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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

Referenceopen allclose all

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.

 

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/cm2

%

Cells/cm2

%

Cells/cm2

%

Cells/cm2

%

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.

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.