N,N'-(ethoxymethylsilylene)bis[N-methylbenzamide]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key study: Study according to OECD 471. GLP study. 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.

Key study: Study according to OECD 473. GLP study. The test item does not induce chromosomal aberration in human lymphocytes. Therefore, the test item can be considered as non-clastogenic and has no genotoxic potential.

Key study: Study according to OECD 490. GLP study. the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Link to relevant study records

Reference

Reference 1

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

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature.

Target gene:

his D (Salmonella typhimurium TA 98)
his C (Salmonella typhimurium TA 1537)
his G (Salmonella typhimurium TA 100 and TA1535)
tryp E (Escherichia coli WP2 uvrA pKM101)

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: Δuvr B, rfa

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: Δuvr B, rfa

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 100

Additional strain / cell type characteristics:

other: Δuvr B, rfa, pKM 101

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: Δuvr A, pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 μg/plate. The preliminary study showed no toxicity of the test item up to 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: the item is completely soluble in the vehicle

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

NaCl 0.9%, Acetone, DMSO, Ethanol

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 1:in agar (plate incorporation) with and without metabolic activation
A stock solution of the test item was prepared at 100 mg / mL. In a test tube, 0.1 mL of the bacterial suspension containing 1-9x109 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 super cooled in 45ºC containing 5% (v/v) of a L-Histidine-D-Biotine solution (0.5 mM) for Salmonella Typhimurium strains or containing 5% (v/v) of nutrient broth nº2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL for Escherichia coli strain.
In the assay with metabolic activation, either a standard plate incorporation method where the protocol is similar to the described above, except that, 500 μL of S9-mix fraction is quickly added, before pouring the mixture onto the plates.
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

Assay 2: preincubation with and without metabolic activation
A stock solution of the test item was prepared at 100 mg / mL. The assay without metabolic activation was performed as mendioned in assay 1.
In the assay with metabolic activation, 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.
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, and the ratio is calculate as mentioned before.
If the first assay is positive, the second one is performed in the same manner.
If the first assay, in presence of the test item, is negative, the pre-incubation test is performed for the second assay.

- Cell density at seeding (if applicable): 1:9x109 bacteria/mL

DURATION
- Preincubation period: 30 min at 37ºC (Assay 2)
- Exposure duration: 48-72 hours at 37ºC

SELECTION AGENT (mutation assays):
Salmonella Thyphimuriun 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 test (Strain TA100): In a test tube 0.1 mL of the bacterial suspension (1-9 x103 bacteria /mL) and 0.1 mL of the stock 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 h at 37ºC, and the colonies counted. A negative control containing the blank alone is run in parallel. In case of bacteriostatic activity
is detected, the highest concentration to be retined 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:
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.

PREPARATION OF THE METABOLIC ACTIVATION SYSTEM:
Obtention of S9 fraction: S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOX (POB Box 1189 - 157 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): The final concentration of co-factors and salts is as follows: S9 fraction 10%; MgCl2-6H2O 8 mM; KCl 33 mM; Glucose-6-Phophate Na2 5 mM; NADP Na2 4mM; Phosphate buffer pH 7.4 0.1 M.

CONTROL OF STRAINS
- Histidine and tryptophane requirements with cultrues in presence and in absence of L-histidine and L-triptophane for Samonella 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.
- Δuvr B mutation i.e. U.V.B. sensitivity for Salmonella typhimurium and Δuvr A mutation i.e. U.V.A. sensitivity for Escherichia coli.
- Spontaneous revertant rate.
- Sensitivity to reference mutagens.

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.
Values and frequency ranges from the laboratory's historical control

Evaluation criteria:

The result of the test is considered as negative if the revertant number is below 3-fold the number of spontaneous reversions for TA 1535 and TA 1537 strains, and below 2-fold the number of spontaneous reversions for TA 98, TA 100 and E. coli WP2 (uvrA-) (pKM 101) strains with and without metabolic activation.
The result of the test is considered as 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 2-fold that of spontaneous revertant colonies for TA 98, TA 100 and E. coli WP2 (uvrA-) (pKM 101), and 3-fold for TA 1535 and TA 1537.

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:
Neither original solution nor dilutions have bacteriostatic effect. The test item is tested at these doses: 5000, 1500, 500, 150 and 50 μg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: (2007-2016)
S. typhimurium TA 1535: without metab. activation (Sodium Azide): n= 975; 702.3 ± 203.4 (mean ± SD); 190 / 1481 (min / max); with metab. activation (without pre-incubation) (2-Anthramine): n= 525; 104.0 ± 53.0 (mean ± SD); 26 / 269 (min / max); with metab. activation (with pre-incubation) (2-Anthramine): n= 519; 73.2 ± 34.9 (mean ± SD); 25 / 185 (min / max)
S. typhimurium TA 1537: without metab. activation (9-Aminoacridine): n=975; 851.2 ± 428.1 (mean ± SD); 219 / 1967 (min / max); with metab. activation (without pre-incubation) (2-Anthramine): n= 525; 55.8 ± 23.4 (mean ± SD); 24 / 170 (min / max); with metab. activation (with pre-incubation) (2-Anthramine): n= 519; 49.5 ± 22.5 (mean ± SD); 21 / 182 (min / max)
S. typhimurium TA 98: without metab. activation (2-Nitrofluorene): n= 975; 512.6 ± 219.5 (mean ± SD); 187 / 1667 (min / max); with metab. activation (without pre-incubation) (2-Anthramine): n=525; 574.5 ± 209.5 (mean ± SD); 219 / 1499.0 (min / max); with metab. activation (with pre-incubation) (2-Anthramine): n= 519; 474.6 ± 196.5 (mean ± SD); 174 / 1370 (min / max)
S. typhimurium TA 100: without metab. activation (Sodium Azide): n=975; 934.4 ± 325.2 (mean ± SD); 381 / 1690 (min / max); with metab. activation (without pre-incubation) (2-Anthramine): n=522; 862.1 ± 359.1 (mean ± SD); 361 / 2163.0 (min / max); with metab. activation (with pre-incubation) (2-Anthramine): n=522; 682.4 ± 290.0 (mean ± SD); 309 / 1889 (min / max)
E. coli WP2 (pKM 101) (uvr A-): without metab. activation (cis-Platinium (II) Diamine Dichloride): n= 717; 502.8 ± 168.1 (mean ± SD); 248 / 1089 (min / max); with metab. activation (without pre-incubation) (Dimethyl Benzanthracene): n= 372; 707.3 ± 248.0 (mean ± SD); 378 / 1680 (min / max); with metab. activation (with pre-incubation) (Dimethyl Benzanthracene): n=372; 701.8 ± 229.7 (mean ± SD); 397 / 1680 (min / max)

- Negative (solvent/vehicle) historical control data: (2007-2016)
S. typhimurium TA 1535: without metab. activation: n= 975; 10.9 ± 3.6 (mean ± SD); 4 / 23 (min / max); with metab. activation (without pre-incubation): n= 525; 12.3 ± 4 (mean ± SD); 3 / 23 (min / max); with metab. activation (with pre-incubation): n= 519; 12.7 ± 4.2 (mean ± SD); 5 / 25 (min / max)
S. typhimurium TA 1537: without metab. activation: n= 975; 6.0 ± 2.4 (mean ± SD); 1 / 14 (min / max); with metab. activation (without pre-incubation): n= 525, 8.0 ± 3.1 (mean ± SD); 1 / 24 (min / max); with metab. activation (with pre-incubation): n= 519; 8.3 ± 3.2 (mean ± SD); 1 / 19 (min / max)
S. typhimurium TA 98: without metab. activation: n= 975; 16.0 ± 3.8 (mean ± SD); 6 / 29 (min / max); with metab. activation (without pre-incubation): n= 525; 23.2 ± 4.8 (mean ± SD); 12 / 38 (min / max); with metab. activation (with pre-incubation): n= 519; 23.3 ± 5.2 (mean ± SD); 11 / 36 (min / max)
S. typhimurium TA 100: without metab. activation: n= 975; 62.2 ± 14.3 (mean ± SD); 41 / 126 (min / max); with metab. activation (without pre-incubation): n= 522; 101.7 ± 22.9 (mean ± SD); 58 / 189 (min / max); with metab. activation (with pre-incubation): n=519; 101.3 ± 24.8 (mean ± SD); 51 / 189 (min / max)
E. coli WP2 (pKM 101) (uvr A-): without metab. activation: n= 717; 75.0 ± 29.2 (mean ± SD); 41 / 188 (min / max); with metab. activation (without pre-incubation): n= 372; 154.8 ± 33.6 (mean ± SD); 80 / 264 (min / max); with metab. activation (with pre-incubation): n= 372; 157.5 ± 35.4 (mean ± SD); 69 / 250 (min / max)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects of the test item were observed in any of the five tested strains used up to the highest dose (with and without metabolic activation) in assay 1 and 2.

Table 1. Sterility control

Serie	Doses	Colony number/plate
Control nº 1		1	2	3
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA191216-S1)	5000 µg / plate	0	0	0
	1500 µg / plate	0	0	0
	500 µg / plate	0	0	0
	150 µg / plate	0	0	0
	50 µg / plate	0	0	0
S9-mix	500µL / plate	0	0	0
Control nº 2		1	2	3
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA020117-S1)	5000 µg / plate	0	0	0
	1500 µg / plate	0	0	0
	500 µg / plate	0	0	0
	150 µg / plate	0	0	0
	50 µg /plate	0	0	0
S9-mix	500 µL / plate	0	0	0

 

Table 2. Bacteriostatic activity control nº2

		Doses (/plate)
		0 (negative control)	DMSO	50 µg	150 µg	500 µg	1500 µg	2500 µg	5000 µg
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA081216-S4)	N1	259	249	289	334	304	238	303	311
	N2	288	293	302	320	301	297	312	287
	N3	304	308	302	302	218	312	287	260
	N	284±23	283± 31	298±8	319± 16	274± 49	282± 39	301±13	286± 26
	%	-	100%	105%	112%	97%	100%	106%	101%

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 3. Bacteriostatic activity control nº3

		Doses (/plate)
		0 (negative control)	DMSO	50 µg	150 µg	500 µg	1500 µg	5000 µg
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA191216-S1)	N1	221	239	235	239	241	250	239
	N2	241	237	234	235	237	241	230
	N3	223	264	216	185	227	215	237
	N	228± 11	247± 15	228± 11	220± 30	235±7	235±18	235± 5
	%	-	108%	100%	96%	103%	103%	103%

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 4. TA 1535 - Assay nº1 – without metabolic activation (-S9-mix)

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	7	6	7	6.67	0.58	-
Positive control solvent	5 µL	6	8	6	6.67	1.15	-
Positive control: Sodium azide	5 µg  in 5 µL	513	616	583	570.67	52.60	85.60
Vehicle	50 µL	8	7	8	7.67	0.58	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA191216-S1)	5000 µg	4	5	6	5.00	1.00	0.65
	1500 µg	8	17	8	11.00	5.20	1.43
	500 µg	11	9	5	8.33	3.06	1.09
	150 µg	5	7	8	6.67	1.53	0.87
	50 µg	7	5	8	6.67	1.53	0.87

 

Table 5. 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	100 µL	14	8	5	9.00	4.58	-
Positive control solvent	20 µL	7	6	5	6.00	1.00	-
Positive control: 2-Anthramine	2 µg  in 20 µL	90	71	61	74.00	14.73	12.33
Vehicle	50 µL	10	8	7	8.33	1.53	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA191216-S1)	5000 µg	9	9	10	9.33	0.58	1.12
	1500 µg	7	14	14	11.67	4.04	1.40
	500 µg	4	5	8	5.67	2.08	0.68
	150 µg	9	6	5	6.67	2.08	0.80
	50 µg	7	10	13	10.00	3.00	1.20

 

Table 6. TA 1535 - Assay nº2 – without metabolic activation (-S9-mix)

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	14	16	7	12.33	4.73	-
Positive control solvent	5 µL	11	12	12	11.67	0.58	-
Positive control: Sodium azide	5 µg  in 5 µL	735	642	692	689.67	46.54	59.11
Vehicle	50 µL	18	10	14	14.00	4.00	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA020117-S1)	5000 µg	14	10	14	12.67	2.31	0.90
	1500 µg	15	12	18	15.00	3.00	1.07
	500 µg	16	14	13	14.33	1.53	1.02
	150 µg	12	20	17	16.33	4.04	1.17
	50 µg	7	16	14	12.33	4.73	0.88

Table 7. 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	100 µL	13	17	11	13.67	3.06	-
Positive control solvent	10 µL	12	12	10	11.33	1.15	-
Positive control: 2-Anthramine	1 µg  in 10 µL	64	49	67	60.00	9.64	5.29
Vehicle	50 µL	11	19	19	16.33	4.62	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA020117-S1)	5000 µg	10	17	12	13.00	3.61	0.80
	1500 µg	12	15	13	13.33	1.53	0.82
	500 µg	14	15	13	14.00	1.00	0.86
	150 µg	14	15	20	16.33	3.21	1.00
	50 µg	17	15	16	16.00	1.00	0.98

 

Table 8. TA 1537 - Assay nº1 – without metabolic activation (-S9-mix)

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	5	5	7	5.67	1.15	-
Positive control solvent	20 µL	4	3	6	4.33	1.53	-
Positive control: 9-Aminoacridine	50 µg  in 20 µL	1055	1110	933	1032.67	90.59	238.31
Vehicle	50 µL	7	2	9	6.00	3.61	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA191216-S1)	5000 µg	7	4	4	5.00	1.73	0.83
	1500 µg	8	13	12	11.00	2.65	1.83
	500 µg	8	7	8	7.67	0.58	1.28
	150 µg	13	7	11	10.33	3.06	1.72
	50 µg	2	4	7	4.33	2.52	0.72

 

Table 9. 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	100 µL	7	4	6	5.67	1.53	-
Positive control solvent	20 µL	12	8	10	10.00	2.00	-
Positive control: 2-Anthramine	2 µg  in 20 µL	25	39	30	31.33	7.09	3.13
Vehicle	50 µL	7	10	6	7.67	2.08	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA191216-S1)	5000 µg	14	7	14	11.67	4.04	1.52
	1500 µg	10	8	16	11.33	4.16	1.48
	500 µg	12	10	10	10.67	1.15	1.39
	150 µg	12	7	9	9.33	2.52	1.22
	50 µg	3	12	7	7.33	4.51	0.96

 

Table 10. TA 1537 - Assay nº2 – without metabolic activation (-S9-mix)

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	7	4	4	5.00	1.73	-
Positive control solvent	20 µL	3	2	4	3.00	1.00	-
Positive control: 9-Aminoacridine	50 µg  in 20 µL	1716	1860	1415	1663.67	227.07	554.56
Vehicle	50 µL	1	4	5	3.33	2.08	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA020117-S1)	5000 µg	2	6	3	3.67	2.08	1.10
	1500 µg	3	3	2	2.67	0.58	0.80
	500 µg	2	3	3	2.67	0.58	0.80
	150 µg	4	4	3	3.67	0.58	1.10
	50 µg	4	2	3	3.00	1.00	0.90

 

Table 11. 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	100 µL	8	5	9	7.33	2.08	-
Positive control solvent	20 µL	2	3	5	3.33	1.53	-
Positive control: 2-Anthramine	1 µg  in 10 µL	28	25	22	25.00	3.00	7.50
Vehicle	50 µL	6	10	4	6.67	3.06	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA020117-S1)	5000 µg	5	3	4	4.00	1.00	0.60
	1500 µg	3	4	4	3.67	0.58	0.55
	500 µg	5	6	5	5.33	0.58	0.80
	150 µg	6	7	6	6.33	0.58	0.95
	50 µg	5	4	5	4.67	0.58	0.70

 

Table 12. 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: 2-Nitrofluorene	2 µg  in 20 µL	191	229	217	212.33	19.43	9.80
Vehicle	50 µL	19	29	17	21.67	6.43	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA191216-S1)	5000 µg	14	15	23	17.33	4.93	0.80
	1500 µg	26	22	23	23.67	2.08	1.09
	500 µg	22	21	22	21.67	0.58	1.00
	150 µg	24	27	28	26.33	2.08	1.22
	50 µg	28	25	21	24.67	3.51	1.14

Table 13. 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: 2-Anthramine	2 µg  in 20 µL	253	233	239	241.67	10.26	8.33
Vehicle	50 µL	32	24	30	28.67	4.16	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA191216-S1)	5000 µg	21	16	20	19.00	2.65	0.66
	1500 µg	32	36	22	30.00	7.21	1.05
	500 µg	35	32	28	31.67	3.51	1.10
	150 µg	32	31	29	30.67	1.53	1.07
	50 µg	33	33	30	32.00	1.73	1.12

 

Table 14. 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: 2-Nitrofluorene	2 µg  in 20 µL	252	266	312	276.67	31.39	16.60
Vehicle	50 µL	21	20	18	19.67	1.53	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA020117-S1)	5000 µg	10	14	10	11.33	2.31	0.58
	1500 µg	16	17	10	14.33	3.79	0.73
	500 µg	21	18	16	18.33	2.52	0.93
	150 µg	23	22	18	21.00	2.65	1.07
	50 µg	18	25	21	21.33	3.51	1.08

 

Table 15. 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	20 µL	19	16	32	22.33	8.50	-
Positive control: 2-Anthramine	1 µg  in 10 µL	254	259	237	250.00	11.53	11.19
Vehicle	50 µL	32	25	29	28.67	3.51	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA020117-S1)	5000 µg	25	31	32	29.33	3.79	1.02
	1500 µg	25	35	32	30.67	5.13	1.07
	500 µg	37	13	27	25.67	12.06	0.90
	150 µg	24	33	34	30.33	5.51	1.06
	50 µg	20	35	27	27.33	7.51	0.95

Table 16. 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	73	63	54	63.33	9.50	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA191216-S1)	5000 µg	56	52	58	53.33	3.06	0.87
	1500 µg	57	53	60	56.67	3.51	0.89
	500 µg	55	57	52	54.67	2.52	0.86
	150 µg	58	57	52	55.67	3.21	0.88
	50 µg	55	53	48	52.00	3.61	0.82

 

Table 17. 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: 2-Anthramine	2 µg  in 20 µL	392	373	395	386.67	11.93	5.35
Vehicle	50 µL	81	73	71	75.00	5.29	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA191216-S1)	5000 µg	85	88	82	85.00	3.00	1.13
	1500 µg	74	79	90	81.00	8.19	1.08
	500 µg	78	77	76	77.00	1.00	1.03
	150 µg	71	74	93	79.33	11.93	1.06
	50 µg	79	72	71	74.00	4.36	0.99

 

Table 18. 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	64	74	62	66.67	6.43	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA020117-S1)	5000 µg	48	53	44	48.33	4.51	0.73
	1500 µg	47	52	47	48.67	2.89	0.73
	500 µg	48	52	56	52.00	4.00	0.78
	150 µg	49	47	46	47.33	1.53	0.71
	50 µg	57	52	46	51.67	5.51	0.78

Table 19. 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	20 µL	76	78	75	76.33	1.53	-
Positive control: 2-Anthramine	1 µg  in 10 µL	356	390	370	372.00	17.09	4.87
Vehicle	50 µL	71	77	69	72.33	4.16	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA020117-S1)	5000 µg	58	71	69	66.00	7.00	0.91
	1500 µg	61	73	75	69.67	7.57	0.96
	500 µg	76	72	77	75.00	2.65	1.04
	150 µg	73	64	78	71.67	7.09	0.99
	50 µg	74	68	75	72.33	3.79	1.00

 

Table 20. 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: cis-Platinum (II)	1 µg  in 10 µL	310	324	341	325.00	15.52	2.88
Vehicle	50 µL	94	96	96	95.33	1.15	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA191216-S1)	5000 µg	108	131	114	117.67	11.93	1.23
	1500 µg	101	117	128	115.33	13.58	1.21
	500 µg	136	125	119	126.67	8.62	1.33
	150 µg	102	113	120	111.67	9.07	1.17
	50 µg	137	104	131	124.00	17.58	1.30

 

Table 21. 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: Dimethylbenzanthracene	5 µg  in 5 µL	424	503	436	454.33	42.57	3.28
Vehicle	50 µL	175	182	143	166.67	20.79	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA191216-S1)	5000 µg	171	145	155	157.00	13.11	0.94
	1500 µg	141	157	168	155.33	13.58	0.93
	500 µg	173	136	149	152.67	18.77	0.92
	150 µg	155	162	171	162.67	8.02	0.98
	50 µg	154	162	170	162.00	8.00	0.97

 

Table 22. 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: cis-Platinum (II)	1 µg  in 10 µL	345	332	328	335.00	8.89	2.46
Vehicle	50 µL	142	145	125	137.33	10.79	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA020117-S1)	5000 µg	110	127	130	122.33	10.79	0.89
	1500 µg	118	138	111	122.33	14.01	0.89
	500 µg	132	151	147	143.33	10.02	1.04
	150 µg	130	125	111	122.00	9.85	0.89
	50 µg	136	123	134	131.00	7.00	0.95

 

Table 23. 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: Dimethylbenzanthracene	2.5 µg  in 5 µL	447	438	506	463.67	36.94	2.29
Vehicle	50 µL	160	188	201	183.00	20.95	-
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6)  BATCH: 1000106346 (LEMI code: GGA020117-S1)	5000 µg	167	173	182	174.00	7.55	0.95
	1500 µg	159	168	185	170.67	13.20	0.93
	500 µg	171	182	188	180.33	8.62	0.99
	150 µg	181	186	193	186.67	6.03	1.02
	50 µg	178	188	180	182.00	5.29	0.99

 

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.

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, 1500, 500, 150 and 50 μg/plate) were put in contact with the strains TA 1535, TA 1537, TA98, TA100 of Salmonela typhimurium and Escherichia coli WP2 (uvrA-) (pKM 101), with and without metabolic activation. Two independent assays were performed. For assay nº1, the different concetrations 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 the absence of metabolic activation and with pre-incubation in the presence of metablic 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 the various concentration of the test item, with and without metabolic activation in the strains tested. The different doses prepared from solutions of 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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 20, 2017 - February 22, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Biopredic International (France)
- Sex, age and number of blood donors if applicable: Female donor, 33 years old not under medication
- Modal number of chromosomes: 46

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: HAMF10 supplemented with 15% (v/v) of heat inactivated fetal calf serum, 25 IU/ml of penicillin, 25 µg/ml of strptomycin and 25 µg/ml of phytohemagglutinin

Cytokinesis block (if used):

Demecolcin (0.15 microgram/ml)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

93.75, 187.5, 375 and 750 µg/ml.
The highest concentration chosen was the one which does not induce cell death of more than 50%.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: was selected in accordance with the information supplied by the sponsor

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
The culture flasks (25 cm3) containing 4.5 ml of the complete medium were prepared, 0.5 ml of complete blood was added and then the flasks were incubated in an upright position 48 hours ± 2 hours at 37ºC, 5% CO2.

Without metabolic activation (contact time: 4 h 30'): the cells were transferred into sterile tubes and centrifuged. The cell pellets were washed once. After further centrifugation, the pellets were resuspended in 4.5 ml HAS F10 medium with 25 µg/ml of phytohemagglutinin 500 µl of each dilution of the test item, the negative and positive control, in their vehicles, were then added to the cell suspension in order to obtain the desired final concentrations. The tubes were incubated at 37ºC, 5%CO2 for 4 h ± 30 minutes. After incubation, the tubes were centrifuged and the cell pellets were washed twice. After incubation, cell pellets were resuspended in 5 ml of complete medium and then transferred to 25 cm2 culture flasks and incubated for 18 hours ± 1 hour (about 1.5 times the normal cell cycle after the treatment start, that is to say, 22 hours ± hour total) at 37ºC, 5% CO2.

Without metabolic activation (contact time: 22 ± 1 h): 500 µl of each dilution of the test item, the negative control and positive control (mytomicin C at a final concentration of 0.25 µg/ml of culture medium) were added directly into culture flasks. Culture flasks were incubated for 22 h ± 1 h at 37ºC, 5% CO2.

With metabolic activation S9 at 10% (v/v) (contact time: 3 h to 3 h 30'): The cells were transferred into sterile tubes and centrifuged. The cell pellets were washed once. After further centrifugation, the pellets were resuspended in 4.5 ml of culture medium containing 3.75 ml of HAM F10 and 25 µg/ml phytohemagglutinin and 750 µl S9 mix solution at 10%. 500 µl of each dilution of the test item, the negative and positive control, in their vehicles, were then added to the cell suspension. The tubes were incubated at 37ºC, 5% CO2 for 3 hours to 3 hours and 30 minutes. At the end of the incubation, tubes were centrifuged and the cell pellets were washed twice. The two washes made, the cell pellets were resuspended in 5 ml of complete medium and then transferred to 25 cm2 culture flasks and incubated for 18 h ± 1 h (about 1.5 times the normal cell cycle after the treatment start, that is to say, 22 h ± 1 h total) at 37ºC, 5% CO2.

SPINDLE INHIBITOR (cytogenetic assays): demecolcin 0.15 µg/ml

STAIN (for cytogenetic assays): Giemsa 0.4%

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Each culture was separately harvested and treated for the preparation of chromosomes, i.e. a hypotonic treatment (KCl 0.075 M), a fixation (acetic/methanol) and a staining (Giemsa 0.4%).

NUMBER OF CELLS EVALUATED: 300 cells

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Negative control and 3 test item concentrations: 300 well-spread metaphase cells. Positive control: 50 well-spread metaphase cells analysed).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity:
The test item was tested on cells Balb/c 3T3 clone 31 at 2000, 1000, 500, 250, 100 and 50 µg/ml. The mitotic index is based on the observation of at least 1000 cells for the negative control and for each concentration of the test item. For positive control al least 500 cells were observed.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

Evaluation criteria:

Criteria based on historical data: the negative control mitotic index has to be between 5% and 15%; the highest test item concentration studied must induce a maximum reduction of 50% of the mitotic index compared to the negative control mitotic index; the negative control should have a percentage of cells with at least one aberration lower or equal to 5%; the positive control should have a percentage of cells with at least one aberration over 10%.
Criteria based on OECD guideline: negative control data are within the historical negative control data distribution range within 95% control limits of this distribution; positive controls induce responses consistent with those generated in the historical positive control database and produce a statistically significant increase over the negative controls; all experimental conditions have been tested, unless one of them has yielded positive results; an adequate number of cells and concentration are analyzable; the criteria for selecting the maximum concentration are consistent.

Statistics:

The number of cells with at least one chromosomal aberration in each studied series was tested with the Chi-2 or Fisher exact test using SPSS software, version 14.
The risk of error chosen for our analysis was 5%. The results are considered significant for a probability threshold equal to p<0.05, comparing the treated series and the positive control with the negative control.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Whatever the concentration studied, the reduction of the mitotic index is always below 50% compared to the mitotic index of the negative control.
The cells treatment with the test item shows a percentage of metaphases containing at least one chromosomal aberration equal to a maximum of 4%, and whatever the condition studied or the concentration tested.

Preliminaty cytotoxicity results:

Table 1.Mortality percentage according to the concentration studied.

Concentration (µg/ml)	2000	1000	500	250	100	50
% of mortality	71	68	34	10	0	0

 

The test item is considered as cytotoxic at the concentrations 2000 and 1000 µg/ml.

The highest concentration chosen for the chromosome aberration test was: 750 µg/ml. This value was calculated by graphic extrapolation.

Mitotic index table.

Table 2.Without S9, short time (4 h)

Series		Observed cells	Metaphase cells	Mitotic index	IM reduction %
Negative control (vehicle)		35858	3201	8.93%
Positive control Mitomycin C (1 µg/ml)		65256	6334	9.71%	-8.73%
Concentration ID-16/09987	750 µg/ml	129095	11335	8.78%	1.64%
	375 µg/ml	104339	8921	8.55%	4.22%
	187.5 µg/ml	69985	5345	7.64%	14.45%
	93.75 µg/ml	119670	8620	7.20%	19.31%

 

Table 3.With S9 10%, short time (3 h)

Series		Observed cells	Metaphase cells	Mitotic index	IM reduction %
Negative control (vehicle)		163125	12019	7.37%
Positive control Cyclophosphamide (50 µg/ml)		193985	14452	7.45%	-1.11%
Concentration ID-16/09987	750 µg/ml	28735	2694	9.38%	-27.24%
	375 µg/ml	70645	6273	8.88%	-20.52%
	187.5 µg/ml	128027	11001	8.59%	-16.62%
	93.75 µg/ml	159298	13569	8.52%	-15.61%

 

Table 4.Without S9, long time (22 h)

Series		Observed cells	Metaphase cells	Mitotic index	IM reduction %
Negative control (vehicle)		204153	16989	8.32%
Positive control Mitomycin C (0.25 µg/ml)		257700	20638	8.01%	3.76%
Concentration ID-16/09987	750 µg/ml	324074	24453	7.55%	9.33%
	375 µg/ml	389761	28849	7.40%	11.06%
	187.5 µg/ml	235013	17409	7.41%	10.98%
	93.75 µg/ml	97517	7753	7.95%	4.46%

Metaphase analysis and statistical calculation table

Table 5.Without S9, short time (4 h)

		Number of analysed metaphase	G*	ga*	C	Di	CR	R	M	ca	de	ic	tr	qr	CP	cepu	Pp	Er	Total number of aberrations	Mean number of aberration /metaphase	Number of metaphases including at least 1 aberration (%)	Exact Ficher test value	Evaluation
Negative control (vehicle)		300	4	14	0	0	0	0	0	3	2	0	0	0	0	0	0	1	6	0.02	2.0%	-	-
Positive control Mitomycin C (1 µg/ml)		50	0	0	3	0	6	2	5	14	3	1	7	6	0	2	0	0	49	0.98	52.0%	0	Significant
Concentration ID-16/09987	750 µg/ml	300	1	17	0	0	0	0	0	8	0	0	0	0	0	0	0	0	8	0.03	2.7%	0.603	Non significant
	375 µg/ml	300	1	8	1	0	0	0	0	6	1	0	0	0	0	0	0	0	8	0.03	2.7%	0.603	Non significant
	187.5 µg/ml	300	0	6	1	0	0	0	1	10	0	0	0	0	0	0	0	0	12	0.04	4.0%	0.231	Non significant

 

Table 6.With S9 10%, short time (3 h)

		Number of analysed metaphase	G*	ga*	C	Di	CR	R	M	ca	de	ic	tr	qr	CP	cepu	Pp	Er	Total number of aberrations	Mean number of aberration /metaphase	Number of metaphases including at least 1 aberration (%)	Exact Ficher test value	Evaluation
Negative control (vehicle)		300	0	6	0	0	0	0	1	3	2	0	0	0	0	0	0	0	4	0.01	1.3%	-	-
Positive control Cyclosphosphamide (50 µg/ml)		50	2	1	4	1	0	0	5	11	3	0	0	1	0	2	0	0	22	0.44	40.0%	0	Significant
Concentration ID-16/09987	750 µg/ml	300	1	9	1	0	0	0	1	3	0	1	0	0	0	0	0	2	8	0.03	2.7%	0.262	Non significant
	375 µg/ml	300	3	5	0	0	0	0	0	9	1	1	0	0	0	0	0	0	10	0.03	3.3%	0.174	Non significant
	187.5 µg/ml	300	2	11	0	0	0	0	0	5	0	0	0	0	0	0	0	0	5	0.02	1.7%	1	Non significant

 

Table 7.Without S9, long time (22 h)

		Number of analysed metaphase	G*	ga*	C	Di	CR	R	M	ca	de	ic	tr	qr	CP	cepu	Pp	Er	Total number of aberrations	Mean number of aberration /metaphase	Number of metaphases including at least 1 aberration (%)	Exact Ficher test value	Evaluation
Negative control (vehicle)		300	3	9	2	0	0	0	0	6	0	0	0	0	0	0	0	0	8	0.03	2.7%	-	-
Positive control Mitomycin C (0.25 µg/ml)		50	2	2	3	0	2	0	0	11	1	0	2	1	0	0	0	0	20	0.40	34.0%	0	Significant
Concentration ID-16/09987	750 µg/ml	300	0	9	3	0	0	0	0	3	0	0	0	0	0	0	0	3	9	0.03	2.7%	1	Non significant
	375 µg/ml	300	1	8	0	0	0	0	0	7	0	0	0	0	0	0	0	2	9	0.03	3.0%	0.812	Non significant
	187.5 µg/ml	300	1	4	0	0	0	0	1	8	0	0	0	0	0	0	0	0	10	0.03	3.3%	0.643	Non significant

 

Conclusions:

The test item does not induce chromosomal aberration in human lymphocytes. Therefore, the test item can be considered as non-clastogenic and has no genotoxic potential.

Executive summary:

A study to determine the ability of the test item to induce chromosomal aberrations in human lymphocytes was performed according to OECD 473, following GLP. A preliminary test was conducted to determine the maximum dose, and 750 µg/ml was chosen to be the one that does not induce cell death of more than 50%. The test was performed at the concentrations 750, 375, 187.5 and 93.75 µg/ml, with and without metabolic activation system (S9 mix). A determination of the mitotic index was performed to assess the cytotoxicity. Three concentrations were then selected for the chromosomal aberrations analysis, 750, 375 and 187.7 µg/ml, and 3 different assays were performed: without S9, short time incubation (4 h) and long time incubation (22 h) and with 10% of S9, short time incubation (3 h). Duplicate cultures were performed for each concentration as well as for the negative (vehicle) and positive (mitomycin and monohydrated cyclophosphamide) controls. Three hundred well-spread metaphases were analysed for each concentration of the test item and negative control. For the positive control, at least 50 well-spread metaphase cells were examined. In the selected experimental conditions, the test item does not present a number of structural aberrations statistically different from the one detected for the negative control. Positive controls have a statistically significant increase in the number of chromosomal aberrations compared to the negative control, which enable to validate the test (p<0.05). The test item does not induce chromosomal aberration in human lymphocytes. Therefore, the test item can be considered as non-clastogenic and has no genotoxic potential.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 11, 2016 - December 28, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: JHSF (Japan Health Science Fundation)
- Number of passages if applicable: 27 (Test 1 without metabolic activation); 32 (Test 1 with metabolic activation); 42 (Test 2)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Test 1:
Without metabolic activation: 250, 500, 1000 and 2000 µL. The highest concentration corresponded to the mother solution diluted 10 times.
With metabolic activation: 125, 250, 500 and 1000 µL.
Test 2:
Without metabolic activation:125, 250, 500 and 1000 µL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none
- Justification for choice of solvent/vehicle: the test item is soluble in water and it is dissoved in culture medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.
TEST 1
Short treatment (4 h) without metabolic activation: Day 0: A series of tubes containing 6x10E6 cells in suspension/tube in R5 medium was seeded. 1mL of the different concentrations of the test item were added directly into the tubes. At the end of the incubation period of 4 hours, the tubes were centrifuged and the medium was aspirated. 2 washes were performed and 10 ml of R10 medium were deposited in each culture. A count of the cells was performed and 10 ml of R10 emdium were deposited in each culture. A count of the cells was performed on each tube (N0)*, then treated cells were transferred into flasks and incubated 24 hours (± 1 hour) at 37ºC (± 0.5ºC), 5% CO2.
Day 1: After incubation period, cell counting was performed on each condition (N24h). Cells culture were adjusted if necessary to 2x10E5 cells/ml and incubated for 24 hours (± 1 hour) at 37ºC (± 0.5ºC), 5% CO2.
Day 2: After incubation period, cell counting was performed again on each condition (N48h), i.e. 48 hours after the end of the treatment. These counts allowed assessing the cytotoxicity expressed as a percentage of relative cell growth (RSG). Gene mutation test was performed by treated cells cloning on 4 concentrations of the test item as determined by cytoxicity. The cells were seeded in 96-well plate at 2 cells/200 µl in non-selective culture medium (2 plates/condition) to determine the viability. Plate were incubated at 37ºC (± 0.5ºC), 5% CO2 for 10 to 12 days. Empty wells were recorded to calculate the survival rate (cloning efficiency, CE) to determine the viability. On the other hand, 96-well plates were seeded at 2x10E3 cells/200 µl in selective culture medium to determine the mutation frequency (4 plates/condition). Plates are incubated at 37ºC (± 0.5ºC), 5% CO2 for 10 to 12 days. Empty wells were recorded to calculate the mutation frequency.
Short treatment (4 h) with metabolic activation: The test is the same as described in the short treatment without metabolic activation except the culture medium which was replaced by medium containing 20% of S9 mix (2% final).
TEST 2
Long treatment without metabolic activation (24 h): Day 0: A series of flasks containing 6x10E6 cells in suspension/tube in R5 medium was seeded. 1mL of the different concentrations of the test item were added directly into the tubes and then incubated for 24 hours (± 1 hour) at 37ºC (± 0.5ºC), 5% CO2.
Day 1: After incubation period, the cultures were transferred to 50 ml sterile tubes (each identified) and were centrifuged and the medium was aspirated. 2 washes were performed and 10 ml of R10 medium were deposited in each culture. A count of the cells was performed on each tube (N0). The treated cells were placed in new 25 cm2 culture flasks at 2x10E5 cells/ml and incubated for 24 hours (± 1 hour) at 37ºC (± 0.5ºC), 5% CO2.
Day 2 and Day 3: The test was carried out as Day 1 and Day 2 in the "Short treatment without metabolic activation" assay.

DURATION
- Exposure duration: Test 1: 4 hours ± 10 min, Test 2: 24 hours ± 1 hour
- Expression time (cells in growth medium): 2 days at 37ºC (± 0.5ºC), 5% CO2.
- Selection time (if incubation with a selection agent): 10 to 12 days, at 37ºC (± 0.5ºC), 5% CO2.

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 replicates per dose (Negative control: 2 replicates; Positive control: 2 replicates)

NUMBER OF CELLS EVALUATED: 2000 cells/well

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth and relative cloning efficiency:
Cytotoxicity assessment was integrated in each test. The concentration tested were: 2000, 1000, 500, 250, 125, 62.5, 31.25 and 15.625 µg/ml. Cell cultures were treated with the different concentrations of the test item in duplicate. Positive and negative controls were carried out in parallel.
Cytotoxicity was determined by calculating the percentage of relative growth of cells (RSG: Relative Survival Growth) with SG (Suspension Growth)
- Relative Survival Growth (RSG)= SG test item/ SG Neg x D0 factor x 100; where D0 factor=(N0 test item/ N0 neg)
- Suspension Growth (SG)= Number of cells at 24 hours x Number of cells at 48 hours = N24h x N48h
Viability was determined by calculating the percentage of relative total growth of cells (RTG) with Cloning efficiency (CE)
- Relative Total Growth (RTG) = RSG x SR / 100
- Cloning Efficiency (CE) = -Ln(P0) / Number of cells/ well; where P0 = total number of empty wells / total number of seeded (192 for the viability); and, Number of cells/well= 2 in non-selective medium (R20) (CE in non-selctive medium is calculated using a Poisson distribution).
- Relative survival (RS) = CE Test item / CE Neg x 100 (RS was expressed as a percentage and was calculated from the cloning efficiency)
- Mutant Frequency (MF) = CE in the selective medium (mutant) / CE in nonselective medium (viability); with CE = -Ln(P0) / Number of cells/ well; where P0 = total number of empty wells / total number of seeded (384 for the mutation test); and, Number of cells/well= 2000 in selective medium (R20 + TFT)
4 concentrations, within the limits of 80 to 90% of cytotoxicity (equivalent to 10 to 10% of RSG), have been retained for cloning for the evaluation of mutations.

OTHER EXAMINATIONS:
Clastogenicity: The size of the colonies were estimated, a small colony is estimate to less than 25% of the diameter of the well and the size of a large colony greater than 25% of the diameter of the well. The presence of small colonies reflects a clastogenic effect indicating an alteration of chromosomes (chromosome aberrations). Large colonies reflect point mutations or deletions.
In the case of a negative result, the frequency of mutants in small and large colonies will be given for negative and positive controls. In the case of a positive result, the frequency of mutants in small and large colonies will be given for at least one concentration of the test item and negative and positive controls.

Evaluation criteria:

The test item should be considered potentially mutagenic if the following criteria are met: the mutation rate induced is significantly higher than the negative control, the observed effect is dose-dependent, the observed effect is reproducible, the rate of mutation is greater than the value of the GET (126) + MF negative control.

Statistics:

The mutant frequency for each series studied was subjected to a Chi-2 test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

doses were selected based on the cytotoxicity observed

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- confounding effects: Not observed

RANGE-FINDING/SCREENING STUDIES:
The cytoxicity of the test item was determined. As cytotoxicity was observed, the doses forthe gene mutation test were determined base on these results.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was observed after a short treatment (4 hours) with and without metabolic

VALIDATION TEST
The negative control shows a cloning efficacy (CE) between 65 and 120%, a mutation frequency (MF) between 50x10-6 and 170x10-6 cells and a suspension growth (SG) between 8 and 32 (4h treatment) and between 32 and 180 (24h treatment).
The positive control shows a mutation frequency (MF) higher than 300x10-6 with at least 40% in small colony.
Theses controls are consistent with the validity criteria. These results validate the test.

Concentration determination for the mutation test (viability determination: % RSG)

Table 1. Test 1, viability without S9 after a short treatment, 4 hours.

4H-S9	Number of cells x 10E4			SG	D0 Factor	RSG	%RSG
	N0*	24h	48h
Negative Control	55.25	85.25	104.5	22.27	-	-	-
Positive Control MMS 10 µg/ml	51.25	70.75	102.25	18.09	0.93	0.76	76.00
2000 µg/ml	49.75	49.5	98	12.13	0.90	0.49	49.00
1000 µg/ml	48	72.25	109.75	19.82	0.87	0.77	77.00
500 µg/ml	53.25	77.5	118	22.86	0.96	0.99	99.00
250 µg/ml	50	78.25	103.75	20.30	0.90	0.82	82.00
125 µg/ml	52.75	79.25	-	-	-	-	-
62.5 µg/ml	57.5	78.5	-	-	-	-	-
31.25 µg/ml	56.5	78.75	-	-	-	-	-
15.625 µg/ml	60	79	-	-	-	-	-

RSG: relative growth suspension

Table 2. Test 1, viability with S9 after a short treatment, 4 hours.

4H+S9	Number of cells x 10E4			SG	D0 Factor	RSG	%RSG
	N0*	24h	48h
Negative Control	61	77.25	114.5	22.11	-	-	-
Positive Control CP 1.5 µg/ml	59.75	68.25	102	17.40	0.98	0.77	77.00
2000 µg/ml	15	-	-	-	-	-	-
1000 µg/ml	40	45.5	85.25	9.70	0.66	0.29	29.00
500 µg/ml	46	52.25	102.5	13.39	0.75	0.45	45.00
250 µg/ml	46.25	76.75	101.5	19.48	0.76	0.67	67.00
125 µg/ml	47	81.75	100.5	20.54	0.77	0.72	72.00
62.5 µg/ml	51.75	74.75	-	-	-	-	-
31.25 µg/ml	50.75	87.75	-	-	-	-	-
15.625 µg/ml	41.5	72.75	-	-	-	-	-

RSG: relative growth suspension

Table 3. Test 2, viability without S9 after a long treatment, 24 hours.

4H-S9	Number of cells x 10E4			SG	D0 Factor	RSG	%RSG
	N0*	24h	48h
Negative Control	78.5	92.5	126.75	153.39	-	-	-
Positive Control MMS 2 µg/ml	65.5	95.25	122.25	127.12	0.83	0.69	69.00
2000 µg/ml	13	-	-	-	-	-	-
1000 µg/ml	39.5	45.25	100.5	29.94	0.50	0.10	10.00
500 µg/ml	48.75	77.25	121.5	76.26	0.62	0.31	31.00
250 µg/ml	67.5	81.25	145	132.54	0.86	0.74	74.00
125 µg/ml	72.5	91.25	118.5	130.66	0.92	0.78	78.00
62.5 µg/ml	81.5	92.25	-	-	-	-	-
31.25 µg/ml	79.75	88.5	-	-	-	-	-
15.625 µg/ml	83.75	88.75	-	-	-	-	-

RSG: relative growth suspension

 

Gene mutation test (%RS and MF)

Table 4. Test 1, relative survival without S9 after a short treatment (4 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE	%RS	%RTG
Negative Control	15	192	2	0.96	-	-
	13
	28
Positive Control MMS 10 µg/ml	33	192	2	0.57	59.38	45.13
	28
	61
2000 µg/ml	18	192	2	0.88	91.67	44.92
	15
	33
1000 µg/ml	11	192	2	1.11	115.63	89.04
	10
	21
500 µg/ml	10	192	2	1.11	115.63	114.47
	11
	21
250 µg/ml	8	192	2	1.24	129.17	105.92
	8
	16

RS: Relative survival

CE: Cloning efficacy

 

Table 5. Test 1, relative survival with S9 after a short treatment (4 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE	%RS	%RTG
Negative Control	17	192	2	0.96	-	-
	9
	26
Positive Control CP 1.5 µg/ml	28	192	2	0.57	59.38	45.13
	21
	49
1000 µg/ml	13	192	2	0.88	91.67	44.92
	10
	23
500 µg/ml	22	192	2	1.11	115.63	89.04
	15
	37
250 µg/ml	20	192	2	1.11	115.63	114.47
	21
	41
125 µg/ml	21	192	2	1.24	129.17	105.92
	19
	40

RS: Relative survival

CE: Cloning efficacy

 

Table 6. Test 2, relative survival without S9 after a long treatment (24 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE	%RS	%RTG
Negative Control	15	192	2	0.96	-	-
	13
	28
Positive Control MMS 2 µg/ml	26	192	2	0.57	59.38	45.13
	26
	52
1000 µg/ml	12	192	2	0.88	91.67	44.92
	15
	27
500 µg/ml	15	192	2	1.11	115.63	89.04
	14
	29
250 µg/ml	20	192	2	1.11	115.63	114.47
	14
	34
125 µg/ml	11	192	2	1.24	129.17	105.92
	17
	28

RS: Relative survival

CE: Cloning efficacy

 

Table 7. Test 1, frequency of mutants without S9 after a short treatment (4 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE (x 10^-6)	MF (x 10^-6)	Chi 2	%RS	%RTG
Negative Control	82	384	2000	77.29	80.51	-	-	-
	79
	88
	80
	329
Positive Control MMS 10 µg/ml	45	384	2000	343.98	603.47	101.22	P ≤0.05	Significant
	43
	51
	54
	193
2000 µg/ml	78	384	2000	84.95	96.53	0.39	P> 0.05	Non significant
	82
	83
	81
	324
1000 µg/ml	75	384	2000	80.34	72.38	0.10	P> 0.05	Non significant
	85
	82
	85
	327
500 µg/ml	81	384	2000	91.16	82.13	-0.11	P> 0.05	Non significant
	82
	74
	83
	320
250 µg/ml	81	384	2000	68.26	55.05	2.35	P> 0.05	Non significant
	84
	84
	86
	335

RS: Relative survival

CE: Cloning efficacy

MF: Mutation frecuency

 

Table 8. Test 1, frequency of mutants with S9 after a short treatment (4 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE (x 10^-6)	MF (x 10^-6)	Chi 2	%RS	%RTG
Negative Control	84	384	2000	80.34	80.34	-	-	-
	87
	77
	79
	327
Positive Control CP 1.5 µg/ml	65	384	2000	269.50	396.32	63.50	P ≤0.05	Significant
	63
	50
	46
	224
1000 µg/ml	82	384	2000	95.87	90.44	0.35	P> 0.05	Non significant
	80
	78
	77
	317
500 µg/ml	72	384	2000	95.87	116.91	2.43	P> 0.05	Non significant
	79
	85
	81
	317
250 µg/ml	80	384	2000	88.05	114.35	2.50	P> 0.05	Non significant
	82
	78
	82
	322
125 µg/ml	83 79	384	2000	86.50	110.90	1.81	P> 0.05	Non significant
	80
	81
	323

RS: Relative survival

CE: Cloning efficacy

MF: Mutation frecuency

 

Table 9. Test 2, frequency of mutants without S9 after a long treatment (24 hours)

Treatment without TFT	Empty wells	Total wells	Number of cell/wells	CE (x 10^-6)	MF (x 10^-6)	Chi 2	%RS	%RTG
Negative Control	82	384	2000	74.26	77.35	-	-	-
	84
	82
	83
	331
Positive Control MMS 2 µg/ml	59	384	2000	260.65	401.00	67.23	P ≤0.05	Significant
	54
	56
	59
	228
1000 µg/ml	85	384	2000	81.87	83.54	-0.11	P> 0.05	Non significant
	75
	79
	87
	326
500 µg/ml	82	384	2000	84.95	89.42	0.20	P> 0.05	Non significant
	74
	86
	82
	324
250 µg/ml	88	384	2000	55.00	63.22	0.58	P> 0.05	Non significant
	85
	83
	88
	344
125 µg/ml	83 86	384	2000	63.80	66.46	0.19	P> 0.05	Non significant
	85
	84
	338

RS: Relative survival

CE: Cloning efficacy

MF: Mutation frecuency

 

 

Small and large colonies

Table 10. Distribution of small (SC) and large colonies (LC)

4H-S9		Negative Control	Positive Control MMS 10 µg/ml
Test 1	SC	13	96
	LC	42	95
	% SC	24	50

 

4H+S9		Negative Control	Positive Control CP 1.5 µg/ml
Test 1	SC	16	85
	LC	41	75
	% SC	28	53

 

24H-S9		Negative Control	Positive Control MMS 2 µg/ml
Test 2	SC	6	74
	LC	49	82
	% SC	11	47

 

Table 11. Mutation frequencies obtained in each test condition

Test conditions	Dose 1 (µg/ml)	Dose 2 (µg/ml)	Dose 3 (µg/ml)	Dose 4 (µg/ml)	Negative Control	Positive Control
4 hours without S9	2000	1000	500	250	Vehicule	MMS 10 µg/ml
	97	72	82	55	81	603

 

Test conditions	Dose 1 (µg/ml)	Dose 2 (µg/ml)	Dose 3 (µg/ml)	Dose 4 (µg/ml)	Negative Control	Positive Control
4 hours with S9	1000	500	250	125	Vehicule	CP 1.5 µg/ml
	90	117	114	111	80	396

 

Test conditions	Dose 1 (µg/ml)	Dose 2 (µg/ml)	Dose 3 (µg/ml)	Dose 4 (µg/ml)	Negative Control	Positive Control
24 hours without S9	1000	500	250	125	Vehicule	MMS 2 µg/ml
	84	89	63	66	77	401

Conclusions:

Under the experimental conditions used, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

The potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus was studied using the cell line L5178Y according to OECD 490, following GLP. A cytotoxicity test included in the gene mutation test was performed on 8 doses. As cytotoxicty was observed, the doses for main tests were selected based on these results. Four doses were selected, in the short treatment (4 h) with metabolic activation the doses tested were 1000, 500, 250 and 125 µg/ml, and without metabolic activation 2000, 1000, 500 and 250 µg/ml. In the long treatment (24 h) without metabolic activation the doses tested were 1000, 500, 250 and 125 µg/ml. Negative (vehicle) and a positive controls ( Methylmethanesulfonate and Cyclophosphamide monohydrate) were performed in parallel. Cells were incubated with test item or controls and then they were analysed for frequency of mutants in

small and large colonies. Positive controls led to an increase in mutation frequency at least 300 x 10E-6 with at least 40% in small colony, which validate the test. The frequencies of mutations observed for the test item is not statistically different from those observed for the negative control. Under these experimental conditions, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation study in bacteria

Key study: The determination of the mutagenic potential of the test item has been assessed 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, 1500, 500, 150 and 50 μg/plate) were put in contact with the strains TA 1535, TA 1537, TA98, TA100 of Salmonela typhimurium and Escherichia coli WP2 (uvrA-) (pKM 101), with and without metabolic activation. Two independent assays were performed. For assay nº1, the different concetrations 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 the absence of metabolic activation and with pre-incubation in the presence of metablic activation system. For both assays, negative and positive controls were carried out in parallel. There is no evidence of any increase in the number of revertant colonies in the presence of the various concentration of the test item, with and without metabolic activation in the strains tested. The different doses prepared from solutions of 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.

In vitro chromosome aberration study in mammalian cells

Key study: A study was performed according to OECD 473, following GLP. A preliminary test was conducted to determine the maximum dose, and 750 µg/ml was chosen to be the one that does not induce cell death of more than 50%. The test was performed at the concentrations 750, 375, 187.5 and 93.75 µg/ml, with and without metabolic activation system. A determination of the mitotic index was performed to assess the cytotoxicity. Three concentrations were then selected for the chromosomal aberrations analysis, 750, 375 and 187.7 µg/ml, and 3 different assays were performed: without S9, short time incubation (4 h) and long time incubation (22 h) and with 10% of S9, short time incubation (3 h). Duplicate cultures were performed for each concentration as well as for the negative and positive controls. In the selected experimental conditions, the test item does not present a number of structural aberrations statistically different from the one detected for the negative control. Positive controls have a statistically significant increase in the number of chromosomal aberrations compared to the negative control, which enable to validate the test (p<0.05).The test item does not induce chromosomal aberration in human lymphocytes. Therefore, the test item can be considered as non-clastogenic and has no genotoxic potential.

In vitro gene mutation study in mammalian cells

Key study: The potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus was studied using the cell line L5178Y according to OECD 490, following GLP. A cytotoxicity test included in the gene mutation test was performed on 8 doses. As cytotoxicty was observed, the doses for main tests were selected based on these results. Four doses were selected, in the short treatment (4 h) with metabolic activation the doses tested were 1000, 500, 250 and 125 µg/ml, and without metabolic activation 2000, 1000, 500 and 250 µg/ml. In the long treatment (24 h) without metabolic activation the doses tested were 1000, 500, 250 and 125 µg/ml. Negative and a positive controls were performed in parallel. Cells were incubated with test item or controls and then they were analysed for frequency of mutants in small and large colonies. Positive controls led to an increase in mutation frequency at least 300 x 10E-6 with at least 40% in small colony, which validate the test. The frequencies of mutations observed for the test item is not statistically different from those observed for the negative control. Under these experimental conditions, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Justification for classification or non-classification

Based on the available information, the test item has not to be classified as mutagen, according to CLP Regulation 1272/2008.