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Diss Factsheets

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:
2017
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

Constituent 1
Chemical structure
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
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.