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Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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:
cytidine 3'-(dihydrogen phosphate)
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: Δuvr B, rfa, pKM 101
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: Δuvr B, rfa
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: Δuvr B, rfa, pKM 101
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: Δuvr B, rfa
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
other: Δuvr B, pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 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 resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: 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.