Cytidine 3'-(dihydrogen phosphate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria: Key study: 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 with and without metabolic activation up to 5000 µg/plate.

In vitro cytogenicity study in mammalian cells: Key study: OECD 473. GLP study. The test item does not induce chromosomal aberration in Chinese Hamster Ovary (CHO) cells. 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: 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:

July 17, 2017 - August 24, 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
- Stability under test conditions: yes

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 98

Additional strain / cell type characteristics:

other: Δuvr B, rfa, pKM 101

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: Δuvr B, rfa

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: Δuvr B, rfa, pKM 101

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: Δuvr B, rfa

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: Δuvr B, pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 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: water
- Justification for choice of solvent/vehicle: the item is completely soluble in the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-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 1-9E09 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 at 45ºC containing 10% (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): 1-9E09 bacteria/mL

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

SELECTION AGENT (mutation assays):
Salmonella Typhimurium 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 (1-9E03 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-11101-5-3805 validated on 7.2017 - expiry date: 25.05.2019).
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: Presence of precipitates not hindering the scoring were observed at the highest concentration (5000 µg/plate).

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
Suspension of Cytidine –(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: M16017C (LEMI code: GNE070817-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
Suspension of Cytidine –(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: M16017C (LEMI code: GNE210817-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 nº1

		Doses (/plate)
		0 (negative control)	DMSO	50 µg	150 µg	500 µg	1500 µg	2500 µg	5000 µg
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE170717-S1)	N1	143	171	180	162	161	163	153	110
	N2	176	156	159	176	142	173	149	151
	N3	178	161	175	177	148	177	146	109
	N	166± 20	163± 8	171± 11	172± 8	150± 10	171± 7	149± 4	123± 24
	%	-	98%	103%	104%	91%	103%	90%	74%

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 nº2

		Doses (/plate)
		0 (negative control)	Water	50 µg	150 µg	500 µg	1500 µg	2500 µg	5000 µg
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE240717-S1)	N1	112	114	107	110	111	107	104	86
	N2	106	121	118	123	124	116	83	119
	N3	101	111	124	108	120	108	89	84
	N	106± 6	115± 5	116±9	114± 8	118± 7	110± 5	92±11	96± 20
	%	-	108%	109%	107%	111%	104%	87%	91%

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. Bacteriostatic activity control nº3

		Doses (/plate)
		0 (negative control)	DMSO	50 µg	150 µg	500 µg	1500 µg	5000 µg
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE070817-S1)	N1	224	221	227	211	224	210	218
	N2	219	223	213	208	214	211	220
	N3	218	205	218	217	220	189	213
	N	220± 3	216± 10	219± 7	212± 5	219± 5	203±12	217± 4
	%	-	98%	100%	96%	100%	92%	98%

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

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	16	10	13	13.00	3.00	-
Positive control solvent	5 µL	13	14	13	13.33	0.58	-
Positive control: Sodium azide	5 µg  in 5 µL	1070	1073	1087	1076.67	9.07	80.75
Vehicle	100 µL	12	24	8	14.67	8.33	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE070817-S1)	5000 µg*	19	16	14	16.33	2.52	1.11
	1500 µg	19	16	25	20.00	4.58	1.36
	500 µg	18	16	10	14.67	4.16	1.00
	150 µg	17	17	19	17.67	1.15	1.20
	50 µg	15	17	11	14.33	3.06	0.98

*presence of precipitates not hindering the scoring.

 

Table 6. 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	11	11	8	10.00	1.73	-
Positive control solvent	20 µL	15	13	13	13.67	1.15	-
Positive control: 2-Anthramine	2 µg  in 20 µL	205	203	213	207.00	5.49	15.15
Vehicle	100 µL	8	10	12	10.00	2.00	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE070817-S1)	5000 µg*	11	13	11	11.67	1.15	1.17
	1500 µg	16	13	17	15.33	2.08	1.53
	500 µg	9	15	16	13.33	3.79	1.33
	150 µg	10	17	17	14.67	4.04	1.47
	50 µg	17	17	15	16.33	1.15	1.63

*presence of precipitates not hindering the scoring.

 

Table 7. TA 1535 - Assay nº2 – without metabolic activation (-S9-mix)

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	15	11	17	14.33	3.06	-
Positive control solvent	5 µL	11	15	10	12.00	2.65	-
Positive control: Sodium azide	5 µg  in 5 µL	948	1008	961	972.33	31.56	81.03
Vehicle	100 µL	13	14	10	12.33	2.08	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE210817-S1)	5000 µg*	16	13	10	13.00	3.00	1.05
	1500 µg	12	14	14	13.33	1.15	1.08
	500 µg	13	15	10	12.67	2.52	1.03
	150 µg	13	15	12	13.33	1.53	1.08
	50 µg	15	16	14	15.00	1.00	1.22

*presence of precipitates not hindering the scoring.

 

Table 8. 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	16	11	13.33	2.52	-
Positive control solvent	10 µL	17	10	11	12.67	3.79	-
Positive control: 2-Anthramine	1 µg  in 10 µL	135	121	119	125.00	8.72	9.87
Vehicle	100 µL	12	8	14	11.33	3.06	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE210817-S1)	5000 µg*	10	11	10	10.33	0.58	0.91
	1500 µg	7	14	8	9.67	3.79	0.85
	500 µg	9	14	9	10.67	2.89	0.94
	150 µg	7	10	14	10.33	3.51	0.91
	50 µg	9	13	10	10.67	2.08	0.94

*presence of precipitates not hindering the scoring.

 

Table 9. TA 1537 - Assay nº1 – without metabolic activation (-S9-mix)

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	11	10	5	8.67	3.21	-
Positive control solvent	20 µL	14	10	16	13.33	3.06	-
Positive control: 9-Aminoacridine	50 µg  in 20 µL	1301	1556	1510	1455.67	135.91	109.18
Vehicle	100 µL	12	8	20	13.33	6.11	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE070817-S1)	5000 µg*	6	9	7	7.33	1.53	0.55
	1500 µg	13	12	3	9.33	5.51	0.70
	500 µg	10	7	5	7.33	2.52	0.55
	150 µg	9	11	14	11.33	2.52	0.85
	50 µg	6	13	14	11.00	4.36	0.83

*presence of precipitates not hindering the scoring.

 

Table 10. 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	11	8	10	9.67	1.53	-
Positive control solvent	20 µL	14	5	10	9.67	4.51	-
Positive control: 2-Anthramine	2 µg  in 20 µL	59	76	62	65.67	9.07	6.79
Vehicle	100 µL	12	11	14	12.33	1.53	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE070817-S1)	5000 µg*	8	13	8	9.67	2.89	0.78
	1500 µg	11	6	14	10.33	4.04	0.84
	500 µg	10	9	12	10.33	1.53	0.84
	150 µg	16	11	13	13.33	2.52	1.08
	50 µg	9	15	10	11.33	3.21	0.92

*presence of precipitates not hindering the scoring.

 

Table 11. 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	6	8	7.00	1.00	-
Positive control solvent	20 µL	6	6	6	6.00	0.00	-
Positive control: 9-Aminoacridine	50 µg  in 20 µL	1845	1552	1837	1744.67	166.90	290.78
Vehicle	100 µL	13	14	10	12.33	2.08	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNEF210817-S1)	5000 µg*	7	6	8	7.00	1.00	0.57
	1500 µg	9	11	7	9.00	2.00	0.73
	500 µg	5	4	8	5.67	2.08	0.46
	150 µg	12	3	6	7.00	4.58	0.57
	50 µg	13	9	7	9.67	3.06	0.78

*presence of precipitates not hindering the scoring.

 

Table 12. 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	5	12	6	7.67	3.79	-
Positive control solvent	20 µL	3	4	7	4.67	2.08	-
Positive control: 2-Anthramine	1 µg  in 10 µL	36	30	34	33.33	3.06	7.14
Vehicle	100 µL	12	8	14	11.33	3.06	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE210817-S1)	5000 µg*	9	10	8	9.00	1.00	0.79
	1500 µg	7	11	4	7.33	3.51	0.65
	500 µg	12	10	7	9.67	2.52	0.85
	150 µg	8	8	10	8.67	1.15	0.76
	50 µg	9	6	9	8.00	1.73	0.71

*presence of precipitates not hindering the scoring.

Table 13. TA 98 - Assay nº1 – without metabolic activation (-S9-mix)

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	18	17	17	17.33	0.58	-
Positive control solvent	20 µL	21	22	27	23.33	3.21	-
Positive control: 2-Nitrofluorene	2 µg  in 20 µL	454	441	447	447.33	6.51	19.17
Vehicle	100 µL	23	20	24	22.33	2.08	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE070817-S1)	5000 µg*	17	15	14	15.33	1.53	0.69
	1500 µg	16	18	18	17.33	1.15	0.78
	500 µg	16	18	14	16.00	2.00	0.72
	150 µg	18	10	15	14.33	4.04	0.64
	50 µg	17	11	20	16.00	4.58	0.72

*presence of precipitates not hindering the scoring.

 

Table 14. 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	26	27	23.67	4.93	-
Positive control solvent	20 µL	26	28	29	27.67	1.53	-
Positive control: 2-Anthramine	2 µg  in 20 µL	967	683	648	766.00	174.95	27.69
Vehicle	100 µL	33	29	31	31.00	2.00	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE070817-S1)	5000 µg*	17	29	19	21.67	6.43	0.70
	1500 µg	18	24	16	19.33	4.16	0.62
	500 µg	22	17	26	21.67	4.51	0.70
	150 µg	19	21	20	20.00	1.00	0.65
	50 µg	26	16	16	19.33	5.77	0.62

*presence of precipitates not hindering the scoring.

 

Table 15. TA 98 - Assay nº2 – without metabolic activation (-S9-mix)

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	14	13	12	13.00	1.00	-
Positive control solvent	20 µL	12	15	11	12.67	2.08	-
Positive control: 2-Nitrofluorene	2 µg  in 20 µL	471	506	492	489.67	17.62	38.66
Vehicle	100 µL	15	20	21	18.67	3.21	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE210817-S1)	5000 µg*	19	22	18	19.67	2.08	1.05
	1500 µg	12	16	21	16.33	4.51	0.88
	500 µg	17	19	21	19.00	2.00	1.02
	150 µg	13	15	13	13.67	1.15	0.73
	50 µg	12	18	16	15.33	3.06	0.82

*presence of precipitates not hindering the scoring.

 

Table 16. 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	22	29	26	25.67	3.51	-
Positive control solvent	20 µL	24	16	22	20.67	4.16	-
Positive control: 2-Anthramine	1 µg  in 10 µL	467	688	614	589.67	112.49	28.53
Vehicle	100 µL	29	25	28	27.33	2.08	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE210817-S1)	5000 µg*	21	23	18	20.67	2.52	0.76
	1500 µg	19	29	21	23.00	5.29	0.84
	500 µg	24	32	28	28.00	4.00	1.02
	150 µg	29	31	26	28.67	2.52	1.05
	50 µg	23	20	19	20.67	2.08	0.76

*presence of precipitates not hindering the scoring.

 

Table 17. TA 100 - Assay nº1 – without metabolic activation (-S9-mix)

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	82	64	59	68.33	12.10	-
Positive control solvent	20 µL	66	61	69	65.33	4.04	-
Positive control: Sodium azide	20 µg  in 20 µL	1156	999	1192	1115.67	102.63	17.08
Vehicle	100 µL	57	62	65	61.33	4.04	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE070817-S1)	5000 µg*	63	69	91	74.33	14.74	1.21
	1500 µg	73	66	70	69.67	3.51	1.14
	500 µg	103	88	78	89.67	12.58	1.46
	150 µg	81	95	80	85.33	8.39	1.39
	50 µg	82	80	79	80.33	1.53	1.31

*presence of precipitates not hindering the scoring.

 

Table 18. 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	58	63	77	66.00	9.85	-
Positive control solvent	20 µL	58	54	58	56.67	2.31	-
Positive control: 2-Anthramine	2 µg  in 20 µL	1621	1127	1265	1337.67	254.89	23.61
Vehicle	100 µL	61	64	58	61.00	3.00	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE070817-S1)	5000 µg*	52	64	59	58.33	6.03	0.96
	1500 µg	71	59	69	66.33	6.43	1.09
	500 µg	90	71	70	77.00	11.27	1.26
	150 µg	63	67	82	70.67	10.02	1.16
	50 µg	68	52	60	60.00	8.00	0.98

*presence of precipitates not hindering the scoring.

 

Table 19. TA 100 - Assay nº2 – without metabolic activation (-S9-mix)

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	49	83	69	67.00	17.09	-
Positive control solvent	20 µL	53	61	59	57.67	4.16	-
Positive control: Sodium azide	20 µg  in 20 µL	1227	1055	1128	1136.67	86.33	19.71
Vehicle	100 µL	80	69	63	70.67	8.62	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE210817-S1)	5000 µg*	81	63	68	70.67	9.29	1.00
	1500 µg	47	62	60	56.33	8.14	0.80
	500 µg	90	80	60	76.67	15.28	1.08
	150 µg	58	76	81	71.67	12.10	1.01
	50 µg	82	57	68	69.00	12.53	0.98

*presence of precipitates not hindering the scoring.

 

Table 20. 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	82	52	61	65.00	15.39	-
Positive control solvent	20 µL	66	53	58	59.00	6.56	-
Positive control: 2-Anthramine	1 µg  in 10 µL	556	601	584	580.33	22.72	9.84
Vehicle	100 µL	66	55	60	60.33	5..51	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE210817-S1)	5000 µg*	86	73	60	73.00	13.00	1.21
	1500 µg	59	60	63	60.67	2.08	1.01
	500 µg	106	64	70	80.00	22.72	1.33
	150 µg	66	74	75	71.67	4.93	1.19
	50 µg	57	61	59	59.00	2.00	0.98

*presence of precipitates not hindering the scoring.

 

Table 21. E. COLI - Assay nº1 – without metabolic activation (-S9-mix)

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	121	122	131	124.67	5.51	-
Positive control solvent	10 µL	124	136	128	129.33	6.11	-
Positive control: cis-Platinum (II)	1 µg  in 10 µL	401	437	492	443.33	45.83	3.43
Vehicle	100 µL	127	132	117	125.33	7.64	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE070817-S1)	5000 µg*	122	113	132	122.33	9.50	0.98
	1500 µg	131	124	123	126.00	4.36	1.01
	500 µg	117	134	136	129.00	10.44	1.03
	150 µg	118	125	137	126.67	9.61	1.01
	50 µg	138	127	129	131.33	5.86	1.05

*presence of precipitates not hindering the scoring.

 

Table 22. 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	150	204	221	191.67	37.07	-
Positive control solvent	5 µL	169	203	217	196.33	24.68	-
Positive control: Dimethylbenzanthracene	5 µg  in 5 µL	617	642	654	637.67	18.88	3.25
Vehicle	100 µL	191	214	201	202.00	11.53	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE070817-S1)	5000 µg*	191	202	183	192.00	9.54	0.95
	1500 µg	201	212	204	205.67	5.69	1.02
	500 µg	187	153	170	170.00	17.00	0.84
	150 µg	214	209	218	213.67	4.51	1.06
	50 µg	195	20	217	206.00	11.00	1.02

*presence of precipitates not hindering the scoring.

Table 23. E. COLI - Assay nº2 – without metabolic activation (-S9-mix)

Serie	Dose/plate	Plate			Mean	Standard deviation	R
		nº1	nº2	nº3
Negative control	100 µL	138	142	160	146.67	11.72	-
Positive control solvent	10 µL	145	146	134	141.67	6.66	-
Positive control: cis-Platinum (II)	1 µg  in 10 µL	298	292	303	297.67	5.51	2.10
Vehicle	100 µL	139	156	129	141.33	13.65	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE210817-S1)	5000 µg*	165	150	149	154.67	8.96	1.09
	1500 µg	172	159	138	156.33	17.16	1.11
	500 µg	161	155	169	161.67	7.02	1.14
	150 µg	153	150	138	147.00	7.94	1.04
	50 µg	149	146	157	150.67	5.69	1.07

*presence of precipitates not hindering the scoring.

Table 24. 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	174	184	185	181.00	6.08	-
Positive control solvent	5 µL	201	183	209	197.67	13.32	-
Positive control: Dimethylbenzanthracene	2.5 µg  in 5 µL	324	831	856	670.33	300.19	3.39
Vehicle	100 µL	205	209	193	202.33	8.33	-
Suspension of Cytidine 3’-(dihydrogen phosphate) synonym CYTIDINE-5’-MONOPHOSPHATE ACIDE Code: 3066078 Batch: MP16017C   (LEMI code: GNE210817-S1)	5000 µg*	189	175	183	182.33	7.02	0.90
	1500 µg	179	210	193	194.00	15.52	0.96
	500 µg	173	211	220	201.33	24.95	1.00
	150 µg	180	205	182	189.00	13.89	0.93
	50 µg	179	196	172	182.33	12.34	0.90

*presence of precipitates not hindering the scoring.

Conclusions:

The test item do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 with and without metabolic activation up to 5000 µg/plate.

Executive summary:

The determination of the mutagenic potential of the test item has been assessed by the bacterial reverse mutation test, performed according to OECD 471 method and under GLP conditions. A preliminary study showed no toxicity up to 5000 µg/plate. A stock solution of the test item was prepared at 100 mg/mL and various concentrations of the test item (5000, 1500, 500, 150 and 50 µg/plate) were put in contact with the strains TA 1535, TA 1537, TA98, TA100 of Salmonella typhimurium and Escherichia coli WP2 (uvrA-) (pKM 101), with and without metabolic activation. Two independent assays were performed. For assay nº1, the different 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 signifiacnt 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:

August 16, 2017 - September 21, 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
- Stability under test conditions: yes

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC CCL 61, ECACC 85051005
- Suitability of cells: CHO cells are currently used in standard protocols for in vitro cytogenetic tests.
- Cell cycle length, doubling time or proliferation index: 13 h 30
- Number of passages if applicable: 29-31
- Modal number of chromosomes: 20

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Mc Coy's + 10% FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

2000, 800, 320, 128 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:

yes

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
- Cell density at seeding: 10E03 cells/cm2

Before exposure to test item, cells were seeded in a 25 cm2 culture flask at the starting density of 10E03 cells/cm2 into 5 ml of complete culture medium (Mc Coy's supplemented with 10% (v/v) Fetal Calf Serum (FCS)). Cells cultures were incubated at 37ºC in a humid atmosphere containing 5% (v/v) CO2, for 40 hours.
Selected concentrations of test item solutions are placed in contact with the test system. Two independent tests are carried out: Assay 1 (short-term treatment, without and with metabolic activation) and Assay 2 (long-term treatment without metabolic activation).

Short-term treatment (Assay 1):
Without metabolic activation: 4 hours later at 37ºC in a humidified atmosphere containing 5% (v/v) CO2, the culture medium is discarded and the cells washed twice with culture medium. 5 mL of fresh complete culture medium are added and the cells incubated, 18 hours at 37ºC in a humidified atmosphere containing 5% (v/v) CO2.

With metabolic activation: 40 hours after seeding, the complete cell culture is discarded, cells layer washed with culture medium and incubated with reaction mixture composed by culture medium supplemented with 10% (v/v) S9-mix. 3 hours later at 37ºC in a humidified atmosphere containing 5% (v/v) CO2, the culture medium is discarded and the cells layer incubated, 18 hours at 37ºC in a humidified atmosphere containing 5% (v/v) CO2.

Long-term treatment (Assay 2):
Without metabolic activation: the cells are incubated, 20 hours at 37ºC in a humidified atmosphere containing 5% (v/v) CO2.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (SIGMA - Ref:D1925 - Batch: RNBF6984) 0.15 µg/ml

STAIN (for cytogenetic assays): Giemsa at 0.4% (w/v) in phosphate buffer (0.01 M, pH 6.8).

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end ofthe incubation period (18 h and 20 h), Colcemid was added in each flask and cells incubated for 2.5 hours at 37ºC, 5% CO2. Then the cells were collected, they were detached with 0.5 mL trypsin, living cells were counted, hypotonic treatment (KCl 0.075 M at 37ºc for 10 minutes), fixed with Carnoy mixture (methanol:acetic acid, 3:1), spread on coded microscopic slides and stained using Giemsa stain at 0.4% (w/v) in phosphate buffer (0.01 M, pH 6.8).

NUMBER OF CELLS EVALUATED: 300 cells

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Negative control and the highest non-cytotoxic concentration of each solution (if necessary) of test item: 300 well-spread metaphase cells. Positive control: 25 well-spread metaphase cells analysed). Methaphases are analysed under a microscope (Zeiss), magnification x1000 for the detection of chromosomal aberrations, polyploidy and endoreduplications.

DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in Cell Count (RICC)
- Any supplementary information relevant to cytotoxicity:
The test item was tested on cells Balb/c 3T3 at 2000, 1400, 800 and 560 µg/ml. Any of the tested solutions did not show any inhibition of cell growth statistically significant. According to the evaluation criteria of the cytotoxicity, the solution prepared from the test item is not cytotoxic at 2000 µg/mL. Positive control (phenol 0.64 mg/mL) induces a 62% (P < 0.001) inhibition on cell growth which validates the study.

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

Evaluation criteria:

The test item will be considered as clastogen in vitro with regards to CHO cells according to the following criteria:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent absolute negative control,
- the increase is dose-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.
The test item for which the results do not meet the all above criteria is considered non-clastogenic in this system.
Positive results indicate that the test item induces structural chromosome aberrations in CHO cell cultures.
The positive control shall largely fulfil to all three criteria.
The number of cells with chromosomal aberrations in the negative control shall be less than 5%.
Equivocal or discutable results may not allow a clear positive response. Results shall be clarified by further testing using modification of experimental conditions (concentration spacing and metabolic activation conditions).

Statistics:

The number of cells presenting one, or more, aberration is considered as a direct response and evaluated statistically using the Chi squared trend test.
The results were consider significant if P <0.05 comparing cultures treated with different solutions of test item with their corresponding negative control, positive control and negative control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No
- Precipitation: No

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) Historical control (2007 to 2016)
- Positive historical control data: Short-treatment, without metabolic activation (Mitomycin): 40.74 ± 8.45 [min/max : 28 / 61] (n=23); Short-treatment, with metabolic activation (Cyclosphosphamide): 35.57 ± 5.01 [min/max : 24 / 48] (n=23); Long-treatment, without metabolic activation (Mitomycin): 41.83 ± 7.70 [min/max : 28 / 56] (n=23)
- Negative (solvent/vehicle) historical control data: Short-treatment, without metabolic activation: 1.97 ± 1.46 [min/max : 0.5 / 7.32] (n=21); Short-treatment, with metabolic activation: 1.92 ± 1.57 [min/max : 0.3 / 7.73] (n=21); Long-treatment, without metabolic activation: 1.73 ± 0.62 [min/max : 0.7 / 3.5] (n=21)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC

Table 1a: PH and osmolality

Serie		Assay	pH	Osmolality (mosm/Kg H2O)
Absolute negative control		4 h without S9	7.4	303
		3 h with S9	7.4	303
		20 h without S9	7.2	304
Solution obtained from 3’-(dihydrogen phosphate) Code: 3066078 Batch. M16017C	LEMI code: GNE310817-S1	4 h without S9	7.4	306
	LEMI code: GNE310817-S2	3 h with S9	7.4	306
	LEMI code: GNE070917-S1	20 h without S9	7.2	309

 

Table 1b: Preliminary cytotoxicity

SERIE	2000 µg/mL		1400 µg/mL		800 µg/mL		560 µg/mL
	Cells/cm^2	%	Cells/cm^2	%	Cells/cm^2	%	Cells/cm^2	%
Solution of Cytidine 3’-(dihydrogen phosphate) Code: 3066078 Batch: M16017C LEMI code: GNE-170817-S1	101565 ± 21785	86	108280 ± 19070	92	109845 ± 13725	93	105155 ± 8545	89
Negative control (complete culture medium)	118280± 7760	-
Positive control (phenol 0.64 mg/mL)	45155 ± 6235	38 P < 0.001

  Table 2a: RICC without metabolic activation

Serie		Assay	Concentration	Cells number	RICC reduction (%)***
Without metabolic activation
Pre-incubation control****		1*	-	3025000	-
		2**	-	2975000	-
Absolute negative control		1*	-	5950000	-
		2**	-	6675000	-
Positive control (mitomycin C)		1*	0.25 µg/mL	4625000	45%
		2**	0.125 µg/mL	5200000	40%
Solution obtained from Cytidine 3’-(dihydrogen phosphate) Code: 3066078 Batch: M16017C	LEMI code: GNE310817-S1	1*	128 µg/mL	5600000	12%
			320 µg/mL	6575000	-
			800 µg/mL	6750000	-
			2000 µg/mL	6575000	-
	LEMI code: GNE070917-S1	2**	128 µg/mL	7150000	-
			320 µg/mL	6475000	5%
			800 µg/mL	7075000	-
			2000 µg/mL	7100000	-

* 1: Assay 1 (short treatment, 4 hours without metabolic activation)

**2: Assay 2 (continuous treatment, 20 hours without metabolic activation)

*** RICC reduction % (solution): 100 – RICC

RICC: Relative increase in the number of cells in exposed cultures versus increase in non-treated cultures, a ratio expressed as a percentage.

RICC (solution): [Increase in number of cells in treated cultures (final – starting)/ Increase in number of cells in negative control cultures (final – starting)] x 100

RICC (positive control): [Increase in number of cells in positive control cultures (final – starting)/ Increase in number of cells in negative control cultures (final – starting)] x 100

RICC (solvent control): [Increase in number of cells in solvent control cultures (final – starting)/ Increase in number of cells in negative control cultures (final – starting)] x 100

“-“: no reduction in RICC

**** Pre-incubation control (initial number of cells): number of cells before incubation

Table 2b: RICC with metabolic activation

Serie		Assay	Concentration	Cells number	RICC reduction (%)**
With metabolic activation (S9-mix)
Pre-incubation control***		1*	-	3025000	-
Absolute negative control		1*	-	5625000	-
Positive control (mitomycin C)		1*	10 µg/mL	4650000	38%
Solution obtained from Cytidine 3’-(dihydrogen phosphate) Code: 3066078 Batch: M16017C	LEMI code: GNE310817-S2	1*	128 µg/mL	6500000	-
			320 µg/mL	5800000	-
			800 µg/mL	5625000	0%
			2000 µg/mL	5825000	-

* 1: Assay 1 (short treatment, 3 hours treatment with S9-mix (10% v/v)

** RICC reduction % (solution): 100 – RICC

RICC: Relative increase in the number of cells in exposed cultures versus increase in non-treated cultures, a ratio expressed as a percentage.

RICC (solution): [Increase in number of cells in treated cultures (final – starting)/ Increase in number of cells in negative control cultures (final – starting)] x 100

RICC (positive control): [Increase in number of cells in positive control cultures (final – starting)/ Increase in number of cells in negative control cultures (final – starting)] x 100

RICC (solvent control): [Increase in number of cells in solvent control cultures (final – starting)/ Increase in number of cells in negative control cultures (final – starting)] x 100

“-“: no reduction in RICC

*** Pre-incubation control (initial number of cells): number of cells before incubation

Table 3a: Type and number of chromosome aberrations (without metabolic activation)

Serie		Assay	Conc.	Cells observed	Aberrations (type and number)
					Chromosomal						Chromatidic						Other
					G***	C	M	D	CR	R	g***	c	ic	tr	qr	d	CP	cp
Without metabolic activation
Absolute negative control		1*	-	300	2	1	0	0	0	0	1	1	0	0	0	0	0	0
		2**	-	300	2	1	0	0	0	0	0	1	0	0	0	0	0	0
Positive control (mitomycin C)		1*	0.25 µg/mL	26	0	0	0	0	0	0	0	5	0	2	0	1	0	0
		2**	0.125 µg/mL	52	3	5	0	0	0	0	1	4	0	2	1	0	0	0
Solution obtained from Cytidine 3’-(dihydrogen phosphate) Code: 3066078 Batch: M16017C	LEMI code: GNE310817-S1	1*	320 µg/mL	300	2	1	0	0	0	0	2	0	0	0	0	0	0	0
			800 µg/mL	301	2	1	0	0	0	0	3	1	1	0	0	0	0	0
			2000 µg/mL	300	1	2	0	0	0	0	4	3	0	0	0	0	0	0
	LEMI code: GNE070917-S1	2**	320 µg/mL	300	3	1	0	0	0	0	3	2	0	0	0	0	0	0
			800 µg/mL	300	3	0	0	0	0	0	3	1	0	0	0	0	0	0
			2000 µg/mL	300	6	4	0	0	0	0	4	2	0	0	0	0	0	0

*1: Assay 1

**2: Assay 2

***not taken into account.

Type of aberration: G: gap or achromatic lesion; C: break or terminal delection; D: dicentric chromosome; CR: complex rearrangement; R: ring; g: gap; c: break; d: median delection; ci: chromosome interchange; tr: triradial; qr: quadriradial; PC: pulverised chromosome; pc: pulverised cell.

Table 3b: Number and percentage of cells with aberrations (without metabolic activation)

Serie		Assay	Conc.	Cells observed	Total aberrations	Aberrations/cell	Cells with aberration	Cells with aberration (%)	ꭓ2	P***
Without metabolic activation
Absolute negative control		1*	-	300	2	0.007	2	0.7%	-	-
		2**	-	300	2	0.007	2	0.7%	-	-
Positive control (mitomycin C)		1*	0.25 µg/mL	26	8	0.308	8	30.8%	55.34	< 0.001
		2**	0.125 µg/mL	52	12	0.231	12	23.1%	46.96	< 0.001
Solution obtained from Cytidine 3’-(dihydrogen phosphate) Code: 3066078 Batch: M16017C	LEMI code: GNE310817-S1	1*	320 µg/mL	300	1	0.003	1	0.3%	0.33	NS****
			800 µg/mL	301	3	0.010	3	1.0%	0.20	NS****
			2000 µg/mL	300	5	0.017	4	1.3%	0.66	NS****
	LEMI code: GNE070917-S1	2**	320 µg/mL	300	3	0.010	3	1.0%	0.20	NS****
			800 µg/mL	300	1	0.003	1	0.3%	0.33	NS****
			2000 µg/mL	300	6	0.020	6	2.0%	1.97	NS****

*1: Assay 1

**2: Assay 2

***P: Statistical significance.

****NS: Not statistically Significant, P≥ 0.005

Table 4a: Type and number of chromosome aberrations (with metabolic activation)

Serie		Assay	Conc.	Cells observed	Aberrations (type and number)
					Chromosomal						Chromatidic						Other
					G**	C	M	D	CR	R	g**	c	ic	tr	qr	d	CP	cp
Without metabolic activation
Absolute negative control		1*	-	300	1	0	0	0	0	0	5	1	0	0	0	1	0	0
Positive control (cyclophosphamide)		1*	10 µg/mL	25	0	0	0	0	0	0	0	5	0	4	0	0	0	0
Solution obtained from Cytidine 3’-(dihydrogen phosphate) Code: 3066078 Batch: M16017C	LEMI code: GNE310817-S2	1*	320 µg/mL	300	2	0	0	0	0	0	2	1	0	0	0	0	0	0
			800 µg/mL	300	3	1	0	0	0	0	0	0	0	0	0	0	0	0
			2000 µg/mL	300	2	0	0	0	0	0	5	2	0	0	0	0	0	0

*1: Assay 1

**not taken into account.

Type of aberration: G: gap or achromatic lesion; C: break or terminal delection; D: dicentric chromosome; CR: complex rearrangement; R: ring; g: gap; c: break; d: median delection; ci: chromosome interchange; tr: triradial; qr: quadriradial; PC: pulverised chromosome; pc: pulverised cell.

 

 Table 4b: Number and percentage of cells with aberrations (with metabolic activation)

Serie		Assay	Conc.	Cells observed	Total aberrations	Aberrations/cell	Cells with aberration	Cells with aberration (%)	ꭓ2	P**
Without metabolic activation
Absolute negative control		1*	-	300	2	0.007	2	0.7%	-	-
Positive control (cyclophosphamide)		1*	10 µg/mL	25	9	0.360	8	32.0%	57.12	< 0.001
Solution obtained from Cytidine 3’-(dihydrogen phosphate) Code: 3066078 Batch: M16017C	LEMI code: GNE310817-S2	1*	320 µg/mL	300	1	0.003	1	0.3%	0.33	NS***
			800 µg/mL	300	1	0.003	1	0.3%	0.33	NS***
			2000 µg/mL	300	2	0.007	2	0.7%	0.00	NS***

*1: Assay 1

**P: Statistical significance.

***NS: Not statistically Significant, P≥ 0.005

Conclusions:

The test item does not induce chromosomal aberration in Chinese Hamster Ovary (CHO) cells. 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 Chinese Hamster Ovary (CHO) cells was performed according to OECD 473, following GLP. A preliminary test was conducted to determine the maximum dose, and 2000 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 2000, 800, 320 and 128 µg/ml, with and without metabolic activation system (S9 mix). A determination of the Relative Increase in Cell Count (RICC) was performed to assess the cytotoxicity. Three concentrations were then selected for the chromosomal aberrations analysis, 2000, 800 and 320 µg/ml, and 3 different assays were performed: without S9, short time incubation (4 h) and long time incubation (20 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. Three hundred well-spread metaphases were analysed for each concentration of the test item and negative control. For the positive control, at least 25 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 CHO cells. 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:

March 1, 2017 - March 28, 2017

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
- Stability under test conditions: yes

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

clone 3.7.2C

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: JHSF (Japan Health Science Fundation)
- Number of passages if applicable: 62 (Test 1); 71 (Test 2)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Culture medium RPMI 1640, Penicillin/Streptomycin, sodium pyruvate 100 mM and horse serum heat inactivated
- 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: water
- Justification for choice of solvent/vehicle: the test item is soluble in water and it is dissoved in culture medium.

Untreated negative controls:

yes

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. The different concentrations of the test item were added directly into the tubes (2 cultures per concentration). The negative control (vehicle) was added to the same volume as for the test item (2 cultures per concentration). In parallel and in the same conditions, two cultures were treated with the positive controls. 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 1.5x10E6 cells in suspension/tube in R10 medium was seeded. 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. The negative control (vehicle) was added to the same volume as for the test item (2 cultures per concentration). In parallel and in the same conditions, two cultures were treated with the positive controls.
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

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: MMS 10 µg/ml (-S9 4h): 848 ± 258.5 (min. 491 - max. 1368) (n=15); MMS 2 µg/ml (-S9 24h): 521 ± 206.2 (min. 258 - max. 1059) (n=12); CP 1.5 µg/ml (+S9 4h): 572 ± 172.3 (min. 305 - max. 1009) (n=23)
- Negative (solvent/vehicle) historical control data: -S9 4h: 98 ± 38 (min. 63 - max. 167) (n=15); -S9 24h: 99 ± 25.7 (min. 70 - max. 152) (n=12); +S9 4h: 104 ± 39.8 (min. 53 - max. 198) (n=23)

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

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	70.25	80.5	102.25	20.58	-	-	-
Positive Control MMS 10 µg/ml	56.5	70	90.75	15.88	0.80	0.62	62.00
2000 µg/ml	67.5	79.75	107.75	21.48	0.96	1.00	100.00
1000 µg/ml	68.75	74.75	114.25	21.35	0.98	1.02	102.00
500 µg/ml	67.25	73.5	106.75	19.62	0.96	0.92	92.00
250 µg/ml	68	93.75	112.75	26.43	0.97	1.25	125.00
125 µg/ml	69	91.25	-	-	-	-	-
62.5 µg/ml	67.25	91	-	-	-	-	-
31.25 µg/ml	67.25	93.5	-	-	-	-	-
15.625 µg/ml	72.25	85.75	-	-	-	-	-

RSG: relative growth suspension

N₀in the count carried out just after the contact time (here 4 hours ± 10 minutes) with the test item

 

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	55.5	89.75	110.5	24.79	-	-	-
Positive Control CP 1.5 µg/ml	53.5	68.75	99.25	17.06	0.96	0.66	66.00
2000 µg/ml	64.25	92.5	101.75	23.53	1.16	1.10	110.0
1000 µg/ml	61.75	81	104.25	21.11	1.11	0.95	95.00
500 µg/ml	53.75	81.75	114.5	23.40	0.97	0.92	92.00
250 µg/ml	64.5	73	111.5	20.35	1.16	0.95	95.00
125 µg/ml	62.75	54.5	-	-	-	-	-
62.5 µg/ml	57	65.75	-	-	-	-	-
31.25 µg/ml	57.5	80.75	-	-	-	-	-
15.625 µg/ml	61.75	80.5	-	-	-	-	-

RSG: relative growth suspension

N₀in the count carried out just after the contact time (here 4 hours ± 10 minutes) with the test item

 

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

24H-S9	Number of cells x 10E4			SG	D0 Factor	RSG	%RSG
	N0*	24h	48h
Negative Control	82.25	99.25	117.5	159.86	-	-	-
Positive Control MMS 2 µg/ml	64.5	92	97.5	96.43	0.78	0.47	47.00
2000 µg/ml	82.75	101.75	111	155.77	1.01	0.98	98.00
1000 µg/ml	78	99.25	116	149.67	0.95	0.89	89.00
500 µg/ml	75.75	98.75	110.5	137.76	0.92	0.79	79.00
250 µg/ml	79.25	102.25	106.75	144.17	0.96	0.87	87.00
125 µg/ml	81.5	103.5	-	-	-	-	-
62.5 µg/ml	78.75	102	-	-	-	-	-
31.25 µg/ml	81.75	102.5	-	-	-	-	-
15.625 µg/ml	81.75	100.75	-	-	-	-	-

RSG: relative growth suspension

N₀in the count carried out just after the contact time (here 24 hours ± 1 hour) with the test item

 

 

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	14	192	2	0.95	-	-
	15
	29
Positive Control MMS 10 µg/ml	28	192	2	0.57	60.00	37.20
	34
	62
2000 µg/ml	14	192	2	1.06	111.58	111.58
	9
	23
1000 µg/ml	15	192	2	0.95	100.00	102.00
	14
	29
500 µg/ml	14	192	2	1.00	105.26	96.84
	12
	26
250 µg/ml	17	192	2	0.88	92.63	115.79
	16
	33

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	18	192	2	0.98	-	-
	9
	27
Positive Control CP 1.5 µg/ml	26	192	2	0.61	62.24	41.08
	31
	57
2000 µg/ml	14	192	2	0.98	100.00	110.00
	13
	27
1000 µg/ml	14	192	2	0.96	97.96	93.06
	14
	28
500 µg/ml	6	192	2	1.21	123.47	113.59
	11
	17
250 µg/ml	11	192	2	0.95	96.24	92.09
	18
	29

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	10	192	2	1.04	-	-
	14
	24
Positive Control MMS 2 µg/ml	28	192	2	0.66	63.46	29.83
	23
	51
2000 µg/ml	6	192	2	1.27	122.12	119.68
	9
	15
1000 µg/ml	4	192	2	1.27	122.12	108.69
	11
	15
500 µg/ml	6	192	2	1.35	129.81	102.55
	7
	13
250 µg/ml	9	192	2	1.02	98.08	85.33
	16
	25

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	75	384	2000	120.11	126.43	-	-	-
	76
	74
	77
	302
Positive Control MMS 10 µg/ml	37	384	2000	480.10	842.28	119.80	P ≤0.05	Significant
	37
	33
	40
	147
2000 µg/ml	67	384	2000	196.91	185.76	3.66	P> 0.05	Non significant
	64
	65
	63
	259
1000 µg/ml	74	384	2000	143.84	151.41	0.56	P> 0.05	Non significant
	72
	69
	73
	288
500 µg/ml	74	384	2000	126.77	126.77	-0.09	P> 0.05	Non significant
	77
	75
	72
	298
250 µg/ml	73	384	2000	150.83	171.40	2.26	P> 0.05	Non significant
	73
	67
	71
	284

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	77	384	2000	118.45	120.87	-	-	-
	71
	80
	75
	303
Positive Control CP 1.5 µg/ml	46	384	2000	341.39	559.66	78.99	P ≤0.05	Significant
	53
	38
	57
	194
2000 µg/ml	75	384	2000	161.51	164.81	2.18	P> 0.05	Non significant
	68
	68
	67
	278
1000 µg/ml	64	384	2000	174.26	181.52	3.76	P> 0.05	Non significant
	71
	70
	66
	271
500 µg/ml	67	384	2000	166.94	137.97	0.30	P> 0.05	Non significant
	72
	70
	66
	275
250 µg/ml	75 76	384	2000	143.84	151.41	1.10	P> 0.05	Non significant
	67
	70
	288

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	76	384	2000	149.08	143.35	-	-	-
	73
	68
	68
	285
Positive Control MMS 2 µg/ml	38	384	2000	526.40	797.58	97.39	P ≤0.05	Significant
	31
	28
	37
	134
2000 µg/ml	65	384	2000	194.98	153.53	-0.24	P> 0.05	Non significant
	66
	65
	64
	260
1000 µg/ml	66	384	2000	216.59	170.54	0.48	P> 0.05	Non significant
	60
	63
	60
	249
500 µg/ml	68	384	2000	187.35	138.78	-0.32	P> 0.05	Non significant
	66
	70
	60
	264
250 µg/ml	68 66	384	2000	156.14	153.08	0.08	P> 0.05	Non significant
	73
	74
	281

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	19	138
	LC	63	99
	% SC	23	58

 

4H+S9		Negative Control	Positive Control CP 1.5 µg/ml
Test 1	SC	13	107
	LC	68	83
	% SC	16	56

 

24H-S9		Negative Control	Positive Control MMS 2 µg/ml
Test 2	SC	21	122
	LC	79	128
	% SC	21	49

 

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
	186	151	127	171	126	842

 

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	2000	1000	500	250	Vehicule	CP 1.5 µg/ml
	165	182	138	151	121	560

 

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	2000	1000	500	250	Vehicule	MMS 2 µg/ml
	154	171	139	153	143	798

Conclusions:

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. According to the 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. 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 in bacteria: Key study: 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 Salmonella typhimurium and Escherichia coli WP2 (uvrA-) (pKM 101), with and without metabolic activation. Two independent assays were performed. For assay nº1, the different concentrations 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 metabolic activation system. For both assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no evidence of any increase in the number of revertant colonies in the presence of 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 cytogenicity study in mammalian cells: Key study: A study to determine the ability of the test item to induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells was performed according to OECD 473, following GLP. A preliminary test was conducted to determine the maximum dose, and 2000 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 2000, 800, 320 and 128 µg/ml, with and without metabolic activation system (S9 mix). A determination of the Relative Increase in Cell Count (RICC) was performed to assess the cytotoxicity. Three concentrations were then selected for the chromosomal aberrations analysis, 2000, 800 and 320 µg/ml, and 3 different assays were performed: without S9, short time incubation (4 h) and long time incubation (20 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. Three hundred well-spread metaphases were analysed for each concentration of the test item and negative control. For the positive control, at least 25 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 inCHO cells. Therefore, the test item can be considered as non-clastogenic and has no genotoxic potential.

In vitro gene mutation study in mamalian 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. According to the 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. 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.

Based on the available data, there are no safety concerns related to genotoxicity for this substance.

Justification for classification or non-classification

Based on the available data (negative results in vitro), the test item is not classified for mutagenicity in accordance with CLP Regulation (EC) No. 1272/2008.