Cytidine 3'-(dihydrogen phosphate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 17, 2017 - August 24, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cytidine 3'-(dihydrogen phosphate)

EC Number:

200-556-6

EC Name:

Cytidine 3'-(dihydrogen phosphate)

Cas Number:

63-37-6

Molecular formula:

C9H14N3O8P

IUPAC Name:

4-amino-1-(5-O-phosphonopentofuranosyl)pyrimidin-2(1H)-one

Test material form:

solid

Details on test material:

Synonyms: 5-CMP acid, 5'-Cytidylic acid, Cytidine 5'-monophosphate

Specific details on test material used for the study:

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

Method

Target gene:

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

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: Δuvr B, rfa, pKM 101

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: Δuvr B, rfa

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: Δuvr B, rfa, pKM 101

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: Δuvr B, rfa

Species / strain 5

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.

Controls

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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: 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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

The determination of the mutagenic potential of the test item has been assessed by the bacterial reverse mutation test, performed according to OECD 471 method and under GLP conditions. A preliminary study showed no toxicity up to 5000 µg/plate. A stock solution of the test item was prepared at 100 mg/mL and various concentrations of the test item (5000, 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.