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EC number: 210-047-0 | CAS number: 603-52-1
- 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 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 diphenylcarbamate
- EC Number:
- 210-047-0
- EC Name:
- Ethyl diphenylcarbamate
- Cas Number:
- 603-52-1
- Molecular formula:
- C15H15NO2
- IUPAC Name:
- ethyl N,N-diphenylcarbamate
- Test material form:
- solid: particulate/powder
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. non hygroscopic
- Stability under test conditions: stable under normal storage conditions
- Solubility and stability of the test substance in the solvent/vehicle: Solutions are prepared. A stock solution for each assay was prepared at 100 mg/ml in DMSO. The item is completely soluble. Stability: prepared extemporaneously.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no
Method
- Target gene:
- Histidine-requiring gene for Salmonella Typhinurium and Tryptophane-requiring gene for Escherochia Coli.
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- other: Δuvr B, rfa, pKM 101
- Species / strain / cell type:
- S. typhimurium TA 1537
- Additional strain / cell type characteristics:
- other: Δuvr B, rfa
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- other: Δuvr B, rfa, pKM 101
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- other: Δuvr B, rfa
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Additional strain / cell type characteristics:
- other: Δuvr B, pKM 101
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction prepared from Sprague Dawley rat liver homogenate (10% v/v)
- Test concentrations with justification for top dose:
- Doses of Assay 1: 5.000, 4.000; 1.500, 500, 150 and 50 µg/plate; Doses of Assay 2: 5.000, 1.500, 500, 150 and 50 µg/plate.
The preliminaty citotoxicity 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. - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Solvent is compatible with the survival of the bacteria and the S9 activity.
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- 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:
Assay nº1: For the first assay direct incorporation method is used. Various concentrations of test item were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)); Assay nº2: various concentrations of test item were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system.
Assay without metabolic activation: Salmonella Typhimurium strains: for each strain, 0.1 mL of the bacterial suspension containing 1-9x10^9 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar, maintained supercooled at 45ºC, containing 10% (v/v) of a L-Histidine-D-Biotine solucion (0.5 mM). - Escherichia coli strain: in a test tube 0.1 mL of the bacterial suspension containing 1-9x10^9 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled in 45ºC containing 5% (v/v) of nutrient broth nº 2 to which are added 5µL of a L-Tryptophane solution at 2 mg/mL. Plates are incubated at 37ºC over a 48-72 hour period. The number of revertant colonies per plate is counted. Moreover the following controls are carried out: Negative controls: Absolute negative control containing no test item corresponding to the spontaneous reversion rate, solvent used to solubilize positive controls: NaCL 0.9%, Acetone, DMSO; vehicle used to solubilize test item: DMSO; positive control.
Assay with metabolic activation: two protocols can be used: either a standar plate incorporation method where the protocol is similar to that descrived above, except that, 500 µL of S9-mix fraction is quickly added, before pouring the mixture onto the plates; or the pre-incubation assay where the solution of the test item solution with the test strain, and 500 µL of S9-mix fraction are preincubated with shaking for 30 min., at 37ºC prior to mixing with the overlay agar and pouring onto the minimal agar plate.
Presentation of data: After a 48-72 hour incubation period at 37ºC, revertant colonies are counted in each plate. Data are presented as the number of revertant colonies (mean +/- standard deviation) per plate. The following ratio is calculated:
R= number of revertant colonies in the presence of the test item / number of revertant colonies in the absence of the test item.
- Cell density at seeding: 1-9x10^9 bacteria/mL
DURATION
- Preincubation period: 30 min. at 37ºC (Assay 2)
- Exposure duration:48-72 hour incubation period at 37ºC
SELECTION AGENT (mutation assays):
Salmonella Typhomurium strains: lack of Histidine in the media.
Escherichia coli: lack of Tryptophane in the media.
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity:
Preliminary cytotoxicity testing (strain TA100): in a test tube 0.1 mL of the bacterial suspension (1-9x10^3 bacteria/mL) and 0.1 mL of the stock solution and dilutions, are successively added to 2 mL of top agar at 45ºC, 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). 3 plates per concentration are incubated for 48-72 hours at 37ºC, and the colonies counted, A negative control containing the blank alone is run in parallel.
In case becteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75% or less. The precipitate, if present, should not interfere with the scoring. The following four dilutions studied are distributed according to a semi-logarithmic progression.
OTHER EXAMINATIONS:
Sterility tests: test item and the corresponding dilutions are added to 2 mL of top agar maintained at 45ºC, 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 37ºC and then examined. There should be no bacterial growth on any plate. S9-mix sterility is checked using the same protocol.
Control of strains: The genotype of bacterial strains is checked: Histidine and tryptophane requirements with cultures in presenca and in absence of L-histidine and L-triptophane for Salmonella typhimurium and Escherichia coli strains respectively; loss of cell wall LPS (rfa mutation) measuring crystal violet inhibition for Salmonella typhimurium strains; Ampicillin resistance for the strains which have the pKM 101 plasmide; uvr Bmutation i.e. U.V.B sensitivity for Salmonella typhimurium and uvr A mutation i.e. U.V.A sensitivity for Escherichia coli; spontaneous revertant rate; sensitivity to reference mutagens.
- OTHER:
Solutions preparation: a stock solution for each assay was prepared at 100 mg/mL in DMSO. The highest dose tested was 5000 µg/plate. Solution for assay nº1= LEMI code: GGA191216-S2; Aspect-Color: clear colorless; Solubility: completely soluble; Stability: prepared extemporaneously; pH: NA. Solution for assay nº2: LEMI code: GGA020117-S2; Aspect-Color: clear colorless; Solubility: completely soluble; Stability: prepared extemporaneously; pH: NA.
Preparation of the metabolic activation system: S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provide by MOLTOX (POB Box 1189 - 457 Industrial Park Dr - Boone, NC 28607 - USA) (S9 Moltox-11101-5-3607 validated on 04.2016 - expiry date: 24.03.2018). Preparation of S9-mix 10% (v/v). For final concentration of co-factors and salts see Table 2.
Assay repetition: if the first assay is positive, the second one is performed in the same manner. If the fist assay, in presence of test item, is negative, the pre-incubation test is performed for the second assay. - Rationale for test conditions:
- Results of sterility controls show the absence of any bacterial growth in the presence of the various concentrations of the test item and in the presence of S9-mix.
Results of the bacteriostatic activity control show toxicity consistent with the maximum tolerated 75% in presence of 5000 µg/plate. A supplementary dose at 4000 µg/plate (80% of the maximal dose) is added on the first mutagenic assay to be sure this high toxicity no hindering the scoring of the revertants.
Values and frequency ranges from the laboratory's historical control. - Evaluation criteria:
- Criteria conclusion: 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 (uvr A) (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 itm 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.
All results must be confirmed in an independent experiment.
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 of the test item was 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%. The highest dose used in the preliminary study was also used for main experiments and 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
- Positive historical control data: Without metabolic activation: TA1535 (n 975) = 702.3 +/- 203.4 (mean), 190.0 / 1481.0 (min / max); TA1537 (n 975) = 851.2 +/- 428.1 (mean), 219.0 / 1967.0 (min / max); TA 98 (n 975) = 512.6 +/- 219.5 (mean), 187.0 / 1667.0 (min / max); TA 100 (n 975) = 934.4 +/- 325.2 (mean), 381.0 / 1690.0 (min / max); WP2 (pKM101) (uvr A) (n 717) = 502.8 +/- 168.1 (mean), 248.0 / 1089.0 (min / max). With metabolic actibation (without pre-incubation): TA 1535 (n 525) = 104.0 +/- 53.0 (mean), 26.0 / 269.0 (min / max); TA 1537 (n525) = 55.8 +/- 23.4 (mean), 24.0 / 170.0 (min / max); TA 98 (n 525) = 574.5 +/- 209.5 (mean), 219.0 / 1499.0 (min / max); TA 100 (n 522) = 862.1 +/- 359.1 (mean), 361.0 / 2163.0 (min / max); WP2 (pKM101) (uvr A) (n 372) = 707.3 +/- 248.0 (mean), 378.0 / 1680.0 (min / max). With metabolic activation (with pre-incubation): TA 1535 (n 519) = 73.2 +/- 34.9 (mean), 25.0 / 185.0 (min / max); TA 1537 (n519) = 49.5 +/- 22.5 (mean), 21.0 / 182.0 (min / max); TA 98 (n 519) = 474.6 +/- 196.5 (mean), 174.0 / 1370.0 (min / max); TA 100 (n 522) = 682.4 +/- 290.0 (mean), 309.0 / 1889.0 (min / max); WP2 (pKM101) (uvr A) (n 372) = 701.8 +/- 229.7 (mean), 397.0 / 1680.0 (min / max).
- Negative (solvent/vehicle) historical control data: Without metabolic activation: TA1535 (n 975) = 10.9 +/- 3.6 (mean), 4.0 / 23.0 (min / max); TA1537 (n 975) = 6.0 +/- 2.4 (mean), 1.0 / 14.0 (min / max); TA 98 (n 975) = 16.0 +/- 3.8 (mean), 6.0 / 29.0 (min / max); TA 100 (n 975) = 62.2 +/- 14.3 (mean), 41.0 / 126.0 (min / max); WP2 (pKM101) (uvr A) (n 717) = 75.0 +/- 29.2 (mean), 41.0 / 188.0 (min / max). With metabolic actibation (without pre-incubation): TA 1535 (n 525) = 12.3 +/- 4.0 (mean), 3.0 / 23.0 (min / max); TA 1537 (n525) = 8.0 +/- 3.1 (mean), 1.0 / 24.0 (min / max); TA 98 (n 525) = 23.2 +/- 4.8 (mean), 12.0 / 38.0 (min / max); TA 100 (n 522) = 101.7 +/- 22.9 (mean), 58.0 / 189.0 (min / max); WP2 (pKM101) (uvr A) (n 372) = 154.8 +/- 33.6 (mean), 80.0 / 264.0 (min / max). With metabolic activation (with pre-incubation): TA 1535 (n 519) = 12.7 +/- 4.2 (mean), 5.0 / 25.0 (min / max); TA 1537 (n519) = 8.3 +/- 3.2 (mean), 1.0 / 19.0 (min / max); TA 98 (n 519) = 23.3 +/- 5.2 (mean), 11.0 / 36.0 (min / max); TA 100 (n 522) = 101.3 +/- 24.8 (mean), 51.0 / 189.0 (min / max); WP2 (pKM101) (uvr A) (n 372) = 157.5 +/- 35.4 (mean), 69.0 / 250.0 (min / max).
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects of the test item were noted in any of the five tested strains used up to the highest dose (with and without metabolic activation) in assay 1 and 2.
Any other information on results incl. tables
Sterility control: the colony number in the presence of the various concentrations of the test item and in the presence of S9 -mix for every replicate = 0.
Bacteriostatic activity control nº1
|
|
Doses (/plate) |
|||||||
|
|
0 (negative control) |
DMSO |
50µg |
150µg |
500µg |
1500µg |
2500µg |
5000µg |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA081216-S2 |
N1
N2
N3
N
% |
259
288
304
284+23
- |
311
307
255
291+31
103% |
279
311
301
297+16
105% |
304
309
287
300+12
106% |
298
297
284
293+8
103% |
261
272
304
279+22
98% |
279
278
302
286+14
101% |
68
74
76
73+4
26% |
Bacteriostatic activity control nº2
|
|
Doses (/plate) |
|||||
|
|
0 (negative control) |
DMSO |
2500µg |
3000µg |
4000µg |
5000µg |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA121216-S1 |
N1
N2
N3
N
% |
388
330
394
371+35
- |
428
329
401
386+51
104% |
341
334
370
348+19
94% |
358
301
326
328+29
89% |
205
229
158
197+36
53% |
89
88
95
91+4
24% |
Bacteriostatic activity control nº3
|
|
Doses (/plate) |
|||||||
|
|
0 (negative control) |
DMSO |
50µg |
150µg |
500µg |
1500µg |
2500µg |
5000µg |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
N1
N2
N3
N
% |
221
241
223
228+11
- |
220
241
248
236+15
104% |
263
246
238
249+13
109% |
232
234
198
221+20
97% |
248
245
231
241+9
106% |
178
218
168
188+26
82% |
150
148
139
146+6
64% |
59
64
66
63+4
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
TA 1535 – Assay nº1 – without metabolic activation (-S9-mix)
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100µL |
7 |
6 |
7 |
6.67 |
0.58 |
- |
Positive control solvent |
5µL |
6 |
8 |
6 |
6.67 |
1.15 |
- |
Positive control: Sodium azide |
5µg in 5µL |
513 |
616 |
583 |
570.67 |
52.60 |
85.60 |
Vehicle |
50µL |
8 |
10 |
5 |
7.67 |
2.52 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000µg
4000µg
1500µg
500µg
150µg
50µg |
2
10
9
4
14
6 |
2
18
8
11
8
14 |
2
5
6
6
8
10 |
2.00
11.00
7.67
7.00
10.00
10.00 |
0.00
6.56
1.53
3.61
3.46
4.00 |
0.26
1.43
1.00
0.91
1.30
1.30 |
TA 1535 – Assay nº1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100µL |
14 |
8 |
5 |
9.00 |
4.58 |
- |
Positive control solvent |
20µL |
7 |
6 |
5 |
6.00 |
1.00 |
- |
Positive control: Sodium azide |
2µg in 20µL |
90 |
71 |
61 |
74.00 |
14.73 |
12.33 |
Vehicle |
50µL |
15 |
8 |
11 |
11.33 |
3.51 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000µg
4000µg
1500µg
500µg
150µg
50µg |
5
5
5
10
15
17 |
5
6
13
9
21
13 |
5
5
9
10
16
6 |
5.00
5.33
9.00
9.67
17.33
12.00 |
0.00
0.58
4.00
0.58
3.21
5.57 |
0.44
0.47
0.79
0.85
1.53
1.06 |
TA 1535 – Assay nº2 – without metabolic activation (-S9-mix)
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100µL |
14 |
16 |
7 |
12.33 |
4.73 |
- |
Positive control solvent |
5µL |
11 |
12 |
12 |
11.67 |
0.58 |
- |
Positive control: Sodium azide |
5µg in 5µL |
735 |
642 |
692 |
689.67 |
46.54 |
59.11 |
Vehicle |
50µL |
14 |
14 |
16 |
14.67 |
1.15 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000µg
1500µg
500µg
150µg
50µg |
7
15
8
14
17 |
12
10
7
13
8 |
15
11
13
12
12 |
11.33
12.00
9.33
13.00
12.33 |
4.04
2.65
3.21
1.00
4.51 |
0.77
0.82
0.64
0.89
0.84 |
TA 1535 – Assay nº2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100µL |
13 |
17 |
11 |
13.67 |
3.06 |
- |
Positive control solvent |
10µL |
12 |
12 |
10 |
11.33 |
1.15 |
- |
Positive control: Sodium azide |
1µg in 10µL |
64 |
49 |
67 |
60.00 |
9.64 |
5.29 |
Vehicle |
50µL |
14 |
16 |
14 |
14.67 |
1.15 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000µg*
1500µg*
500µg
150µg
50µg |
6
5
10
23
9 |
10
3
15
14
10 |
1
3
20
14
10 |
5.67
3.67
15.00
17.00
9.67 |
4.51
1.15
5.00
5.20
0.58 |
0.39
0.25
1.02
1.16
0.66 |
*Moderate thinning of the background bacterial lawn
TA 1537 – Assay nº1 – without metabolic activation (-S9-mix)
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100µL |
5 |
5 |
7 |
5.67 |
1.15 |
- |
Positive control solvent |
20µL |
4 |
3 |
6 |
4.33 |
1.53 |
- |
Positive control: Sodium azide |
50µg in 20µL |
1055 |
1110 |
933 |
1032.67 |
90.59 |
238.31 |
Vehicle |
50µL |
4 |
5 |
2 |
3.67 |
1.53 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000µg
4000µg
1500µg
500µg
150µg
50µg |
2
2
4
5
6
6 |
2
5
3
5
6
3 |
1
5
4
5
3
2 |
1.67
4.00
3.67
5.00
5.00
3.67 |
0.58
1.73
0.58
0.00
1.73
2.08 |
0.45
1.09
1.00
1.36
1.36
1.00 |
TA 1537 – Assay nº1 – with metabolic activation (10% S9-mix) – without pre-incubation
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100µL |
7 |
4 |
6 |
5.67 |
1.53 |
- |
Positive control solvent |
20µL |
12 |
8 |
10 |
10.00 |
2.00 |
- |
Positive control: Sodium azide |
2µg in 20µL |
25 |
39 |
30 |
31.33 |
7.09 |
3.13 |
Vehicle |
50µL |
5 |
6 |
6 |
5.67 |
0.58 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000µg
4000µg
1500µg
500µg
150µg
50µg |
2
1
3
3
9
13 |
3
1
5
6
10
9 |
1
2
3
9
8
10 |
2.00
1.33
3.67
6.00
9.00
10.67 |
1.00
0.58
1.15
3.00
1.00
2.08 |
0.35
0.24
0.65
1.06
1.59
1.88 |
TA 1537 – Assay nº2 – without metabolic activation (-S9-mix)
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100µL |
7 |
4 |
4 |
5.00 |
1.73 |
- |
Positive control solvent |
20µL |
3 |
2 |
4 |
3.00 |
1.00 |
- |
Positive control: Sodium azide |
50µ in 20µL |
1716 |
1860 |
1415 |
1663.67 |
227.07 |
554.56 |
Vehicle |
50µL |
3 |
2 |
2 |
2.33 |
0.58 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000µg
1500µg
500µg
150µg
50µg |
5
5
3
2
3 |
4
5
2
2
2 |
3
3
1
2
4 |
4.00
4.33
2.00
2.00
3.00 |
1.00
1.15
1.00
0.00
1.00 |
1.71
1.86
0.86
0.86
1.29 |
TA 1537 – Assay nº2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100µL |
8 |
5 |
9 |
7.33 |
2.08 |
- |
Positive control solvent |
10µL |
2 |
3 |
5 |
3.33 |
1.53 |
- |
Positive control: Sodium azide |
1µg in 10µL |
28 |
25 |
22 |
25.00 |
3.00 |
7.50 |
Vehicle |
50µL |
5 |
4 |
3 |
4.00 |
1.00 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000µg
1500µg
500µg
150µg
50µg |
5
7
7
7
4 |
5
5
4
6
5 |
4
6
5
6
7 |
4.67
6.00
5.33
6.33
5.33 |
0.58
1.00
1.53
0.58
1.53 |
1.17
1.50
1.33
1.58
1.33 |
TA 98 – Assay nº1 – without metabolic activation (-S9-mix)
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
22 |
11 |
22 |
18.33 |
6.35 |
- |
Positive control solvent |
20 µL |
22 |
25 |
18 |
21.67 |
3.51 |
- |
Positive control: Sodium azide |
2 µg in 20 µL |
191 |
229 |
217 |
212.33 |
19.43 |
9.80 |
Vehicle |
50 µL |
35 |
24 |
16 |
25.00 |
9.54 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000* µg
4000* µg
1500 µg
500 µg
150 µg
50 µg |
27
15
22
11
26
18 |
23
25
26
24
19
18 |
24
19
24
32
28
21 |
24.67
19.67
24.00
22.33
24.33
19.00 |
2.08
5.03
2.00
10.60
4.73
1.73 |
0.99
0.79
0.96
0.89
0.97
0.76 |
*Moderate thinning of the background bacterial lawn
TA 98 – Assay nº1 – with metabolic activation (10% S9-mix) – without pre-incubation
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
18 |
22 |
23 |
21.00 |
2.65 |
- |
Positive control solvent |
20 µL |
30 |
23 |
34 |
29.00 |
5.57 |
- |
Positive control: Sodium azide |
2 µg in 20 µL |
253 |
233 |
239 |
241.67 |
10.26 |
8.33 |
Vehicle |
50 µL |
24 |
26 |
19 |
23.00 |
3.61 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000* µg
4000* µg
1500 µg
500 µg
150 µg
50 µg |
26
26
18
30
31
35 |
16
15
16
33
36
31 |
22
19
18
26
24
26 |
21.33
20.00
17.33
29.67
30.33
30.67 |
5.03
5.57
1.15
3.51
6.03
4.51 |
0.93
0.87
0.75
1.29
1.32
1.33 |
*Moderate thinning of the background bacterial lawn
TA 98 – Assay nº2 – without metabolic activation (-S9-mix)
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
27 |
13 |
22 |
20.67 |
7.09 |
- |
Positive control solvent |
20 µL |
17 |
17 |
16 |
16.67 |
0.58 |
- |
Positive control: Sodium azide |
20 µg in 20 µL |
252 |
266 |
312 |
276.67 |
31.39 |
16.60 |
Vehicle |
50 µL |
21 |
20 |
20 |
20.33 |
0.58 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000* µg
1500* µg
500 µg
150 µg
50 µg |
7
16
14
28
22 |
19
16
17
18
13 |
15
14
14
13
17 |
13.67
15.33
15.00
19.67
17.33 |
6.11
1.15
1.73
7.64
4.51 |
0.67
0.75
0.74
0.97
0.85 |
*Moderate thinning of the background bacterial lawn
TA 98 – Assay nº2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
28 |
26 |
26 |
26.67 |
1.15 |
- |
Positive control solvent |
10 µL |
19 |
16 |
32 |
22.33 |
8.50 |
- |
Positive control: Sodium azide |
1 µg in 10 µL |
254 |
259 |
237 |
250.00 |
11.53 |
11.19 |
Vehicle |
50 µL |
31 |
24 |
26 |
27.00 |
3.61 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000* µg
1500* µg
500 µg
150 µg
50 µg |
8
12
14
11
12 |
9
9
29
18
14 |
6
13
16
22
23 |
7.67
11.33
19.67
17.00
16.33 |
1.53
2.08
8.14
5.57
5.86 |
0.28
0.42
0.73
0.63
0.60 |
*Moderate thinning of the background bacterial lawn
TA 100 – Assay nº1 – without metabolic activation (-S9-mix)
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
54 |
53 |
51 |
52.67 |
1.53 |
- |
Positive control solvent |
20 µL |
57 |
52 |
50 |
53.00 |
3.61 |
- |
Positive control: Sodium azide |
20 µg in 20 µL |
1334 |
1134 |
1256 |
1241.33 |
100.80 |
23.42 |
Vehicle |
50 µL |
74 |
65 |
53 |
64.00 |
10.54 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000* µg
4000* µg
1500 µg
500 µg
150 µg
50 µg |
42
29
49
72
76
68 |
47
30
52
62
60
55 |
30
26
65
73
67
73 |
39.67
28.33
55.33
69.00
67.67
65.33 |
8.74
2.08
8.50
6.08
8.02
9.29 |
0.62
0.44
0.86
1.08
1.06
1.02 |
*Moderate thinning of the background bacterial lawn
TA 100 – Assay nº1 – with metabolic activation (10% S9-mix) – without pre-incubation
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
71 |
72 |
91 |
78.00 |
11.27 |
- |
Positive control solvent |
20 µL |
72 |
73 |
72 |
72.33 |
0.58 |
- |
Positive control: Sodium azide |
2 µg in 20 µL |
392 |
373 |
395 |
386.67 |
11.93 |
5.35 |
Vehicle |
50 µL |
81 |
85 |
75 |
80.33 |
5.03 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000* µg
4000* µg
1500 µg
500 µg
150 µg
50 µg |
27
54
71
84
84
72 |
21
24
87
79
72
79 |
30
46
72
70
70
70 |
26.00
41.33
76.67
77.67
75.33
73.67 |
4.58
15.53
8.96
7.09
7.57
4.73 |
0.32
0.51
0.95
0.97
0.94
0.92 |
*Moderate thinning of the background bacterial lawn
TA 100 – Assay nº2 – without metabolic activation (-S9-mix)
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
54 |
67 |
60 |
60.33 |
6.51 |
- |
Positive control solvent |
20 µL |
60 |
51 |
60 |
57.00 |
5.20 |
- |
Positive control: Sodium azide |
20 µg in 20 µL |
1531 |
1505 |
1569 |
1535.00 |
32.19 |
26.93 |
Vehicle |
50 µL |
46 |
48 |
55 |
49.67 |
4.73 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000* µg
1500 µg
500 µg
150 µg
50 µg |
46
57
43
66
64 |
48
57
62
60
56 |
42
48
65
58
49 |
45.33
54.00
56.67
61.33
56.33 |
3.06
5.20
11.93
4.16
7.51 |
0.91
1.09
1.14
1.23
1.13 |
*Moderate thinning of the background bacterial lawn
TA 100 – Assay nº2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
120 |
95 |
88 |
101.00 |
16.82 |
- |
Positive control solvent |
10 µL |
76 |
78 |
75 |
76.33 |
1.53 |
- |
Positive control: Sodium azide |
1 µg in 10 µL |
356 |
390 |
370 |
372.00 |
17.09 |
4.87 |
Vehicle |
50 µL |
73 |
72 |
79 |
74.67 |
3.79 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000* µg
1500 µg
500 µg
150 µg
50 µg |
10
65
69
82
83 |
15
70
81
75
73 |
18
65
69
71
72 |
14.33
66.67
73.00
76.00
76.00 |
4.04
2.89
6.93
5.57
6.08 |
0.19
0.89
0.98
1.02
1.02 |
*Moderate thinning of the background bacterial lawn
E. COLI – Assay nº1 – without metabolic activation (-S9-mix)
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
107 |
109 |
105 |
107.00 |
2.00 |
- |
Positive control solvent |
10 µL |
131 |
109 |
99 |
113.00 |
16.37 |
- |
Positive control: Sodium azide |
1 µg in 10 µL |
310 |
324 |
341 |
325.00 |
15.52 |
2.88 |
Vehicle |
50 µL |
92 |
102 |
105 |
99.67 |
6.81 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000 µg
4000 µg
1500 µg
500 µg
150 µg
50 µg |
52
52
73
98
134
132 |
35
55
70
113
129
131 |
30
59
90
120
144
119 |
39.00
55.33
77.67
110.33
135.67
127.33 |
11.53
3.51
10.79
11.24
7.64
7.23 |
0.39
0.56
0.78
1.11
1.36
1.28 |
E. COLI – Assay nº1 – with metabolic activation (10% S9-mix) – without pre-incubation
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
182 |
158 |
171 |
170.33 |
12.01 |
- |
Positive control solvent |
5 µL |
152 |
124 |
140 |
138.67 |
14.05 |
- |
Positive control: Sodium azide |
5 µg in 5 µL |
424 |
503 |
436 |
454.33 |
42.57 |
3.28 |
Vehicle |
50 µL |
123 |
128 |
143 |
131.33 |
10.41 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000 µg
4000 µg
1500 µg
500 µg
150 µg
50 µg |
62
115
105
161
180
176 |
53
84
106
162
171
160 |
42
76
124
158
169
147 |
52.33
91.67
111.67
160.33
173.33
161.00 |
10.02
20.60
10.69
2.08
5.86
14.53 |
0.40
0.70
0.85
1.22
1.32
1.23 |
E. COLI – Assay nº2 – without metabolic activation (-S9-mix)
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
137 |
144 |
122 |
134.33 |
11.24 |
- |
Positive control solvent |
10 µL |
125 |
156 |
128 |
136.33 |
17.10 |
- |
Positive control: Sodium azide |
1 µg in 10 µL |
345 |
332 |
328 |
335.00 |
8.89 |
2.46 |
Vehicle |
50 µL |
108 |
129 |
99 |
112.00 |
15.39 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000 µg
1500 µg
500 µg
150 µg
50 µg |
134
123
133
133
141 |
113
133
134
144
126 |
131
131
138
118
116 |
126.00
129.00
135.00
131.67
127.67 |
11.36
5.29
2.65
13.05
12.58 |
1.13
1.15
1.21
1.18
1.14 |
E. COLI – Assay nº2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie |
Dose/Plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
215 |
198 |
186 |
199.67 |
14.57 |
- |
Positive control solvent |
5 µL |
211 |
192 |
204 |
202.33 |
9.61 |
- |
Positive control: Sodium azide |
2.5 µg in 5 µL |
447 |
438 |
506 |
463.67 |
36.94 |
2.29 |
Vehicle |
50 µL |
204 |
181 |
187 |
190.67 |
11.93 |
- |
Solution of Ethyl ethyl(phenyl)carbamate (CAS number 1013-75-8) BATCH: 1000106375
LEMI code: GGA191216-S2 |
5000* µg
1500* µg
500 µg
150 µg
50 µg |
42
85
187
182
221 |
39
65
175
172
220 |
31
91
169
170
166 |
37.33
80.33
177.00
174.67
202.33 |
5.69
13.61
9.17
6.43
31.47 |
0.20
0.42
0.93
0.92
1.06 |
*Moderate thinning of the background bacterial lawn
Applicant's summary and conclusion
- Conclusions:
- The test item do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 with and without metabolic activation up to 5000 µg/plate.
- Executive summary:
The determination of the mutagenic potential of the test item has been assessed by the bacterial reverse mutation test, performed according to OECD 471 method and under GLP conditions. A preliminary study showed no toxicity up to 5000 µg/plate. A stock solution of the test item was prepared at 100 mg/mL and various concentrations of the test item (5000, 4000, 1500, 500, 150 and 50 µg/plate) were put in contact with the strains TA 1535, TA 1537, TA 98, TA 100 of Salmonella typhimurium and Escherichia coli WP2 (uvrA-) (pKM 101), with and without metabolic activation. Two independent assays were performed. For assay nº1, the different concentratrions of the test item were put in contact with the strains in the absence and presence of a metabolic activation system S9 -mix 10% (v/v) for 48 - 72 h at 37ºC. For assay nº2, the different concentrations of the test item were put in contact with the strains in absence of metabolic activation and with pre-incubation in the presence of metabolic activation system. For both assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no evidence of any increase in the number of revertant colonies in the presence of various concentrations of the test item with and without metabolic activation in the strains tested. The different doses prepared from solutions of the test item, do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 (uvrA-) (pKM 101) with or without metabolic activation.
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Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.