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EC number: 444-960-2 | CAS number: 39148-16-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames (OECD 471); GLP; S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WPA; 1.6 – 5000 µg/plate; negative
Chromosome aberration (OECD 473); GLP; human peripheral lymphocytes; 9.922 – 1320 µg/mL; negative
Gene mutation (OECD 476); GLP; Mouse lymphoma L5178 cells; 7.813 – 250 µg/mL; negative (Read-across to Fosetyl-Al, CAS 39148-24-8)
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:
- 23 Feb - 7 Apr 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- (adopted 1997) and current version of 2020
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- (adopted 1993)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- (adopted 1998)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (for S. typhimurium strains) and trp operon (for E. coli strain)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities). - Test concentrations with justification for top dose:
- Range finding experiment (with and without metabolic activation, performed only in TA100):
1.6, 8, 40, 200, 1000 and 5000 µg/plate
Experiment I: (with and without metabolic activation):
1.6, 8, 40, 200, 1000 and 5000 µg/plate
Experiment II: (with and without metabolic activation):
156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- other: 2-aminoanthracene (AAN)
- Details on test system and experimental conditions:
- Range Finding Experiment and Main Experiments:
METHOD OF APPLICATION: in agar (plate incorporation, range finding experiment, experiment I and experiment II without S9 mix) and pre-incubation (experiment II with S9 mix)
DURATION
- Preincubation period: 1 h
- Exposure duration: 72 h
NUMBER OF REPLICATIONS: triplicates (test item and positive controls), quintuplicate (solvent control) in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method:other: inspection of the background lawn - Rationale for test conditions:
- according to OECD guideline
- Evaluation criteria:
- A test item is considered as mutagenic if:
- the assay is considered as valide
- the results revealed statistical significance (p ≤ 0.01) and a significant dose-relation ship was present
- positive responses were reproducible
Acceptability
The assay is considered valid if the following criteria were met:
1. the mean negative control counts fell within the range of historical controls
2. the positive control substances induce clear increases in revertants confirming discrimination between different strains and an active S9-mix
3. no more than 5% of the plates were lost through contamination or some other unforeseen event - Statistics:
- Individual plate counts were determined separately and the mean and standard deviations of plate counts for each treatment were calculated.
Statistical analysis were performed by m-statistic (to prove Poisson-distribution) and Dunnett's test (comparison of counts from each dose with the control) and linear regression analysis (prove of dose-response). - Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance was completely soluble in water.
- Precipitation: No precipitation was observed at any concentration.
RANGE-FINDING/SCREENING STUDIES:
A range finding study was performed initially in tester strain TA100 to determine cytotoxicity. As the study design of experiment I was similar to the range finding test, the obtained mutagenicity data were included in experiment I (please refer to Table 1).
COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertants obtained for solvent and positive controls lay within the range of historical control data. Thus, the study was considered as valide. - Conclusions:
- Under the conditions chosen, the test substance was not mutagenic in this bacterial reverse mutation assay.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 9 Feb - 17 Apr 2001
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Only 200 metaphases counted, chromatid-and chromosome-type aberrations not reported separately; mycoplasma contamination not checked
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- (adopted 1997)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Version / remarks:
- (adopted 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH Harmonised Tripartite Guideline on Genotoxicity: Specific Aspects Of Regulatory tests (1995)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- 2016
- Deviations:
- yes
- Remarks:
- Only 200 metaphases tested instead of 300 per concentration; Chromatid-and chromosome-type aberrations not reported separately; mycoplasma contamination not checked
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- - Type and identity of media: HEPES-buffered RPMI medium containing 20% FCS and 50 µg/mL gentamycin. Phytohaemagglutinin was used as stimulants for cell division (2%).
- Blood from 3 healthy, non-smoking female donors was used for the experiments (experiment I, trial 1 and 2 of experiment II). - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Experiment I:
exposure 3 h, +/- S9: 9.922 - 1320 µg/mL (9.922, 13.23, 17.64, 23.52, 31.36, 41.81, 55.75, 74.33, 99.11, 132.1, 176.2, 234.9, 313.2, 417.7, 556.9, 742.5, 990.0 and 1320 µg/mL)
Experiment II:
trial 1, exposure 20 h -S9:
37.15 - 1320 µg/mL (37.15, 46.44, 58.05, 72.57, 90.71, 113.4, 141.7, 177.2, 221.5, 276.8, 346.0, 432.5, 540.7, 675.8, 844.8, 1056 and 1320 µg/mL)
trial 1, exposure 3 h +S9: 72.54 - 600.0 µg/mL (72.54, 85.35, 100.4, 118.1, 139.0, 163.5, 192.3, 226.3, 266.2, 313.2, 368.5, 433.5, 510.0 and 600.0 µg/mL)
trial 2, exposure 3 h +S9:
83.31 - 1320 µg/mL (83.31, 98.01, 115.3, 135.7, 159.6, 187.8, 220.9, 259.9, 305.7, 359.7, 423.2, 497.8, 585.7, 689.0, 810.6, 953.7, 1122 and 1320 µg/mL) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- cyclophosphamide
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 and 20 h
- Expression time (cells in growth medium): 20 h (including exposure time)
- Fixation time: 3 and 20 h treatment: 20 h
SELECTION AGENT: 2 h before harvesting, colchicine was added to the cultures (final concentration: 1 µg/mL)
STAIN: 4% solution of Giemsa
NUMBER OF REPLICATIONS: duplicates (test item and positive controls), quadruplicate (solvent control) in two independent experiments
NUMBER OF CELLS EVALUATED: 100 metaphases
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Determination of hyperdiploidy: yes - Rationale for test conditions:
- Test was performed according to OECD guideline that was valide during conduct of the study.
- Evaluation criteria:
- A test item is considered as clastogenic if:
- the proportions of cells with structural aberrations at one or more concentration exceeds the normal range in both replicates, and
- a statistically significant increase in the proportion of aberrant cells (excluding gaps) occurs at these doses
- the results were confirmed in the second experiment.
Increased incidence of cells with gaps or increased proportions of aberrant cells that did not exceed the normal range, or occurring only at very high or very toxic concentrations are likely to be concluded as "equivocal".
Assessment of the biological significance of such increases is likely only to be possible with reference to data from other test systems. Evidence of a dose-related effect is considered useful but not essential in the evaluation of a positive result. As cells with exchange aberrations or cells with greater than one structural aberration occur very infrequently in negative control cultures, their appearance is considered as biologically significant.
Acceptance criteria
The assay is considered valid if the following criteria were met
1. the proportion of cells with structural aberrations, excluding gaps, of the negative controls fall within the range of historical controls
2. the positive control substances induce a statistically significant increase in the number of aberrant cells
3. at least 160/200 intended cells are analysable at each dose level
4. the binomial dispersion test demonstrates acceptable heterogeinity between the replicates. - Statistics:
- Mean values and standard deviations were calculated. Statistical analyses were performed by Fisher´s exact test if the frequency of aberrant cells exceeded the normal range in both replicates at one or more concentrations. Statistical significance was considered for p≤ 0.05.
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Measurements on post treatment media did not reveal any marked effect.
- Effects of osmolality: Measurements on post treatment media did not reveal any marked effect.
- Water solubility: A stock concentration of 93.92 mg test item/mL water was determined as sufficiently soluble and did not result in precipitation at a final concentration of 939.2 µg/mL in culture medium.
- Precipitation: No precipitation was observed.
COMPARISON WITH HISTORICAL CONTROL DATA
The number of aberrant cells obtained for the solvent control lay within the range of historical control data.
VALIDITY OF THE STUDY
The study was considered valide as
1. no evidence of significant heterogeneity between replicate cultures was obtained in the binomial dispersion test and
2. the proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within the range of historical data and
3. at least 160/200 cells were analysed at each dose level and
4. the positive control chemicals induced statistically significant increases in the number of aberrant cells - Conclusions:
- Under the conditions chosen, the test substance did not induce chromosome aberrations and thus, was concluded not to be clastogenic.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 Jul - 5 Aug 1997
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- (guideline adopted 1996)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- (adopted 1996)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2016
- Deviations:
- not applicable
- Remarks:
- Thymidine kinase not used in current guideline (HPRT/XPRT)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- (adopted in June 1996)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Specific details on test material used for the study:
- - Purity test date: 13 Sep 1996 (reanalysis: 13 Sep 1998)
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640 supplemented media
RPMI A
gentamycin (100µg/mL), amphotericin B (2.5 µg/mL) and pluronic (0.5 mg/mL)
RPMI 10 (culture medium)
horse serum (10%, v/v), gentamycin (100µg/mL), amphotericin B (2.5 µg/mL) and pluronic (0.5 mg/mL)
RPMI 20
horse serum (20%, v/v), gentamycin (100µg/mL) and amphotericin B (2.5 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague Dawley rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Range-finding experiment (with and without S9 mix):
31.25, 62.5, 125, 250, 500 and 1000 µg/mL
Experiment 1 and 2 (with and without S9 mix):
7.813, 15.625, 31.25, 62.5, 125 and 250 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Fosetyl Al was sufficiently soluble in water (up to at least 35.4 mg/mL) - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- benzo(a)pyrene
- Details on test system and experimental conditions:
- Range-finding experiment: Cytotoxicity
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 3 days
NUMBER OF REPLICATIONS: single cultures
Main experiments:
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 2 days (sub-culturing was performed as required with the aim of not exceeding 1E+06 cells/mL and, where possible, retaining at least 1E+07 cells/flask); afterwards, cells were plated either for survival or viability in culture medium at a final concentration of 8 cells/mL for 9 or 8 days, respectively, or for mutagenicity in TFT-supplemented selection medium at a final concentration of 1E+04 cells/mL
- Selection time (if incubation with a selection agent): 12 - 13 days (mutagenicity)
SELECTION AGENT (mutation assays): 5-trifluorothymidine (3 µg/mL, TFT)
NUMBER OF REPLICATIONS: two replications each in 2 experiments for the test substance, positive controls were tested in single cultures. For each replication, 2 plates for survival or viability and 4 plates for mutagenicity were counted.
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative survival, relative total growth
OTHER EXAMINATIONS:
- Small and large colonies were counted for the negative and positive controls. - Evaluation criteria:
- Evaluation criteria
The test article was considered to be mutagenic if all the following criteria were met:
1. the assay is considered valide (please refer to acceptance criteria)
2. the mutant frequency at one or more doses was significantly greater than that of the negative control (p<0.05)
3. there was a significant dose-relationship as indictaed by the linear trend analysis (p<0.05)
4. the effects described above were reproducible.
Acceptance criteria
The assay was considered valide if all the following criteria were met:
1. the mutant frequencies in the solvent control cultures fell within the normal range (above 60 mutants per 1E+06 viable cells but not more than 3 times the historical mean value)
2. at least 1 concentration of each of the positive controls induced a clear increase in mutant frequency (the difference between the positive and solvent control mutant frequencies was greater than half the historical mean value)
3. the plating efficiencies of the negative controls from the mutation experiments were between the range of 60 - 140% on Day 0 and 70 - 130% on Day 2. - Statistics:
- Statistical significance of mutant frequencies (total wells with clones) was carried out according to the UKEMS guidelines (Robinson et al., 1990). Thus, the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration based on Dunnett's test for multiple comparisons, and secondly the data was checked for a linear trend in mutant frequency with treatment concentration using weighted regression. The test for linear trend is one-tailed, therefore negative trend was not onsidered as significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Reference:
Robinson WD, Green MHL, Cole J, Garner RC, Healy MJR and Gatehouse D. (1990 ). Statistical evaluation of bacterial/mammalian fluctuation tests. In Statistical Evaluation of Mutagenicity Test Data (Ed Kirkland DJ). Cambridge University Press, pp 102-140. - Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- precipitates starting at 125 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH measurements performed on medium post treatment revealed no marked change in pH at the highest dose tested.
- Effects of osmolality: no measurement required on post-tretament medium as the top dose tested was less than 10 mM (calculated with a molecular weight of 354.11 g/mol)
- Water solubility: soluble in water up to least 35.4 mg/mL following stirring and warming at 37 °C for approx. 2 h
- Precipitation: precipitates were observed starting at 125 µg/mL
RANGE-FINDING/SCREENING STUDIES:
Depressed relative survival was observed in the range-finding experiment starting from 125 µg/mL irrespective from S9 mix supplementation which is most probably due to physical loss of cells during removal of post-treatment precipitate, rather than evidence of genuine toxicity. No marked reduction in cloning efficiency was observed at any dose level tested.
The number of wells containing small and large colonies were scored for the solvent and positive controls. In negative controls, the proportion of small colonies ranged from 54 - 62%. A marked increase in the number of both, small and large colonies were determined after exposure to the positive control substances without altering the ratio of small/large colonies significantly. - Conclusions:
- Under the conditions chosen, the test substance did not induce mutations in mammalian cells.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- refer to analogue justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- In the available in vitro mammalian cell gene mutation study the source substance aluminium tris(ethyl phosphonate) did not induce mutations in mammalian cells. Applying the read-across approach, the target substance Fosetyl-sodium is not expected to differ in its genetic toxicity potential.
Referenceopen allclose all
Table 1. Test results of experiment I
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate ± SD |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 1001 |
TA 1535 |
WP2 uvrA |
TA 98 |
TA 1537 |
||
– |
water (100 µL) |
108 ± 14 |
23 ± 9 |
19 ± 5 |
34 ± 13 |
11 ± 3 |
– |
1.6 |
115 ± 16 |
27 ± 7 |
17 ± 5 |
65 ± 3 |
13 ± 1 |
– |
8 |
96 ± 12 |
23 ± 6 |
15 ± 6 |
63 ± 10 |
15 ± 3 |
– |
40 |
105 ± 10 |
25 ± 6 |
16 ± 4 |
59 ± 18 |
20 ± 7 |
– |
200 |
125 ±32 |
20 ± 4 |
18 ± 10 |
45 ± 7 |
13 ± 3 |
– |
1000 |
108 ± 13 |
17 ± 7 |
20 ± 2 |
61 ± 4 |
12 ± 5 |
– |
5000 |
113 ± 7 |
20 ± 6 |
18 ± 8 |
47 ± 17 |
19 ± 3 |
– |
Positive controls |
NaN3 |
NaN3 |
NQO |
2NF |
AAC |
Mean No. of colonies/plate ± SD |
700± 58 |
598± 17 |
559± 66 |
953± 22 |
194 ± 50 |
|
+ (1.6%) |
water (100 µL) |
112 ± 10 |
21 ± 4 |
25 ± 6 |
36 ± 7 |
14 ± 3 |
+ (1.6%) |
1.6 |
104 ± 14 |
22 ± 8 |
32 ± 6 |
36 ± 9 |
13 ± 2 |
+ (1.6%) |
8 |
106 ± 8 |
20 ± 4 |
25 ± 1 |
50 ± 7 |
12 ± 2 |
+ (1.6%) |
40 |
114 ± 7 |
25 ± 8 |
22 ± 3 |
46 ± 5 |
7 ± 5 |
+ (1.6%) |
200 |
115 ± 23 |
22 ± 2 |
27 ± 8 |
39 ± 6 |
12 ± 2 |
+ (1.6%) |
1000 |
113 ± 1 |
23 ± 5 |
28 ± 3 |
41 ± 4 |
11 ± 4 |
+ (1.6%) |
5000 |
127 ± 8 |
26 ± 5 |
23 ± 3 |
33 ± 8 |
13 ± 4 |
+ (1.6%) |
Positive controls |
AAN |
AAN |
AAN |
B[a]P |
AAN |
Mean No. of colonies/plate ± SD |
2218 ± 71 |
234 ± 10 |
54 ± 20 |
288 ± 40 |
281 ± 14 |
|
1: Mutagenicity data for TA100 were provided by the range finding test.
NaN3 = sodium azide
AAC = 9-aminoacridine
4NQO = 4-nitroquinoline 1-oxide
2NF = 2-nitro-fluorene
B[a]P = Benzo(a)pyrene
AAN = 2-aminoanthracene
SD = standard deviation
Table 2. Test results of experiment II
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate ± SD |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA 1535 |
WP2 uvrA |
TA 98 |
TA 1537 |
||
– |
water (100 µL) |
89 ± 9 |
24 ± 4 |
11 ± 3 |
26 ± 8 |
12 ± 5 |
– |
156.25 |
100 ± 3 |
21 ± 2 |
10 ± 1 |
25 ± 5 |
10 ± 3 |
– |
312.5 |
100 ± 15 |
16 ± 0 |
9 ± 1 |
23 ± 7 |
11 ± 2 |
– |
625 |
100 ± 8 |
17 ± 7 |
11 ± 6 |
34 ± 15 |
12 ± 2 |
– |
1250 |
83 ± 7 |
24 ± 11 |
6 ± 3 |
27 ± 4 |
13 ± 2 |
– |
2500 |
100 ± 10 |
13 ± 5 |
10 ± 5 |
23 ± 5 |
9 ± 7 |
– |
5000 |
80 ± 8 |
17 ± 4 |
10 ± 3 |
36 ± 7 |
13 ± 4 |
– |
Positive controls |
NaN3 |
NaN3 |
NQO |
2NF |
AAC |
Mean No. of colonies/plate ± SD |
683± 36 |
634± 20 |
922± 88 |
1081± 115 |
141 ± 8 |
|
+ (1.6%) |
water (100 µL) |
97 ± 17 |
16 ± 3 |
15 ± 5 |
34 ± 8 |
11 ± 3 |
+ (1.6%) |
156.25 |
111 ± 8 |
15 ± 1 |
17 ± 5 |
36 ± 6 |
13 ± 1 |
+ (1.6%) |
312.5 |
110 ± 4 |
14 ± 2 |
15 ± 6 |
28 ± 12 |
14 ± 3 |
+ (1.6%) |
625 |
111 ± 5 |
19 ± 1 |
9 ± 1 |
31 ± 9 |
16 ± 4 |
+ (1.6%) |
1250 |
100 ± 16 |
15 ± 4 |
10 ± 2 |
39 ± 11 |
19 ± 12 |
+ (1.6%) |
2500 |
93 ± 1 |
17 ± 5 |
11 ± 1 |
30 ± 3 |
15 ± 5 |
+ (1.6%) |
5000 |
88 ± 26 |
19 ± 7 |
11 ± 5 |
35 ± 6 |
14 ± 2 |
+ (1.6%) |
Positive controls |
AAN |
AAN |
AAN |
B[a]P |
AAN |
Mean No. of colonies/plate ± SD |
161 ± 26 |
68 ± 12 |
75 ± 22 |
140 ± 37 |
101 ± 27 |
|
NaN3 = sodium azide
AAC = 9-aminoacridine
4NQO = 4-nitroquinoline 1-oxide
2NF = 2-nitro-fluorene
B[a]P = Benzo(a)pyrene
AAN = 2-aminoanthracene
SD = standard deviation
In experiment II, only a weak positive control response was observed in strain TA100 in the presence of S9 mix for AAN. However, concurrent control treatments with this strain in the absence of S9 (as well as the negative controls in the presence of S9), and other strains in the presence of S9, served to otherwise confirm the correct strain and assay functioning, together with the metabolic activity of the S9 mix. It was therefore considered that this weak positive control response was not indicative of any strain or assay failure, and the data from these treatments were therefore accepted as valid.
Table 3: Historical control values
|
Strain |
S9 |
N |
Revertant colonies per plate ± SD |
Range |
|
Lower |
Upper |
|||||
Solvent controls |
TA98 |
- |
20 |
47.4 ± 10 |
33.6 |
61.4 |
+ |
20 |
44.3 ± 8.4 |
31.6 |
55.0 |
||
TA100 |
- |
20 |
117.1 ± 19.4 |
92.8 |
156.2 |
|
|
+ |
20 |
125.1 ± 21.0 |
97.2 |
153.4 |
|
TA1535 |
- |
20 |
19.5 ± 5.5 |
14.0 |
26.2 |
|
|
+ |
20 |
19.3 ± 5.1 |
14.0 |
25.6 |
|
TA1537 |
- |
19 |
14.4 ± 5.6 |
7.4 |
25.0 |
|
|
+ |
18 |
14.2 ± 5.1 |
7.2 |
19.0 |
|
WP2 uvrA |
- |
20 |
16.4 ± 4.5 |
11.8 |
21.0 |
|
|
+ |
20 |
20.4 ± 7.4 |
10.0 |
27.2 |
|
Positive controls |
TA98 |
- (2NF) |
18 |
303.6 ± 197.0 |
147.1 |
947.5 |
|
+ (B[a]P) |
20 |
141.2 ± 75.5 |
80.0 |
377.1 |
|
TA100 |
- (NaN3) |
20 |
603.4 ± 87.9 |
368.9 |
694.2 |
|
|
+ (AAN) |
19 |
1886.7 ± 368.1 |
1330.7 |
2548.2 |
|
TA1535 |
- (NaN3) |
20 |
604.4 ± 77.4 |
448.5 |
744.9 |
|
|
+ (AAN) |
20 |
157.6 ± 46.6 |
90.2 |
253.0 |
|
TA1537 |
- (AAC) |
19 |
196.2 ± 95.6 |
52.5 |
314.7 |
|
|
+ (AAN) |
18 |
241.6 ± 75.2 |
87.5 |
354.0 |
|
WP2 uvrA |
- (NQO) |
19 |
793.2 ± 131.3 |
601.3 |
1056.8 |
|
|
+ (AAN) |
20 |
127.5 ± 69.1 |
43.9 |
267.3 |
|
1: Mutagenicity data for TA100 were provided by the range finding test.
NaN3 = sodium azide
AAC = 9-aminoacridine
4NQO = 4-nitroquinoline 1-oxide
2NF = 2-nitro-fluorene
B[a]P = Benzo(a)pyrene
AAN = 2-aminoanthracene
SD = standard deviation
Table 1: Results of experiment I – cytotoxicity
Concentration (µg/mL) |
Experiment I: exposure period 3 h, -S9
Mitotic inhibition1(%)
|
Experiment I: exposure period 3 h, +S9
Mitotic inhibition1(%)
|
Water |
0 |
0 |
132.1 |
/ |
0 |
176.2 |
/ |
17 |
234.9 |
/ |
47 |
313.2 |
/ |
63 |
417.7 |
/ |
76 |
742.5 |
16 |
/ |
990.0 |
29 |
/ |
1320 |
35 |
/ |
1: Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100% with T = treatment and C = control
Table 2: Results of experiment II – cytotoxicity
Concentration (µg/mL) |
Experiment II (trial I): exposure period 20 h, -S9
Mitotic inhibition1(%)
|
Experiment II (trial I): exposure period 3 h, +S9
Mitotic inhibition1(%)
|
Experiment II (trial II): exposure period 3 h, +S9
Mitotic inhibition1(%)
|
Water |
0 |
0 |
0 |
433.5 |
/ |
0 |
/ |
510.0 |
/ |
0 |
/ |
600.0 |
/ |
0 |
/ |
675.8 |
0 |
/ |
/ |
810.6 |
/ |
/ |
0 |
844.8 |
7 |
/ |
/ |
953.7 |
/ |
/ |
0 |
1056 |
31 |
/ |
/ |
1122 |
/ |
/ |
12 |
1320 |
33 |
/ |
22 |
1: Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100% with T = treatment and C = control
Due to the marked difference in the susceptibilty determined as cytotoxicity observed between experiment I and II after exposure for 3 h in the presence of S9 mix, a second trial using higher test concentrations was included in experiment II. The reason for the marked difference in susceptibility between lymphocytes in experiment I and II are unclear but could be explained by different sensitivities of lymphocytes obtained from different donors.
Table 3. Test results of experiment I - cytogenicity.
Test item
|
Concentration (µg/mL) |
Mitotic Index (%) |
Aberrant cells (%) |
|
with gaps |
without gaps |
|||
Exposure period 3h, without S9 mix |
||||
Water |
|
100 |
2 |
2 |
NQO |
5 |
/ |
43 |
40 |
Test substance |
742.5 |
84 |
1 |
1 |
990 |
71 |
0 |
0 |
|
1320 |
66 |
4 |
3 |
|
Exposure period 3h, with S9 mix |
||||
Water |
|
100 |
0 |
0 |
CPA |
6.25 |
|
33 |
29 |
Test substance |
132.1 |
108 |
0 |
0 |
176.2 |
82 |
2 |
2 |
|
234.9 |
53 |
0 |
0 |
|
Exposure period 20h, without S9 mix |
||||
Water |
|
100 |
2 |
0 |
NQO |
2.5 |
|
56 |
45 |
Test substance |
844.8 |
93 |
4 |
4 |
1056 |
70 |
8 |
2 |
|
1320 |
68 |
4 |
1 |
|
Exposure period 3h, with S9 mix |
||||
Water |
|
100 |
6 |
4 |
CPA |
6.25 |
|
95 |
79 |
Test substance |
953.7 |
102 |
6 |
5 |
1122 |
88 |
2 |
2 |
|
1320 |
79 |
9 |
5 |
|
NQO: 4-Nitroquinoline 1-oxide; CPA: Cyclophosphamide (positive controls)
Table 4: Historical data (calculated in March 2001)
Sex and S9 treatment |
Category |
Total number of cells scored |
Aberrant cells scored per 100 cells |
|
Mean |
Calculated normal Range |
|||
female, -S9 |
Structural aberrations including gaps |
10399 |
2.04 |
0 - 6 |
|
Structural aberrations excluding gaps |
10399 |
1.13 |
0 -5 |
|
Polyploid cells |
10436 |
0.21 |
0 – 2 |
|
Numerical aberrations |
10436 |
0.35 |
0 – 3 |
female, +S9 |
Structural aberrations including gaps |
9800 |
1.62 |
0 – 6 |
|
Structural aberrations excluding gaps |
9800 |
0.84 |
0 – 4 |
|
Polyploid cells |
9833 |
0.23 |
0 – 2 |
|
Numerical aberrations |
9833 |
0.33 |
0 - 2 |
Table 1a: Range-finding experiment - 3 h exposure - without metabolic activation
Concentration |
Cloning efficiency [%] |
Survival [%] |
Relative Survival[%] |
0 (water) |
64.87 |
64.87 |
100.0 |
31.25 |
79.28 |
82.64 |
127.38 |
62.5 |
63.06 |
60.73 |
93.61 |
125 |
70.65 |
28.97 |
44.65 |
250 |
61.30 |
30.81 |
47.50 |
500 |
59.59 |
31.53 |
48.60 |
1000 |
72.70 |
40.96 |
63.14 |
Table 1b: Range-finding experiment - 3 h exposure - with metabolic activation
Concentration |
Cloning efficiency [%] |
Survival [%] |
Relative Survival[%] |
0 (water) |
84.09 |
84.09 |
100.0 |
31.25 |
70.65 |
72.83 |
86.61 |
62.5 |
63.06 |
63.96 |
76.06 |
125 |
77.01 |
53.72 |
63.89 |
250 |
59.59 |
38.73 |
46.06 |
500 |
70.65 |
27.75 |
33.0 |
1000 |
86.64 |
38.16 |
45.38 |
Table 2a: Experiment I - 3 h exposure - without metabolic activation
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutant frequency |
0 (water) |
127.41 |
100 |
107.53 |
7.813a |
98.82 |
|
|
15.625 |
109.12 |
97 |
132.42 |
31.25 |
103.76 |
101 |
151.52 |
62.5 |
131.28 |
109 |
118.31 |
125 |
117.07 |
96 |
120.27 |
250 |
110.06 |
100 |
115.89 |
NQO (0.05) |
113.97 |
68 |
442.67 |
NQO (0.1) |
96.50 |
77 |
475.15 |
NQO: 4-Nitroquinoline-n-oxide
a: not plated for viability or mutagenicity
Table 2b: Experiment I - 3 h exposure - with metabolic activation
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutant frequency |
0 (water) |
120.33 |
1.0 |
114.19 |
7.813a |
118.14 |
|
|
15.625 |
122.60 |
0.72 |
140.24 |
31.25 |
127.41 |
0.67 |
114.15 |
62.5 |
122.60 |
0.80 |
98.56 |
125 |
126.18 |
0.90 |
107.54 |
250 |
142.94 |
0.80 |
98.18 |
B(a)P (2) |
|
0.58 |
494.82 |
B(a)P (3) |
|
0.32 |
902.47 |
B(a)P: Benzo(a)pyrene
a: not plated for viability or mutagenicity
Table 3a: Experiment II - 3 h exposure - without metabolic activation
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutation frequency |
0 (water) |
82.24 |
1.0 |
219.52 |
7.813a |
87.30 |
|
|
15.625 |
92.80 |
1.04 |
206.07 |
31.25 |
87.96 |
1.23 |
177.53 |
62.5 |
89.99 |
1.32 |
198.31 |
125 |
151.52 |
1.49 |
210.28 |
250 |
132.63 |
1.66 |
195.58 |
NQO (0.05) |
85.35 |
1.32 |
321.77 |
NQO (0.1) |
90.68 |
1.06 |
572.35 |
NQO: 4-Nitroquinoline-n-oxide
a: not plated for viability or mutagenicity
Table 3b: Experiment I - 3 h exposure - with metabolic activation
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutant frequency |
0 (water) |
99.62 |
1.0 |
205.86 |
7.813a |
112.97 |
|
|
15.625 |
104.62 |
1.04 |
177.66 |
31.25 |
106.38 |
0.99 |
149.38 |
62.5 |
123.77 |
1.06 |
187.27 |
125 |
110.06 |
1.09 |
198.12 |
250 |
126.18 |
1.28 |
182.24 |
B(a)P (2) |
84.09 |
0.55 |
1178.82 |
B(a)P (3) |
52.42 |
0.32 |
1305.76 |
B(a)P: Benzo(a)pyrene
a: not plated for viability or mutagenicity
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Mammalian erythrocyte micronucleus test, mouse (OECD 474); GLP; 1063 – 4250 mg/kg bw/day; negative
(Read-across to Fosetyl-Al, CAS 39148-24-8)
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- (guideline adopted 1996)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- (adopted in March 1996)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 2016
- Deviations:
- yes
- Remarks:
- at least 4000 PCEs should be scored; slightly younger animals were used
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
- Version / remarks:
- (adopted in June 1996)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian erythrocyte micronucleus test
- Specific details on test material used for the study:
- - Purity test date: 13 Sep 1996 (reanalysis: 13 Sep 1998)
- Species:
- mouse
- Strain:
- other: CD-1 (outbred)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River UK Ltd., Margate, UK
- Age at study initiation: range-finder: 51 - 66 days, main study: 36 - 43 days
- Weight at study initiation: range-finder: 31 - 39 g (males), 24 - 33 g (females); main study: 24 - 33 g (males), 20 - 26 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: animals were not starved for diet and water prior to dosing
- Housing: animals were housed in groups of no more than 3 animals of the same sex
- Diet: Special Diets Services Ltd, RM1. [E]. SQC., ad libitum
- Water: public water supply, ad libitum
- Acclimation period: range-finder: 15 days, main study: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 42 - 65
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: methyl cellulose (0.5% (w/v))
- Concentration of test material in vehicle:
range-finder: 200, 225 and 250 mg/mL
main study: 53.13, 106.3 and 212.5 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared freshly by suspending Fosetyl Al (with homogenisation) in methyl cellulose. Further dilutions were made in the vehicle to achieve the respective concentrations needed for gavage. Test article preparations were protected from light, mixed by inversion during dosing and used within two hours of formulation.
Analytical analysis verified accuracy of the dosing solutions prepared for the main study (Analytical report CLE 198/112-01F, 1997, Covance). Achieved concentrations ranged from 108 - 130% of the nominal concentrations (low-dose: 108 and 110%, mid-dose: 104 and 112% and high-dose: 127 and 130%) and were thus higher than the nominal concentrations. However, as the signs of toxicity were not unacceptably severe in high-dose animals according to the authors, the study was considered as valid although 4 high-dose females died after administration of the test substance.
RANGE-FINDING STUDY
Initially, a range-finding study was performed to determine the maximum acceptable dose. Groups of 3 animals/sex were exposed once to 4000, 4500 and 5000 mg/kg bw via gavage. Clinical signs of toxicity and body weight development were observed over a time period of 4 days. - Duration of treatment / exposure:
- not applicable
- Frequency of treatment:
- single treatment
- Post exposure period:
- test article and vehicle exposed mice: 24, 48 and 72 h after treatment
CPA exposed mice: 24 h after treatment - Dose / conc.:
- 1 063 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 125 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 4 250 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 5 (plus 5 spare animals/sex exposed to the highest dose)
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- 4 mg cyclophosphamide (CPA)/mL in physiological saline
- Justification for choice of positive control(s): due to the known clastogenic properties, CPA was selected as appropriate positive control
- Route of administration: gavage
- Doses / concentrations: 80 mg/kg bw - Tissues and cell types examined:
- Tissue: bone marrow
Cell type: bone marrow cells - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: range finding study performed to find the maximum tolerated dose.
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): test article and vehicle treated mice were killed 24, 48 and 72 h after gavage. Animals treated with the positive control substance CPA were killed 24 h after substance administration. Mice were killed in the same order as they were dosed.
DETAILS OF SLIDE PREPARATION: bone marrow was isolated from both femurs from each animal except one animal where only a single femur could be sampled. After centrifugation, one drop of cell suspension was used to prepare a smear on a slide, which was allowed to air-dry followed by fixation for 5 min in absolute methanol. Slides were stained with Giemsa (1:6 diluted (v/v)). Afterwards, slides were washed and mounted with coverslips.
METHOD OF ANALYSIS: 2 slides were analysed off-site by a trained analyst and were scored for polychromatic and normochromatic erythrocytes until at least 1000 cells had been analysed. Counting continued until at least 2000 PCE had been observed. - Evaluation criteria:
- Evaluation criteria
The test was considered positive if:
1. a statistically significant increase in the frequency of micronucleated PCE occurred at least at one dose, at one sampling time
2. the frequency of micronucleated PCE at such point exceeded the historical control range.
Increased but statistically insignificant increases in frequencies of micronucleated PCE at one samling time in conjunction with either 1 or 2 of the above concentrations at another sampling time were to be cause for additional testing or analysis.
Acceptability
The assay was considered valid if the following criteria were met:
1. the heterogeinity Chi square test provided evidence of acceptable variability between animals within a group, and
2. the incidence of micronucleated PCE in vehicle control groups fell within or close to the historical vehicle control range
3. at least 8 animals (males plus females) out of each group at each kill time were available for analysis
4. the positive control chemical (CPA) induced a statistically significant increase in the frequency of micronucleated PCE. - Statistics:
- The ratio of PCE/NCE for each animal and the mean for each group was calculated. Further, the individual and group mean frequencies of micronucleated PCE/1000 were determined. For each group, inter-individual variation in the number of micronucleated PCE was estimated by means of a heterogeneity Chi square test. The numbers of micronucleated PCE in each treated group (males and females, separately and combined) were then compared with the numbers in vehicle control groups by using a 2x2 contingency table to determine Chi square. Probability values of p ≤ 0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- 1063 mg/kg bw: 1 unscheduled death, no clinical signs of toxicity; 4250 mg/kg bw: 4/5 females died prior to scheduled sampling and signs of clinical toxicity were observed
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 4000 - 5000 mg/kg bw
- Clinical signs of toxicity in test animals:
5000 mg/kg bw: 1 female was killed in extremis on Day 2 post dosing. Further, several signs of clinical toxicity were obsered including lethargy, prostation, abnormal breathing, eye closure and sunken eyes.
4500 mg/kg bw: 3 males were found dead on Day 1 post dosing. Further, test animals revealed signs of lethargy, tremors, coldness and abnormal breathing.
4000 mg/kg bw: No mortality occured and no clinical signs of toxicity were observed.
-Observation time: Animals were observed for mortality and clinical signs over a period of 4 days.
- Other: Based on the determined mortality and clinical symptoms, 4250 mg/kg bw were established as highest acceptable dose for the main study.
RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals:
1063 mg/kg bw: 1 female died prior to the scheduled sampling time. No clinical signs of toxicity were observed.
2125 mg/kg bw: No mortality occured and no clinical signs of toxicity were observed.
4250 mg/kg bw: 4 females died one day after dosing prior to the scheduled sampling time. Further, test animals showed signs of lethargy, hunched posture, eye closure and tremors. To enable inclusion of at least 8 animals per group (males and females) and each sampling time point in the analysis according to the acceptability criteria, the spare females treated with the highest dose were included in the analysis.
- Induction of micronuclei (for Micronucleus assay): Group mean frequencies of micronucleated PCE were comparable among the groups and were not statistically significant different by Chi square test except the mean frequency of the low-dose group determined at the 24 h sampling time point which was statistically decreased compared to the control value. Moreover, a slight but insignificant increase in micronucleated PCE was observed in females at the 48 h sampling time point (0.2, 0.63, 0.4 and 1.0 for control, low-, mid- and high-dose animals). As no dose-dependency was observed, these effects were interpreted as incidental (please refer to Table 2 and 3).
- Ratio of PCE/NCE (for Micronucleus assay): The ratios of PCE/NCE were similar between test and control animals. A slight but not statistically significant increase in the PCE/NCE ratio was observed in females at the 48- and 72 h sampling time points in all dose groups (please refer to Table 2).
- Statistical evaluation: No statistically significant differences were observed in any evaluated parameter among the test groups except the the mean frequency of micronucleated PCE of the low-dose group determined at the 24 h sampling time point which was statistically decreased compared to the control value (please refer to Table 3). As no dose-dependency was observed, the effect was interpreted as incidental (please refer to Table 3). - Conclusions:
- In this in vivo erythrocytes micronucleus study performed according to OECD 474 and in compliance with GLP, the test substance did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 4250 mg/kg bw/day. Thus, the test substance was concluded not to be clastogenic.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- refer to analogue justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- In the available in vivo erythrocytes micronucleus study performed according to OECD 474 and in compliance with GLP, the source substance aluminium tris(ethyl phosphonate) did not induce micronuclei in erythrocytes of the bone marrow of mice treated up to 4250 mg/kg bw/day. Thus, the test substance was concluded not to be clastogenic. Applying read-across, the target substance Fosetyl-sodium is not expected to be clastogenic under in vivo conditions.
Referenceopen allclose all
Table 1: Results of the in vivo micronucleus assay in male mice.
|
Mean PCE / NCE at sampling time |
Total micronuclei per 1000 PCE at sampling time |
||||||
Exp group |
Number of animals |
Dose [mg/kg bw] |
24 h |
48 h |
72 h |
24 h |
48 h |
72 h |
Vehicle control (methyl cellulose (0.5% (w/v))) |
5 |
0 |
1.03 |
0.86 |
0.90 |
0.5 |
0.5 |
0.6 |
Positive control (CPA) |
5 |
80 |
0.6 |
|
|
24.8 |
|
|
Fosetyl Al |
4 |
1063 |
1.0 |
0.97 |
1.13 |
0.2 |
0.4 |
0.7 |
Fosetyl Al |
5 |
2125 |
1.41 |
0.69 |
0.97 |
0.2 |
0.2 |
0.3 |
Fosetyl Al |
5 |
4250 |
0.85 |
1.0 |
1.23 |
0.5 |
0.5 |
0.1 |
Table 2: Results of the in vivo micronucleus assay in female mice.
|
Mean PCE / NCE at sampling time |
Total micronuclei per 1000 PCE at sampling time |
||||||
Exp group |
Number of animals |
Dose [mg/kg bw] |
24 h |
48 h |
72 h |
24 h |
48 h |
72 h |
Vehicle control (methyl cellulose (0.5% (w/v)) |
5 |
0 |
0.78 |
0.83 |
0.88 |
0.7 |
0.2 |
0.3 |
Positive control (CPA) |
5 |
80 |
0.63 |
|
|
14.9 |
|
|
Fosetyl Al |
5* |
1063 |
0.77 |
1.06 |
1.12 |
0.10 |
0.63 |
0.5 |
Fosetyl Al |
5 |
2125 |
0.85 |
0.97 |
1.43 |
0.2 |
0.4 |
0.3 |
Fosetyl Al |
5 |
4250 |
0.78 |
1.24 |
1.5 |
0.9 |
1.0 |
0.4 |
*: samples from 4 animals were collected for the 48 h sampling time point
Table 3: Results of the in vivo micronucleus assay – mean values per treatment group.
Treatment group |
Dose [mg/kg bw] |
Sampling time [h] |
Mean frequency of micronucleated PCE per 1000
|
PCE/NCE ratio |
Vehicle control |
0 |
24 |
0.6 |
0.91 |
48 |
0.35 |
0.845 |
||
72 |
0.45 |
0.89 |
||
Test substance |
1063 |
24 |
0.15*t |
0.885 |
48 |
0.50 |
1.015 |
||
72 |
0.60 |
1.125 |
||
2125 |
24 |
0.2 |
1.13 |
|
48 |
0.3 |
0.83 |
||
72 |
0.3 |
1.2 |
||
4250 |
24 |
0.7 |
0.815 |
|
48 |
0.75 |
1.12 |
||
72 |
0.25 |
1.365 |
||
Positive control (CPA) |
80 |
24 |
19.85** |
0.615 |
*statistically significant (p ≤ 0.05)
**statistically significant (p ≤ 0.001)
t: represents a statistically decrease in micronucleated cells
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
To characterize the genotoxic potential of the test substance, a gene mutation study in bacteria, a cytogenetic assay in human lymphocytes, and a gene mutation study in mammalian cells with the analogue substance Fosetyl-AL (RL1) are available. No guideline and GLP conform data on gene mutation in mammalian cells are available for the test substance. However, there are reliable data on the analogue substance Fosetyl-Al (CAS 39148-24-8) available, which are considered suitable for read-across. Moreover, in vivo mutagenicity data obtained for the read-across substance Fosetyl-Al are considered in addition to the available in vitro genotoxicity tests. For details on the read-across approach, please refer to the analogue justification (section 13 of the technical dossier).
Gene mutation in bacteria
The gene mutation test in bacteria (Ames assay) was conducted according to OECD Guideline 471 and in compliance with GLP with a standard battery of tester strains including 4 Salmonella typhimurium strains (TA 98, TA 100, TA 1535, TA 1537) and one E.coli strain (WP2 uvrA) (M-207083-01-1).The test substance was completely soluble in water and no precipitation was observed at any concentration tested. Initially, a range finding experiment was performed in tester strain TA100 using final concentrations of 1.6, 8, 40, 200, 1000 and 5000 µg/plate to evaluate toxicity using the plate incorporation assay. The remaining tester strains were tested accordingly in experiment I. As the study design of experiment I was similar to the range finding test, the obtained mutagenicity data for TA100 were included in experiment I. A second experiment using the plate incorporation assay without S9 mix and the preincubation method with S9 mix was performed with all tester strains with concentrations ranging from 156.25 to 5000 µg (156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate). The test substance did not induce relevant increases in revertant colony numbers of any tester strain neither in the absence nor presence of metabolic activation. Further, no cytotoxicity was observed. Solvent and positive controls were considered as valid as the mean numbers of revertant colonies of the solvent controls fell within the range of historical data, and positive control treatments increased the number of revertants significantly. In conclusion, the test substance is considered as non-mutagenic in the Ames test.
Cytogenicity in mammalian cells
Further, a GLP-guideline compliant study according to OECD Guideline 473 was performed to determine clastogenic properties of the test substance (M-207094-01-1). Human lymphocytes obtained from 3 donors were exposed to the test substance in the presence and absence of metabolic activation in two independent experiments. In experiment I, cells were treated with a broad range of doses ranging from 9.9 – 1320 µg/mL (the recommended maximum dose of 10 mM) for 3 hours irrespective of S9 supplementation following a 17 hour recovery period. Chromosome aberrations were analysed at three dose levels (742.5, 990.0 and 1320 µg/mL in the absence and 132.1, 176.2 and 234.9 µg/mL in the presence of S9 mix). The highest chosen concentrations induced approximately 35% and 47% mitotic inhibition in the absence and presence of S9 mix, respectively. In experiment II, the exposure period was elongated to 20 h in the absence of S9 mix whereas the exposure time was kept unchanged in the presence of S9 mix (3 h) followed by a 17 h recovery period. Concentrations ranging from 72.54 – 600 µg/mL and 37.15 – 1320 µg/mL were applied in the presence and absence of S9 mix, respectively. Due to the marked difference in the susceptibility determined as cytotoxicity observed between experiment I and II after exposure for 3 h in the presence of S9 mix, a second trial using higher test concentrations was included in experiment II (83.31 – 1320 µg/mL, 3 h exposure in the presence of S9 mix). The reason for the marked difference in susceptibility between experiment I and II remained unclear but might be attributed to different sensitivities of donors. Chromosome aberrations were analysed at three dose levels (844.8, 1056 and 1320 µg/mL in the absence and 953.7, 1122 and 1320 µg/mL in the presence of S9 mix) including the highest tested concentration of 1320 µg/mL, which induced approximately 33% and 22% mitotic inhibition in the absence and presence of S9 mix respectively. The test substance revealed a frequency of aberrant cells comparable to the respective solvent controls and historical data either in the absence or presence of metabolic activation. Validity of the study was confirmed by the solvent and positive controls as the mean numbers of aberrant cells of the solvent controls fell within the range of historical data, and positive control treatments increased the number of aberrant cells significantly. Therefore, the test substance is not considered to induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested at concentrations up to 10 mM, the recommended maximum concentration according to current regulatory guidelines, in both the absence and presence of S9 mix.
Gene mutations in mammalian cells
No reliable studies on genotoxic properties in mammalian cells are available for the test substance. Therefore, data obtained for Fosetyl-Al are considered following a read-across approach based on similarities in structure and intrinsic properties.
The potential to induce genotoxicity in mammalian cells of Fosetyl-Al was determined in a GLP-compliant study performed according to OECD Guideline 476 (M-184459-01-1). Mouse lymphoma L5178Y cells were treated in the absence and presence of metabolic activation to concentrations ranging from 8 – 250 µg Fosetyl-Al/mL (7.813, 15.625, 31.25, 62.5, 125 and 250 µg/mL) for 3 hours. Precipitates were observed in cultures treated with the 2 top doses tested. The test substance did not induce cytotoxicity in the applied concentrations as cloning efficiency and relative total growth values did not vary between solvent and test substance exposed cultures. No increase in the mutation frequency was determined in the presence or absence of S9 mix in Fosetyl-Al exposed cultures at any dose level tested. The positive control substances Benzo(a)pyrene and 4-nitroquinoline-n-oxide induced a marked increase in the mutation frequency thereby validating the study. Moreover, the number of small and large colonies was increased in positive control treated cultures compared to the solvent controls. In conclusion, Fosetyl-Al is considered to be non-mutagenic in the conducted mouse lymphoma forward mutation assay.
For completness also the data on Fosetyl-sodium are reported, but not reliable and not considered for hazard assessment:
The potential to induce genotoxicity in mammalian cells of the test substance was determined in a study performed similar to OECD Guideline 476, but is performed according to Chinse GLP regulations which differ from the generaly accepted ones (DL1600264, 2017). Chinese hamster lung fibroblasts V79 cells were treated in the absence and presence of metabolic activation to 160, 500, 1600, and 5000 µg/mL for 6 hours. The test substance induced cytotoxicity in the absence of S9 at and above 1600 µg/mL. Here, the relative colony formation rate was reduced to 84.8% and 64.3% at 1600 µg/mL and 5000 µg/mL, respectively. No increase in the mutation frequency was determined in the presence or absence of S9 mix in exposed cultures at any dose level tested.The positive control substances Benzo(a)pyrene and ethyl methane sulfonate induced a marked increase in the mutation frequency, thereby validating the study. In conclusion, the test substance was considered to be non-mutagenic in the conducted gene mutation assay using Chinese hamster lung fibroblasts V79 cells.
The original study was written in Chinese. The translated English version was available and used for assessment. However, according to the newest OECD 476 guideline the following acceptance criteria should be provided:
- The concurrent negative control is considered acceptable for addition to the laboratory historical negative control
- Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
- Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results
- Adequate number of cells and concentrations are analysable
- The criteria for the selection of top concentration are consistent
The first two bullet points cannot be checked as no information on historical controls were provided in the report. Further, the laboratory is a legally established and nationally qualified accreditation institute, but not GLP-certificated. Bullet points 3 and 5 are fulfilled. Another restriction is the number of cells analysed (bullet point 4). In this test, an insufficient number of cells were analysed. Overall, the study is considered as unreliable (RL3).
Genetic toxicity in vivo
No genotoxicity studies in vivo are available for the test substance. However, as reliable data on clastogenic properties in vivo are available for the analogue substance Fosetyl-Al, these data are taken into account for the evaluation of the genotoxic potential of the test substance.
Groups of 5 CD-1 outbred mice per sex were exposed once to 1063, 2125 and 4250 mg/kg bw/day via gavage according to GLP and OECD Guideline 474 (M-178982-01-1). The maximum tolerable dose was determined in a range finding study. Control animals received the concurrent vehicle methyl cellulose (0.5% (w/v) or 80 mg/kg bw/day cyclophosphamide as positive control substance. Five additional animals per sex were exposed with the highest dose applied as spares. Accuracy of dosing solutions was verified by HPLC analysis. Bone marrow samples of vehicle and test animals were collected 24, 48 and 72 hours after test substance administration. Samples from animals treated with the positive substance were collected 24 h after substance administration. Unscheduled mortality was observed in the low- and high-dose group, in which 1 or 4 females died prior to the scheduled sampling time points, respectively. Thus, the inclusion of females from spares to enable analysis of at least eight animals per group (males and females combined) was required. Clinical signs of systemic toxicity were observed in the high-dose group including lethargy, hunched posture, eye closure and tremors. Group mean frequencies of micronucleated polychromatic erythrocytes (PCE) were comparable among the groups and were statistically not significant except the mean frequency of the low-dose group determined at the 24 h sampling time point which was statistically decreased compared to the control value. A slight but not significant increase in micronucleated PCE was observed in low- and high-dose females at the 48 h sampling time point. As no dose-dependency was observed, these effects were interpreted as incidental. Furthermore, the ratios of PCE/NCE were similar between test and control animals although a slight but not statistically significant increase in the PCE/NCE ratio was observed in females at the 48- and 72 h sampling time points in all dose groups. In contrast, treatment with the positive control substance CPA induced a statistically significant increase in micronucleated PCE thereby validating the study. HPLC analysis revealed achieved concentrations ranging from 108 – 130% of the nominal concentration. However, as the signs of toxicity were not unacceptably severe in high-dose animals according to the authors, the study was considered as valid although 4 high-dose females died after administration of the test substance. In conclusion, Fosetyl-Al administered to mice at doses up to 4250 mg/kg bw/day, a dose at which mortality was observed, did not induce clastogenic effects in the conducted study.
Overall conclusion
Taken together, the test substance did not show mutagenic properties in the bacterial reverse mutation test and did not induce chromosomal aberrations in primary human lymphocytes. The analogue substance Fosetyl-Al did not show mutagenic properties in vitro and in vivo. Thus, taken all these data into account, the test substance is not expected to exhibit genotoxic properties.
Justification for classification or non-classification
The available data on genetic toxicity of the test substance do
not meet the criteria for classification according to Regulation (EC)
1272/2008, and are therefore conclusive and do not warrant
classification.
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