(R)-α-phenylethylammonium (-)-(1R, 2S)-(1,2-epoxypropyl)phosphonate monohydrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

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

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Water for injection

Controls 2

Positive controls:

yes

Positive control substance:

other: Hydrazine sulphate (Hyd)

Remarks:

S. typhimurium TA 1535, without metabolic activation (direct test)

Controls 3

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)

Controls 4

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

S. typhimurium TA 102, without metabolic activation (direct test) Migrated to IUCLID6: (Mito)

Controls 5

Positive controls:

yes

Positive control substance:

other: Doxorubicine HCl (Doxo)

Remarks:

S. typhimurium TA 98 and 100, without metabolic activation (direct test)

Controls 6

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 results

Test results 1

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

Test results 2

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.