Ethyl ethyl(phenyl)carbamate

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:

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

Test material information

Constituent 1

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

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 / strain

Species / strain 1

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 2

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.