |
- |
Positive control 2-Anthramine
|
2ug In 20uL |
253 |
233 |
239 |
241.67 |
10.26 |
8.33 |
Vehicle |
50uL |
24 |
26 |
19 |
23.00 |
3.61 |
- |
Solution of test item Lemi code GGA191216-S2 |
5000ug* |
26 |
16 |
22 |
21.33 |
5.03 |
0.93 |
4000ug* |
26 |
15 |
19 |
20.00 |
5.57 |
0.87 |
|
1500ug |
18 |
16 |
18 |
17.33 |
1.15 |
0.75 |
|
500ug |
30 |
33 |
26 |
29.67 |
3.51 |
1.29 |
|
150ug |
31 |
36 |
24 |
30.33 |
6.03 |
1.32 |
|
50ug |
35 |
31 |
26 |
30.67 |
4.51 |
1.33 |
* Moderate thinning of the background bacterial lawn.
Table No.15: TA 98 Assay 2 – without metabolic activation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
27 |
13 |
22 |
20.67 |
7.09 |
- |
Positive control solvent |
20uL |
17 |
17 |
16 |
16.67 |
0.58 |
- |
Positive control 2-Nitrofluorene
|
2 ug In 20 uL |
252 |
266 |
312 |
276.67 |
31.39 |
16.60 |
Vehicle |
50uL |
21 |
20 |
20 |
20.33 |
0.58 |
- |
Solution of test item Lemi code :GGA020117-S2
|
5000ug* |
7 |
19 |
15 |
13.67 |
6.11 |
0.67 |
1500ug* |
16 |
16 |
14 |
15.33 |
1.15 |
0.75 |
|
500ug |
14 |
17 |
14 |
15.00 |
1.73 |
0.74 |
|
150ug |
28 |
18 |
13 |
19.67 |
7.64 |
0.97 |
|
50ug |
22 |
13 |
17 |
17.33 |
4.51 |
0.85 |
Table No.16, TA 98 Assay 2 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
28 |
26 |
26 |
26.67 |
1.15 |
- |
Positive control solvent |
10uL |
19 |
16 |
32 |
22.33 |
8.50 |
- |
Positive control 2-Anthramine
|
1 ug In 10 uL |
254 |
259 |
237 |
250.00 |
11.53 |
11.19 |
Vehicle |
50uL |
31 |
24 |
26 |
27.00 |
3.61 |
- |
Solution of test item Lemi code :GGA020117-S2
|
5000ug* |
8 |
9 |
6 |
7.67 |
1.53 |
0.28 |
1500ug* |
12 |
9 |
13 |
11.33 |
2.08 |
0.42 |
|
500ug |
14 |
29 |
16 |
19.67 |
8.14 |
0.73 |
|
150ug |
11 |
18 |
22 |
17.00 |
5.57 |
0.63 |
|
50ug |
12 |
14 |
23 |
16.33 |
5.86 |
0.60 |
* Moderate thinning of the background bacterial lawn.
Table No.17: TA 100, Assay 1 – without metabolic activation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
54 |
53 |
51 |
52.67 |
1.53 |
- |
Positive control solvent |
20uL |
57 |
52 |
50 |
53.00 |
3.61 |
- |
Positive control Sodium azide
|
20ug In 20uL |
1334 |
1134 |
1256 |
1241.33 |
100.80 |
23.42 |
Vehicle |
50uL |
74 |
65 |
53 |
64.00 |
10.54 |
- |
Solution of test item Lemi code GGA191216-S2 |
5000ug* |
42 |
47 |
30 |
39.67 |
8.74 |
0.62 |
4000ug* |
29 |
30 |
26 |
28.33 |
2.08 |
0.44 |
|
1500ug |
49 |
52 |
65 |
55.33 |
8.50 |
0.86 |
|
500ug |
72 |
62 |
73 |
69.00 |
6.08 |
1.08 |
|
150ug |
76 |
60 |
67 |
67.67 |
8.02 |
1.06 |
|
50ug |
68 |
55 |
73 |
65.33 |
9.29 |
1.02 |
Table No.18: TA 100, Assay 1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
71 |
72 |
91 |
78.00 |
11.27 |
- |
Positive control solvent |
20uL |
72 |
73 |
72 |
72.33 |
0.58 |
- |
Positive control 2-Anthramine
|
2 ug In 20uL |
392 |
373 |
395 |
386.67 |
11.93 |
5.35 |
Vehicle |
50uL |
81 |
85 |
75 |
80.33 |
5.03 |
- |
Solution of test item Lemi code GGA191216-S2 |
5000ug* |
27 |
21 |
30 |
26.00 |
4.58 |
0.32 |
4000ug* |
54 |
24 |
46 |
41.33 |
15.53 |
0.51 |
|
1500ug |
71 |
87 |
72 |
76.67 |
8.96 |
0.95 |
|
500ug |
84 |
79 |
70 |
77.67 |
7.09 |
0.97 |
|
150ug |
84 |
72 |
70 |
75.33 |
7.57 |
0.94 |
|
50ug |
72 |
79 |
70 |
73.67 |
4.73 |
0.92 |
* Moderate thinning of the background bacterial lawn.
Table No.19: TA 100, Assay 2 – without metabolic activation (-S9-mix)
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
54 |
67 |
60 |
60.33 |
6.51 |
- |
Positive control solvent |
20uL |
60 |
51 |
60 |
57.00 |
5.20 |
- |
Positive control Sodium Azide
|
20 ug In 20 uL |
1531 |
1505 |
1569 |
1535.00 |
32.19 |
26.93 |
Vehicle |
50uL |
46 |
48 |
55 |
49.67 |
4.73 |
- |
Solution of test item Lemi code :GGA020117-S2
|
5000ug* |
46 |
48 |
42 |
45.33 |
3.06 |
0.91 |
1500ug |
57 |
57 |
48 |
54.00 |
5.20 |
1.09 |
|
500ug |
43 |
62 |
65 |
56.67 |
11.93 |
1.14 |
|
150ug |
66 |
60 |
58 |
61.33 |
4.16 |
1.23 |
|
50ug |
64 |
56 |
49 |
56.33 |
7.51 |
1.13 |
Table No.20: TA 100, Assay 2 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
120 |
95 |
88 |
101.00 |
16.82 |
- |
Positive control solvent |
10uL |
76 |
78 |
75 |
76.33 |
1.53 |
- |
Positive control 2-Anthramine
|
1 ug In 10 uL |
356 |
390 |
370 |
372.00 |
17.09 |
4.87 |
Vehicle |
50uL |
73 |
72 |
79 |
74.67 |
3.79 |
- |
Solution of test item Lemi code :GGA020117-S2
|
5000ug* |
10 |
15 |
18 |
14.33 |
4.04 |
0.19 |
1500ug |
65 |
70 |
65 |
66.67 |
2.89 |
0.89 |
|
500ug |
69 |
81 |
69 |
73.00 |
6.93 |
0.98 |
|
150ug |
82 |
75 |
71 |
76.00 |
5.57 |
1.02 |
|
50ug |
83 |
73 |
72 |
76.00 |
6.08 |
1.02 |
* Moderate thinning of the background bacterial lawn.
Table No.21: E. COLI, Assay 1 – without metabolic activation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
107 |
109 |
105 |
107.00 |
2.00 |
- |
Positive control solvent |
10uL |
131 |
109 |
99 |
113.00 |
16.37 |
- |
Positive control cis-Platinum (II)
|
1ug In 10uL |
310 |
324 |
341 |
325.00 |
15.52 |
2.88 |
Vehicle |
50uL |
92 |
102 |
105 |
99.67 |
6.81 |
- |
Solution of test item Lemi code GGA191216-S2 |
5000ug |
52 |
35 |
30 |
39.00 |
11.53 |
0.39 |
4000ug |
52 |
55 |
59 |
55.33 |
3.51 |
0.56 |
|
1500ug |
73 |
70 |
90 |
77.67 |
10.79 |
0.78 |
|
500ug |
98 |
113 |
120 |
110.33 |
11.24 |
1.11 |
|
150ug |
134 |
129 |
144 |
135.67 |
7.64 |
1.36 |
|
50ug |
132 |
131 |
119 |
127.33 |
7.23 |
1.28 |
Table No.22: E. COLI, Assay 1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
182 |
158 |
171 |
170.33 |
12.01 |
- |
Positive control solvent |
5uL |
152 |
124 |
140 |
138.67 |
14.05 |
- |
Positive control DimethylbenzanthracEne
|
5ug In 5uL |
424 |
503 |
436 |
454.33 |
42.57 |
3.28 |
Vehicle |
50uL |
123 |
128 |
143 |
131.33 |
10.41 |
- |
Solution of test item Lemi code GGA191216-S2 |
5000ug |
62 |
53 |
42 |
52.33 |
10.02 |
0.40 |
4000ug |
115 |
84 |
76 |
91.67 |
20.60 |
0.70 |
|
1500ug |
105 |
106 |
124 |
111.67 |
10.69 |
0.85 |
|
500ug |
161 |
162 |
158 |
160.33 |
2.08 |
1.22 |
|
150ug |
180 |
171 |
169 |
173.33 |
5.86 |
1.32 |
|
50ug |
176 |
160 |
147 |
161.00 |
14.53 |
1.23 |
Table No.23: E. COLI, Assay 2 – without metabolic activation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
137 |
144 |
122 |
134.33 |
11.24 |
- |
Positive control solvent |
10uL |
125 |
156 |
128 |
136.33 |
17.10 |
- |
Positive control cis-Platinum (II)
|
1 ug In 10 uL |
345 |
332 |
328 |
335.00 |
8.89 |
2.46 |
Vehicle |
50uL |
108 |
129 |
99 |
112.00 |
15.39 |
- |
Solution of test item Lemi code :GGA020117-S2
|
5000ug |
134 |
113 |
131 |
126.00 |
11.36 |
1.13 |
1500ug |
123 |
133 |
131 |
129.00 |
5.29 |
1.15 |
|
500ug |
133 |
134 |
138 |
135.00 |
2.65 |
1.21 |
|
150ug |
133 |
144 |
118 |
131.67 |
13.05 |
1.18 |
|
50ug |
141 |
126 |
116 |
127.67 |
12.58 |
1.14 |
Table No.24: E. COLI, Assay 2 – with metaboblic activation (10 % S9-mix) – with pre-incubation
Serie |
Dose/plate |
Nº1 |
Nº2 |
Nº3 |
mean |
Standart deviation |
R |
Negative control |
100uL |
215 |
198 |
186 |
199.67 |
14.57 |
- |
Positive control solvent |
5uL |
211 |
192 |
204 |
202.33 |
9.61 |
- |
Positive control Dimethylbenzanthracene |
2.5 ug In 5 uL |
447 |
438 |
506 |
463.67 |
36.94 |
2.29 |
Vehicle |
50uL |
204 |
181 |
187 |
190.67 |
11.93 |
- |
Solution of test item Lemi code :GGA020117-S2
|
5000ug* |
42 |
39 |
31 |
37.33 |
5.69 |
0.20 |
1500ug* |
85 |
65 |
91 |
80.33 |
13.61 |
0.42 |
|
500ug |
187 |
175 |
169 |
177.00 |
9.17 |
0.93 |
|
150ug |
182 |
172 |
170 |
174.67 |
6.43 |
0.92 |
|
50ug |
221 |
220 |
166 |
202.33 |
31.47 |
1.06 |
* Moderate thinning of the background bacterial lawn.
Applicant's summary and conclusion
- Conclusions:
- The test item tested at doses 5 000, 1 500, 500, 150 and 50 μg/plate do not induce significant increase in the number of revertants in Salmonella typhimurium and in Escherichia coli, either with or without metabolic activation. Therefore the test item was not mutagenic.
- Executive summary:
In accordance with OECD guideline 471 and GLP study, solutions of test item have ben tested for their capacity to induce reverse mutation in four strain of Salmonella typhimurium and one strain of Escherichia coli. This study was performed in absence and in presence of metabolic activation system S9 obtained from rat livery. Assay 1 (direct incorporation) and Assay 2 (pre-incubation) consist of various concentrations of stock solution were tested. Stock solution was prepared at 100 mg/ml in DMSO. Negative and positive controls were carried out as well. Based of the preliminary test on strain TA100 which showed in presence of 5 000 μg/plate one toxicity consistent with the maximum tolerated 75%, a supplementary dose at 4 000 μg/plate was added to be sure this hight toxicity no hindering the scoring of the revertants. Assay 1 was tested in doses 5 000, 4000, 1 500, 500, 150 and 50 μg/plate and Assay 2 at doses 5 000, 1 500, 500, 150 and 50 μg/plate. Plates were incubated at 37ºC for 48-72 h and after this period colonies were counted. There was no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions without and with metabolic activation. In the presence of the highest dose tested a moderate thinning of the background was observed in strains (TA1535,TA98, TA100). It was consistent with the toxicity measured at this dose. The test item at tested doses do not induce significant increase of revertants in Salmonella typhimurium nor in Escherichia coli, either with and without metabolic activation. Test item is not mutagenic.
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