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EC number: 418-570-8 | CAS number: 25383-07-7
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation in vitro: Key study: Test method according to EU Method B.14 and OECD Guideline 471. GLP study. In the concentrations ranges investigated, the test substance did not show any mutagenic activity.
Chromosome aberrations in vitro: Key study: Test method according to EU Method B.10 and OECD Guideline 473. In the concentrations ranges investigated, the test substance did not show any clastogenic activity.
Link to relevant study records
- 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
- 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
- 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 - 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. - 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.
- 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.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- October 7, 1994 - January 18, 1995
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Test concentrations with justification for top dose:
- Preliminary cytotoxicity test (nominal concentrations): 3, 10, 30, 100, 300, 1000, 2500 and 5000 µg/ml, with and without metabolic activation.
Metaphase analysis at 20 h sampling (nominal concentrations): 300, 1000 and 2500 µg/ml, with and without metabolic activation.
Metaphase analysis at 44 h sampling (nominal concentrations): 300, 1000 and 2500 µg/ml, without metabolic activation and 100, 300 and, 1000 µg/ml, with metabolic activation. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ham's F12 Medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ham's F12 Medium
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation.
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
In the test without metabolic activation 4 ml of fresh medium were put into a flask and 1 ml of the test article solutions, according to the dosage levels, or of the control articles were added. In the test with metabolic activation 4 ml of fresh medium containing the S9 metabolic activation system were put into each flask and then 1 ml of the test article solutions or of the control articles were added.
DURATION
- Exposure duration:
Test article, with metabolic activation: 3 h
Test article, without metabolic activation: 18 h
Positive controls, both with and without metabolic activation: 3 h
- Fixation time (start of exposure up to fixation or harvest of cells): 20 and 44 hours from the initiation of treatment (1st and 2nd harvest)
SPINDLE INHIBITOR (cytogenetic assays): colcemid (0,2 mg/ml)
STAIN (for cytogenetic assays): The slides were stained with Giemsa.
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 200 metaphases/dose (100 metaphases/culture)
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (number of metaphase cells per 1000 cells)
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
OTHER:
POSITIVE CONTROLS: Ehtylmethane sulphonate (EMS): 0.5 µl/ml (605 µg EMS/ml), without metabolic activation.
Cyclophosphamide (CP): 50 µg/ml, with metabolic activation.
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 clastogenic properties in the following criteria are all fulfilled:
- Statistically significant increases in the incidence of cells bearing aberrations are observed at two consecutive dose-levels or at the highest practicable dose-level;
- The increases are reproduced in independent assays;
- The incidence of aberration-bearing cells in the treated cultures exceeds 5.0%. - Statistics:
- Fisher's exact test was used to compare the number of cells with aberrations in control and treated cultures. The analyses were performed using sets of data either including or excluding gaps.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- At 20 h: 1000 µg/ml with and without metabolic activation. At 44 h: 1000 µg/ml with metabolic activation and 2500 µg/ml without metabolic activation.
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The highest dose tested, 5000 µg/ml, affected the pH of the incubation mixture (>0.5 decrease when compared to the solvent controls both without and with metabolic activation). This dose was therefore considered unsuitable for metaphase analysis.
The test article did not affect the pH at the other dosage levels assayed. - Conclusions:
- The test article Fosfomycin PEA salt up to the concentrations of 1000 and 2500 µg/ml, in the presence and in the absence of metabolic activation, respectively, did not induce any statistically significant increase of cells with chromosome aberrations in CHO cells.
- Executive summary:
The clastogenic potential of the test article Fosfomycin PEA salt was investigated by identifying chromosome aberrations in cultured Chinese hamster ovary cells (CHO) according to EU Method B.10 and OECD Guideline 473. The experiment was performed with and without rat liver S9 fraction as metabolizing system. The culture treatment, performed at 24 hours, lasted 3 hours in the test with metabolic activation and 18 hours in the test without metabolic activation. The cell harvesting was carried out at 20 and 44 hours, both with and without metabolic activation. The solvent (Ham’s F12 medium) was used as negative control. Ethylmethane sulfonate (EMS) (0.5 µg/ml) and Cyclophosphamide (CP) (50 µg/ml) were used as positive controls to assess the sensitivity of CHO cells to mutagens as well as the activity of the metabolizing system. The preliminary cytotoxicity test was performed as part of the main study. The test article was tested at the dosage levels of 3, 10, 30, 100, 300, 1000, 2500 and 5000 µg/ml, with and without metabolic activation. On the basis of the mitotic index evaluated (which is indicative of the test article cytotoxicity), 300, 1000 and 2500 µg/ml were selected for metaphase analysis at the 20 hour sampling, both with and without metabolic activation and 300, 1000 and 2500 µg/ml in the absence of metabolic activation and 100, 300 and 1000 µg/ml in the presence of metabolic activation for the metaphase analysis at the 44 hour sampling. The test article Fosfomycin PEA salt up to the concentrations of 1000 and 2500 µg/ml, in the presence and in the absence of metabolic activation, respectively, did not induce any statistically significant increase of cells with chromosome aberrations in CHO cells.
Referenceopen allclose all
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
PRELIMINARY CYTOTOXICITY TEST:
At 20 hour sampling time, at 2500 µg/ml, the reduction of the mitotic index in comparison with the negative controls was about 50%, both in the test without and with metabolic activation. At 1000 µg/ml, the reductions of the mitotic index were 40% and 50% in the absence and in the presence of S9 Mix, respectively. At the other concentrations tested no significant cytotoxicity was detected.
At 44 hour sampling, at the dose of 2500 µg/ml, while it was possible to analyse metaphases without metabolic activation, in the test with metabolic activation no metaphase cells were observed on the slides. At 1000 µg/ml with S9 Mix and 2500 µg/ml without metabolic activation the reductions of the mitotic index in comparison with the negative controls were 44 and 62% respectively. Less relevant cytotoxic effects were observed at the other doses tested.
METAPHASE ANALYSIS:
At neither sampling time, at none of the test article concentrations assayed was there an incidence of cells with chromosome aberrations statistically different from the control group, either in the presence or in the absence of metabolic activation. As expected, the reference mutagens, Ethylmethane sulphonate (EMS) and Cyclophosphamide (CP), produced statistically significant increases in the percentage of cells with chromosome aberrations, both including and excluding gaps (p<0.001). At none of the test article concentrations assayed was observed an incidence of polyploid cells or endoreduplications statistically different from the control group, either in the presence or in the absence of metabolic activation.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Gene mutation in vitro: Key study: Test method according to EU Method B.14 and OECD Guideline 471. GLP study. The test article 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.
Chromosome aberrations in vitro: Key study: Test method according to EU Method B.10 and OECD Guideline 473. The test article up to the concentrations of 1000 and 2500 µg/ml, in the presence and in the absence of metabolic activation, respectively, did not induce any statistically significant increase of cells with chromosome aberrations in CHO cells.
Justification for selection of genetic toxicity endpoint
No study was selected. The available two studies were negative.
Justification for classification or non-classification
Based on the available information on genetic toxicity in vitro, the substance is considered to be non-mutagenic, and therefore, the substance is not classified.
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