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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:
September 4, 1994 - September 23, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(R)-α-phenylethylammonium (-)-(1R, 2S)-(1,2-epoxypropyl)phosphonate monohydrate
EC Number:
418-570-8
EC Name:
(R)-α-phenylethylammonium (-)-(1R, 2S)-(1,2-epoxypropyl)phosphonate monohydrate
Cas Number:
25383-07-7
Molecular formula:
C11H20NO5P
IUPAC Name:
(R)-α-phenylethylammonium (-)-(1R, 2S)-(1,2-epoxypropyl)phosphonate monohydrate
Details on test material:
- Name of test material (as cited in study report): Fosfomycin PEA salt
- Physical state: white crystalline powder
- Lot/batch No.: 4177
- Expiration date of the lot/batch: at least 3 years from manufacturing date
- Storage condition of test material: at room temperature protected from light
- Other: June 10, 1994

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: TA 1535, 1537, 102, 98 ant 100: Defect in the histidine gene (His-), a deep rough (rfa) character and an uvrB deletion (uvrB-). TA98, 100 and 102: ampicillin resistant. TA102 tetracycline resistant.
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Test 1: 50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)
Test 2: 1.5, 5, 15, 50 and 150 µg/plate (with metabolic activation) and 5, 15, 50, 150 and 500 µg/plate (without metabolic activation)
Test 3 (repetition of test 2)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [water for injection]
- Justification for choice of solvent/vehicle: suitable solvent for the test article
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
Water for injection
Positive controls:
yes
Positive control substance:
other: Hydrazine sulphate (Hyd)
Remarks:
S. typhimurium TA 1535, without metabolic activation (direct test)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
S. typhimurium TA 1537, without metabolic activation (direct test) Migrated to IUCLID6: 9-aminoacridine HCL monohydrate (9-AA)
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
S. typhimurium TA 102, without metabolic activation (direct test) Migrated to IUCLID6: (Mito)
Positive controls:
yes
Positive control substance:
other: Doxorubicine HCl (Doxo)
Remarks:
S. typhimurium TA 98 and 100, without metabolic activation (direct test)
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene (2-AF)
Remarks:
S. typhimurium TA 98, 100 and 102, with metabolic activation (indirect test)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
TEST 1): in agar (plate incorporation)
0.1 ml of the test article or negative or positive control solutions was put into sterile test tubes containing 2.5 ml (test without) or 2 ml (test with) of soft agar kept liquid in a thermostatic bath at 45 ºC. A suspension of Salmonella strains in a stationary growth phase (0.1 ml) was rapidly added. For the test with metabolic activation 0.5 ml of S9 Mix was also added. The test tubes were shaken rapidly and the contents poured onto plates containing solid selective growth medium. The plates were incubated at 37 ºC for 72 hours.

Repetition of the assay: The experiment was repeated in an independent assay. Since negative results were obtained with metabolic activation in the first trial, the repeat test followed the pre-incubation method.

TEST 2): preincubation
0.5 ml of S9 Mix (test with metabolic activation) were dispensed in sterile test tubes placed in an ise bath. 0.1 ml of bacterial suspension and 0.1 ml of the test or control article solutions according to the dosage levels, were introduced. The test tubes were gently vortexed and incubated at 37 ºC for 20 min, in a horizontally shaking thermostatic bath. At the end of incubation, 2 ml of soft agar (kept liquid in a thermostatic bath at about 45 ºC were added to each tube. The test tubes were rapidly shaken and the contents poured onto plates containing solid selected growth medium. The plates were incubated at 37 ºC for 72 hours.
TEST 3): A third experiment was carried out in order to confirm the results obtained in the second one.

DURATION
- Preincubation period: 20 min at tests 2 and 3, preincubation tests.
- Exposure duration: 72 h at tests 1, 2 and 3.

NUMBER OF REPLICATIONS: 3 plates per dose at tests 1, 2 and 3.

OTHER:
NEGATIVE AND POSITIVE CONTROL DOSAGE LEVELS:
Negative control: water for injection, 100 µg/plate.
Positive controls without metabolic activation (direct test):
S. typhimurium TA 1535: Hydrazine sulphate (Hyd): 500 µg/plate (solution: 5 mg/ml in water)
S. typhimurium TA 1537:9-amioacridine HCL monohydrate (9-AA): 40 µg/plate (solution: 0.4 mg/ml in DMSO)
S. typhimurium TA 102: Mitomycin C (Mito): 0.5 µg/plate (solution: 0.005 mg/ml in water)
S. typhimurium TA 98 and 100: Doxorubicine HCL (Doxo): 4 µg/plate (solution: 0.04 mg/ml in DMSO)
Positive controls with metabolic activation (indirect test):
S. typhimurium TA 98, 100 and 102: 2-aminofluorene (2-AF): 5 µg/plate (solution: 0.05 mg/ml in water)

PREPARATION OF S9 MIX:
S9 fraction (9000 g supernatant) was prepared from adult male Sprague Dawley rats pretreated by intraperitoneal route at 500 mg/kg (2.5 mg/kg) with Aroclor 1254 (Alltech). S9 Mix consisted of S9 plus cofactors.
Evaluation criteria:
The test article is considered to have elicitated a positive response when:
- The number of reverted colonies is significantly higher when compared with the number of revertants in the solvent controls (as determined by Student's t);
AND
- Either: a dose-response con be verified, that is, a positive correlation between the number of revertants and the dose in an interval of at least 3 doses (linear regression test);
- Or: a statistically significant increase is recorded at one dose only, when confirmed in independent assays.
Statistics:
The mean and standard deviation was calculated for reversions read in each dosage group.
Comparison of the spontaneous reversions (in the negative control) with the ones in the test article plates and ini the positive control plates was done by student's t test.
Dose-effect linear regression was done if statistically significant differences were found.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: At dosage levels of 1500 and 5000 µg/plate, both in the presence and in the absence of metabolic activation and 500 µg/plate with metabolic activation.

Any other information on results incl. tables

Table 1. Mutagenicity test with S. typhimurium strain TA 1535 without metabolic activation.

Compound

Concentration

(µg/p)

Reversions/plate

Mean

SD

Test 1

Control

-

29

27

24

26.67

2.52

Test article

50

25

21

28

24.67

3.51

150

23

20

20

21.00

1.73

500

19

17

19

18.33

1.15

1500

Toxic

-

 

5000

Toxic

-

 

Hydrazine

500

103

127

131

120.33*

15.14

Test 2

Control

-

27

29

24

26.67

2.52

Test article

5

27

27

21

25.00

3.46

15

29

25

24

26.00

2.65

50

21

26

27

24.67

3.21

150

24

22

25

23.67

1.53

500

12

17

14

14.33

2.52

Hydrazine

500

116

106

121

114.33*

7.64

Test 3

Control

-

23

21

28

24.00

3.61

Test article

5

24

26

21

23.67

2.52

15

23

23

22

22.67

0.58

50

27

21

20

22.67

3.79

150

23

23

24

23.33

0.58

500

19

16

20

18.33

2.08

Hydrazine

500

106

127

117

116.67*

10.50

*p < 0.001

 

Table 2. Mutagenicity test with S. typhimurium strain TA 1537 without metabolic activation.

Compound

Concentration

(µg/p)

Reversions/plate

Mean

SD

Test 1

Control

-

12

10

13

11.67

1.53

Test article

50

8

7

10

8.33

1.53

150

13

7

12

10.67

3.21

500

13

11

9

11.0

2.00

1500

2

3

5

3.33

1.53

5000

Toxic

-

 

9-AA

40

40

57

44

47.00*

8.89

Test 2

Control

-

12

7

15

11.33

4.04

Test article

5

9

6

13

9.33

3.51

15

7

14

11

10.67

3.51

50

11

12

8

10.33

2.08

150

13

10

8

10.33

2.52

500

10

5

9

8.00

2.65

9-AA

40

56

47

59

54.00*

6.24

Test 3

Control

-

10

9

9

9.33

0.58

Test article

5

13

9

7

9.67

3.06

15

4

11

9

8.00

3.61

50

8

10

3

7.00

3.61

150

8

9

10

9.00

1.00

500

5

7

3

5.00

2.00

9-AA

40

49

55

58

54.00*

4.58

*p < 0.001

 

Table 3. Mutagenicity test with S. typhimurium strain TA 102 without metabolic activation.

Compound

Concentration

(µg/p)

Reversions/plate

Mean

SD

Control

-

272

322

292

295.33

25.17

Test article

50

268

316

300

294.67

24.44

150

304

248

265

272.33

28.71

500

166

130

148

148.00

18.00

1500

Toxic

-

 

5000

Toxic

-

 

2-Nitrofluorene

2.5

940

869

918

909.0*

36.35

Test 2

Control

-

290

311

271

290.67

20.01

Test article

5

271

284

260

271.67

12.01

15

305

290

271

28..67

17.04

50

280

310

265

285.00

22.91

150

280

320

278

292.67

23.69

500

240

256

211

235.67

22.81

2-Nitrofluorene

2.5

980

1040

1020

1013.33*

30.55

Test 3

Control

-

254

308

320

294.00

35.16

Test article

5

328

306

296

310.00

16.37

15

309

260

272

280.33

25.54

50

322

280

320

307.33

23.69

150

252

260

260

257.33

4.62

500

212

201

218

210.33

8.62

2-Nitrofluorene

2.5

900

1020

940

953.33*

61.10

*p < 0.001

 

Table 4. Mutagenicity test with S. typhimurium strain TA 98 without metabolic activation.

Compound

Concentration

(µg/p)

Reversions/plate

Mean

SD

Control

-

48

37

45

43.33

5.69

Test article

50

44

40

45

43.00

2.65

150

48

41

43

44.0

3.61

500

30

32

26

29.33

3.06

1500

12

7

7

8.67

2.89

5000

Toxic

-

 

Doxorubicine

4

1020

980

1000

1000.00*

20.00

Test 2

Control

-

47

38

41

42.00

4.58

Test article

5

37

43

43

41.00

3.46

15

42

44

41

42.33

1.53

50

45

43

35

41.00

5.29

150

39

41

43

41.00

2.00

500

25

30

37

30.67

6.03

Doxorubicine

4

846

828

864

846.00*

18.00

Test 3

Control

-

32

42

36

36.67

5.03

Test article

5

36

38

25

33.00

7.00

15

37

29

33

33.00

4.00

50

31

28

26

38.33

2.52

150

25

37

26

29.33

6.66

500

33

27

31

30.33

3.06

Doxorubicine

4

804

888

824

850.67*

333.31

*p < 0.001

 

Table 5. Mutagenicity test with S. typhimurium strain TA 100 without metabolic activation.

Compound

Concentration

(µg/p)

Reversions/plate

Mean

SD

Control

-

204

180

201

195.00

13.08

Test article

50

195

186

183

188.00

6.24

150

179

190

185

184.67

5.51

500

130

160

140

143.33

15.28

1500

Toxic

-

 

5000

Toxic

-

 

Doxorubicine

4

560

544

550

551.33*

8.08

Test 2

Control

-

195

180

171

182.00

12.12

Test article

5

188

166

193

182.33

14.36

15

180

170

180

176.67

5.77

50

203

174

182

186.33

14.98

150

186

190

188

188.00

2.00

500

165

140

151

152.00

12.53

Doxorubicine

4

495

510

530

511.67*

17.56

Test 3

Control

-

206

204

212

207.33

4.16

Test article

5

200

206

195

200.33

5.51

15

180

172

208

186.67

18.90

50

200

196

210

202.00

7.21

150

204

202

184

196.67

11.02

500

176

197

198

190.33

12.42

Doxorubicine

4

572

652

570

598.00*

46.78

*p < 0.001

                     

Table 6. Mutagenicity test with S. typhimurium strain TA 1535 with metabolic activation.

Compound

Concentration

(µg/p)

Reversions/plate

Mean

SD

Control

-

24

30

27

27.00

3.00

Test article

50

28

24

25

25.67

2.08

150

26

26

27

26.33

0.58

500

Toxic

-

 

1500

Toxic

-

 

5000

Toxic

-

 

Test 2

Control

-

23

22

28

24.33

3.21

Test article

1.5

23

17

23

21.00

3.46

5

22

22

25

23.00

1.73

15

24

23

26

24.33

1.53

50

19

27

18

21.33

4.93

150

21

16

19

18.67

2.52

Test 3

Control

-

21

25

27

24.33

3.06

Test article

1.5

23

18

20

20.33

2.52

5

15

19

19

17.67

2.31

15

27

18

23

22.67

4.51

50

18

17

22

19.00

2.65

150

18

17

19

18.00

1.00

 

Table 7. Mutagenicity test with S. typhimurium strain TA 1537 with metabolic activation.

Compound

Concentration

(µg/p)

Reversions/plate

Mean

SD

Control

-

12

15

12

13.00

1.73

Test article

50

7

12

5

8.00

3.61

150

7

8

6

7.00

1.00

500

2

3

3

2.67

0.58

1500

Toxic

-

 

5000

Toxic

-

 

Test 2

Control

-

12

10

12

11.33

1.15

Test article

1.5

10

11

12

11.00

1.00

5

13

14

8

11.67

3.21

15

13

11

12

12.00

1.00

50

8

13

9

10.00

2.65

150

8

6

9

7.67

1.53

Test 3

Control

-

10

11

11

10.67

0.58

Test article

1.5

11

10

8

9.67

1.53

 

5

12

6

9

9.00

3.00

 

15

11

12

10

11.00

1.00

 

50

9

10

8

9.00

1.00

 

150

8

8

6

7.33

1.15

 

Table 8. Mutagenicity test with S. typhimurium strain TA 102 with metabolic activation.

Compound

Concentration

(µg/p)

Reversions/plate

Mean

SD

Control

-

252

315

317

294.67

36.96

Test article

50

294

270

280

281.33

12.06

150

307

260

268

278.33

25.15

500

200

164

170

178.00

19.29

1500

Toxic

-

 

5000

Toxic

-

 

2-AF

5

976

1012

988

992.00*

18.33

Test 3

Control

-

304

318

316

312.67

7.57

Test article

1.5

300

272

308

293.33

18.90

 

5

300

302

286

296.00

8.72

 

15

280

277

308

288.33

17.10

 

50

309

310

279

299.33

17.62

 

150

282

263

255

266.67

13.87

2-AF

5

1200

940

1120

1086.67*

133.17

*p < 0.001

 

Table 9. Mutagenicity test with S. typhimurium strain TA 98 with metabolic activation.

Compound

Concentration

(µg/p)

Reversions/plate

Mean

SD

Control

-

48

47

51

48.67

2.08

Test article

50

48

53

44

48.33

4.51

150

31

36

39

35.33

4.04

500

Toxic

-

 

1500

Toxic

-

 

5000

Toxic

-

 

2-AF

5

1128

1164

1112

1134.67*

26.67

Test 2

Control

-

52

44

46

47.33

4.16

Test article

1.5

48

40

50

46.00

5.29

5

41

49

47

45.67

4.16

15

46

46

52

48.00

3.46

50

51

44

47

47.33

3.51

150

40

33

36

36.33

3.51

2-AF

5

1200

1140

1170

1170.00*

30.00

Test 3

Control

-

45

41

43

46.33

4.16

Test article

1.5

39

45

43

42.33

3.06

 

5

41

45

67

51.00

14.00

 

15

39

31

40

36.67

4.93

 

50

39

40

50

43.00

6.08

 

150

35

46

38

39.67

5.69

2-AF

5

1450

1400

1466

1438.67*

34.43

*p < 0.001

 

Table 10. Mutagenicity test with S. typhimurium strain TA 100 with metabolic activation.

Compound

Concentration

(µg/p)

Reversions/plate

Mean

SD

Control

-

186

226

211

207.67

20.21

Test article

50

162

184

184

176.67

12.70

150

190

200

180

190.00

10.00

500

30

48

72

50.00

21.07

1500

Toxic

-

 

5000

Toxic

-

 

2-AF

5

820

810

900

843.33*

49.33

Test 2

Control

-

203

195

207

201.67

6.11

Test article

1.5

188

200

176

188.00

12.00

 

5

195

196

171

187.33

14.15

 

15

206

180

194

193.33

13.01

 

50

181

177

185

181.00

4.00

 

150

194

160

175

176.33

17.04

2-AF

5

940

875

900

905.00*

32.79

Test 3

Control

-

193

185

195

191.00

5.29

Test article

1.5

207

191

195

197.67

8.33

 

5

189

195

206

196.67

8.62

 

15

176

208

1778

187.33

17.93

 

50

200

184

173

185.67

13.58

 

150

173

174

169

172.00

2.65

2-AF

5

940

880

910

910.00

30.00

*p < 0.001

Applicant's summary and conclusion

Conclusions:
The test article Fosfomycin PEA salt did not induce any significant increase in the number of reversions up to the concentrations of 150 and 500 µg/plate in the presence and in the absence of metabolic activation, respectively, in TA 1535, TA 1537, TA 102, TA 98 and TA100 Salmonella typhimurium strains, in two independent experiments.
Executive summary:

The mutagenic potency of Fosfomycin PEA salt was investigate using Salmonella typhimurium TA 1535, TA 1537, TA 102, TA 98 and TA 100 as tester strains. The study was performed using the plate incorporation assay with and without liver homologate (S9 Mix). Three independent experiments were performed, setting up triplicate plates for each experimental point. Hydrazine sulphate, 9-aminoacridine HCl monohydrate, Mitomycin C, Doxorubicine HCl and 2-aminofluorene served as positive controls to test the mutagenicity of the bacterial strains as well as the activity of the metabolizing system. The negative control was the test article solvent, water for injection. In the first experiment, the test article proved to be severely cytotoxic on the test system at the dosage levels of 1500 and 5000 µg/plate, both in the presence and in the absence of metabolic and 500 µg/plate with metabolic activation.

At these dosages, in fact, a complete absence or a strong decrease in the background lawn and in the number of revertant colonies in comparison with the negative control was observed. At 150 and 500 µg/plate with and without metabolic activation, respectively, a slight reduction in the background lawn was detected. The Ames test was therefore repeated assaying test article dosages in the range of concentrations 0.15-150 µg/plate in the trial with metabolic activation and 5-500 µg/plate in the trial without metabolic activation. Then a third experiment was carried out in order to confirm the results obtained in the second one. In the concentrations rages investigated, the test substance did not show any mutagenic activity with or without the addition of S9 liver homogenate fractions. The known reversion properties were determined for the tester strains with the control substances; the positive responses confirmed the good metabolic activity of the liver homogenate fractions.