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EC number: 212-783-8 | CAS number: 868-85-9
- Life Cycle description
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- Endpoint summary
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- 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
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- Toxicological Summary
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- Acute Toxicity
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- Genetic toxicity
- Carcinogenicity
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- Specific investigations
- Exposure related observations in humans
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
IN VITRO STUDIES
-Gene mutation (Ames test): S. typhimurium TA 100: positive with
metabolic activation; S. typhimurium TA 98, TA 1535, TA1537, TA 97:
negative (equivalent to OECD 471); concentrations: 0, 100, 333, 1000,
3333, 5000, 7500, 10000 µg/plate
-Gene mutation (Ames test): S. typhimurium TA 100: positive with
metabolic activation; S. typhimurium TA 98, TA 1535, TA1537: negative
(NTP standard protocol); RL4 (secondary literature)
- Gene mutation (Ames test): S. typhimurium TA 100; TA 98, TA 1535,
TA1537: negative (equivalent to OECD 471); concentrations: 0, 100, 333,
1000, 3333, 10000 µg/plate
- Gene mutation (Ames test): S. typhimurium TA 100; TA 98, TA 1535,
TA1537: negative (equivalent to OECD 471); concentrations: 0, 20, 100,
500, 2500, 12500 µg/plate (first test); 0, 775, 1550, 3100, 6200, 12400
µg/plate (repeat test)
- Gene mutation (Ames test): S. typhimurium TA 1535, TA 1537, TA 98 and
TA 100 (Reevaluation of false positive Ames tests).
- Unscheduled DNA synthesis: hepatocytes from rats untreated and
pretreated with Aroclor or 3-methlycholanthrene. Results: positive in
primary rat hepatocytes pretreated with Aroclor-1254 (Aro) and
3-methylcholanthrene (3-MC); negative in untreated primary rat
hepatocytes (equivalent to OECD TG 482).
- Unscheduled DNA synthesis: primary rat hepatocytes. Results: negative
(RL4; secondary literature).
-Gene mutation (Mouse lymphoma L5178Y cells assay): positive with
metabolic activation (equivalent to OECD guideline 476).
-Gene mutation (Mouse lymphoma L5178Y cells assay): positive with
metabolic activation [NTP standard protocol; RL4 (secondary literature)].
-Chromosome aberration: positive in presence and absence of metabolic
activation (equivalent to OECD guideline 473).
- Sister chromatid exchange assay: positive in presence and absence of
metabolic activation (equivalent to OECD guideline 479).
IN VIVO STUDIES
2 micronucleus assays: one positive (equivalent to OECD 474) and one
negative (GLP, OECD 474).
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with restrictions (No GLP, Only 4 strain tested). However, according to OECD SIDS a reliability of 1 was given.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- No GLP. Only 4 strain tested
- Principles of method if other than guideline:
- According Ames et al (1973), Proc. nat. Acad. Sci. 70, 2281-2285.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- other: S. typhimurium TA 98, 100, 1535, 1537 or TA 97
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix obtained from liver of Aroclor 1254 orally exposed rats and hamsters.
- Test concentrations with justification for top dose:
- 0, 100, 333, 1000, 3333, 5000, 7500, 10000 µg/plate.
- 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:
- sodium azide
- Remarks:
- TA 1535 and TA 100, -S9. The concentration was not reported.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylenediamine (TA 98, -S9). The concentration was not reported.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA 97 and TA 1537, -S9. The concentration was not reported.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene, +S9. The concentration was not reported.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48h
- Selection time (if incubation with a selection agent): 48h
PRELIMINARY DOSE-SETTING EXPERIMENT
Dimethyl phosphonate was initially tested with strain TA100 in the presence and absence of metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less, when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his- pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Non toxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity. As a rule, at least one toxic dose was incorporated into the first mutagenicity test; the repeat test(s) occasionally had the doses adjusted so that and apparent toxic dose was not reached. - Evaluation criteria:
- The criteria used for data evaluation are the following: 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; 2) non-mutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity.
- Key result
- Species / strain:
- other: S. typhimurium TA 98, 100, 1535, 1537 or TA 97
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- S. typhimurium TA 100
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- TA 100 (+S9, 10% HLI, 10000 mg/plate); TA1535 (+S9, 10% HLI, 10000 mg/plate ), TA 98 (+S9, 10% HLI and RLI, 10000 mg/plate )
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The mutagenicity of dimethyl phosphonate was tested in two laboratories. In one laboratory (Case Western University) it was judged equivocal, in the other laboratory (EGG Mason Research Institute, later Microbiological Associates) it was tested with a positive result in strain S. typhimurium TA 100.
Conclusion: dimethyl phosphonate was evaluated to be positive with TA 100, negative with TA 98, 1535, 1537 or 97. - Conclusions:
- Interpretation of results: positive S. typhimurium TA 100.
- Executive summary:
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were treated with dimethyl phosphonate diluted in H2O using the Ames preincubation method equivalent to OECD Guideline No. 471 with deviations (No GLP. Only 4 strain tested), both with and without the addition of a rat and hamster liver homogenate metabolising system. The dose range was determined in a preliminary toxicity assay with upper dose 10 mg/plate. The mutagenicity of dimethyl phosphonate was tested in two laboratories. In one laboratory (Case Western University) it was judged equivocal, in the other laboratory (EGG Mason Research Institute, later Microbiological Associates) it was tested with a positive result in strain TA 100.
Dimethyl phosphonate was evaluated to be positive with TA 100, negative with TA 98, 1535, 1537 or 97.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with restrictions (only 4 strains tested). However, according to OECD SIDS a reliability of 1 was given.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Only 4 strains tested
- Principles of method if other than guideline:
- According Ames et al (1973) Proc. nat. Acad. Sci. 70, 2281-2285.
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- other: S. typhimurium TA 98, 100, 1535, 1537
- Additional strain / cell type characteristics:
- other: All the strains are deep rough (i.e. they are partly deficient in lipopolysaccharide side chains of their cell walls). Secondly, the UV repair ability is reduced.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix obtained from livers of at least 6 adult male Sprague Dawley rats dosed with a single intraperitoneal injection of Aroclor 1254
- Test concentrations with justification for top dose:
- First test: 0, 20, 100, 500, 2500, 12500 µg/plate. Due to the substance’s low toxicity the doses chosen for the repeat tests are: 0, 775, 1550, 3100, 6200, 12400 µg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) DMSO (for positive control) ; ethanol (for dimethyl phosphite).
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- TA1535; without S9; 10 µg per plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: nitrofurantoin (TA100; without S9); 0.2 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-1,2-phenylene diamine (TA 1537, TA98; without S9); 10 µg/plate; 0.5 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (all strains with S-9 mix); 3 µg/plate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 hours
SELECTION AGENT (mutation assays): L-histidine.
Four plates per strain and dose, both with and without S-9 mix, were used for mutant count. The same number of plates made up the negative control; these being filled with the solvent without test substance. Four plates per strain were also used for each positive control.
DETERMINATION OF CYTOTOXICITY
The toxicity of the substance was assessed in three ways:
1) The background growth on the plates for the mutant determination was grossly appraised.
2) A toxic effect of the substance was assumed when the mutant count per plate was clearly lower than the negative control count in dose correlation.
3) The titer was determined (only without S-9 mix). - Evaluation criteria:
- The following criteria were used for the acceptance of an assay:
a) The negative controls have to be within the expected range, as defined by the literature data, and the laboratories´own historical data.
b) The positive controls have to show sufficient effects as defined by the laboratories´experience.
c) Titer determinations must provide sufficient bacterial density in the suspension.
An assay which was not in agreement with at least one the above criteria was not used for assessment. Furthermore, the data generated in this assay have to be confirmed by two additional independent experiments. Even if the criteria for the points (a), (b) and (c) are not met, an assay was accepted if it showed mutagenic activity of the test compound
ASSESSMENT OF RESULTS
A reproducible and dose-related increase in mutant counts for at least one strain is considered positive. For TA 1535, TA 100, and TA 98 in principle a twofold increase compared to the corresponding negative controls should be reached, whereas for TA 1537 at least a threefold should be reached. Otherwise, the result is evaluated as negative.
In case of questionable results, the investigation was continued, until a final evaluation was possible. - Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: >= 6200 µg/plate weak strain-specific bacteriotoxic effect.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- Tests with TA 100 and S-9 mix gave equivocal results. In a first assay the mutant counts were significantly increased. As in a second test the results could not be reproduced, they were regarded as a random result. Dimethyl phosphonate caused no biologically relevant (parameters: dose effect, doubling) variation from the respective negative control. Conclusion: No indications of mutagenic effects of dimethyl phosphonate could be found.
- Conclusions:
- Interpretation of results: negative
- Executive summary:
Evidence of mutagenic activity of dimethylphospite was not found. Neither a dose-related doubling nor a biologically relevant increase in the mutant count, in comparision with the negative controls, was observed.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study. Adopted according to OECD SIDS (public available peer reviewed source). The original source is available and has been reviewed.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- No GLP
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- Laboratory cultures were confirmed as free from mycoplasma by cultivating or Hoechst staining techniques and maintained in Fisher’s medium at 37 °C on gyratory tables. Fisher’s medium (designed F0) was supplemented with 2 mM L-glutamine, sodium pyruvate (110µg/mL), 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v) (designated F10p). On a single occasion, within 1 week of the start of an experiment, cultures were purged of tk+/tk- mutants by exposure for a day to F10p containing THMG (thymidine, 6µg/mL, hypoxanthine, 5µg/mL; glycine, 7.5 µg/mL; and methotrexate, 0.1 µg/mL), then for 3 days to F10 p containing THG only, (ie, THMG without methotrexate).
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix obtained from liver of Aroclor 1254 exposed rats (intraperitoneal exposure).
- Test concentrations with justification for top dose:
- 0, 125, 250, 500, 1000, 2000 µg/mL (first trial without S-9 mix); 0, 600, 1000, 1400, 1800, 2200, 2600 µg/mL (second trial, without S-9 mix); 0, 1700, 1900, 2100, 2300, 2500 µg/mL (first and second trial with S-9 mix).
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: culture medium without serum
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- culture medium without serum
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- Remarks:
- Migrated to IUCLID6: with S-9 mix, 2.5 µg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- culture medium without serum
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- Migrated to IUCLID6: without S9 mix, 15 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in suspension
EXPERIMENTAL DESIGN
Each experiment, other than the initial toxicity test, normally consisted of the following groups: vehicle control, four cultures; positive control, two cultures; at least five test compound concentrations, two cultures per concentration. The first experiment was a toxicity test in which cell population expansion was measured. Ten-fold differences in test compound concentrations were used in the toxicity test, the highest being 5 mg/mL unless a much lower concentration was indicated by the poor solubility of a compound. This test was followed by at least two experiments in the absence of S9 mix. If no clear positive response was observed, the two experiments were performed in the presence of S9 mix. Test compound concentrations were primarily two fold dilutions from the highest testable concentrations, as estimated from the toxicity test.
MUTATION EXPERIMENTS
EXPOSURE: each exposed culture consisted of 6x10*6 cells in a final volume of 10 mL F5p in a 30-mL screw-cap plastic tube. This tube was incubated for 4 hours on a horizontal axis roller drum rotating at 10 rpm. At the end of the incubation time the cells were sedimented by centrifugation at 500 g for 10 min, washed, and finally resuspended in 20 mL F10p. These cell suspensions (3 X 10*5 cells/mL) were incubated for a 2-day expression period, the cell population density being adjusted back to 20 mL of 3 X 10*5 cells/mL after 24 hours. After 48 hours, the cell population densities were estimated and culture volumes containing 3 X 10*6 cells adjusted to 15 mL with F10p, giving a cell population density of 2X 10*5 cells/mL.
CLONING EFFICIENCY: a 0.1-mL sample of cell suspension was withdrawn and diluted 1:100. Three 0.1-mL samples (200 cells) of the diluted cultures were transferred to 30-mL tubes, mixed with 25 mL of cloning medium (Fisher´s medium containing 20% heat-inactivated horse serum, i.e. F20p= containing 0.35% Noble agar at 37°C) and poured into 90-mm Petri plates.
MUTANT SELECTION: three aliquots (each containing 10*6 cells) of the remaining culture were distributed to 30-mL tubes, mixed with 20 mL of cloning medium to give final concentrations of 0.35% Noble agar and 3µg trifluorothymidine/mL at 37 °C, then poured into 90-mm Petri plates.
INCUBATION: the agar was solidified at 4°C for 5-10 min, then the plates were incubated for 1-14 days in 5% CO2:95% air at 37°C.
COLONY COUNTING: colonies were counted using a New Brunswick Biotran III Automated Colony Counter, with colony size discriminator control in the "off" position.
CALCULATIONS: toxicity was expressed as either a reduction of cell population growth in suspension during the expression period, or a reduction in cloning efficiency. A measure of the overall toxicity was the relative total growth (RTG), which is defined as follows:
RTG= (total suspension growth x cloning efficiency) in dosed culture/ (total suspension growth x cloning efficiency) in control culture
Mutant fraction (MF) was calculated as follows:
MF= 200 x mutant clones per plate (usually a mean of 3)/ total clones per plate (usually a mean of 3) = mutants/ 10*6 clonable cells - Evaluation criteria:
- Positive response (+): the dose-related trend and the response at one of the three highest acceptable doses were statistically significant.
Negative response (-): two categories were used. In both there was: a) No dose-related trend; b) No statistically significant response at any dose; c) An acceptable positive control response.
Nontoxic, negative response (=): there was an RTG among the acceptable doses of > 30%, (approximately) higher toxicity being unattainable owing to intrinsic properties of either the compound or the system.
Toxic, negative response (-): there was either an RTG of < 30% (approximately) at the maximum acceptable dose, or the lethal concentration was no greater than 1.5 times a lower concentration at which the RTG was > 30%. - Statistics:
- The statistical analysis was based upon the mathematical model proposed for this system [Lee and Caspary, 1983. Muta Res 113:417-430] and consisted of a dose-trend test [Barlow et al., 1972, New York: John Wiley & Sons] and a variance analysis of pair-wise comparisons of each dose against the vehicle control.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- other: > 2200 µg/mL (without S-9 mix), > 2500 µg/mL (with S-9 mix)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- With S-9 mix: pH decreasing: >= 1700 µg/mL. Without S-9 mix: pH decreasing: >= 500 µg/mL. Positive results were obtained only in the presence of S-9 mix at concentrations >= 2100 µg/mL
There were no significant mutagenic responses in the absence of S9 mix, the RTG in one experiment being reduced to 24%, at a concentration of 2200 µg/mL.
Cells treated with higher dose levels were not plated because of poor growth during the expression period. In the presence of S9 mix, concentrations up to 2500µg/mL were assessed. Significant mutagenic responses were observed in both experiments, the LOED (lowest observed effective dose) being 2100 µg/mL. The responses were not strong in one trial with little more than a twofold increased in mutant fraction being achieved before excessive toxicity prohibited testing of concentrations higher than 2500 µg/mL.
Reductions in pH values were noted at about 500 µg/mL in the absence of S9 mix, and 1700µg/mL in the presence of S9 mix. These reductions in pH may have contributed to some of the toxicity, but the effects upon mutant fraction observed with other activation system [Cifone et al., 1984. 15th Annual Meeting, Environmental Mutagen Society, Montreal] have not been confirmed in the test laboratory, where a lower concentration is used. - Conclusions:
- Interpretation of results: positive with metabolic activation.
- Executive summary:
An in vitro mammalian cell gene mutation assay was performed with a method similar to OECD guideline 476 with restriction (No GLP study). The mutagenic potential of dimethyl phosphonate was tested using the method for induction of the trifluorothymidine resistance in L5178Y mouse lymphoma cells at dose levels 0,125, 250, 500, 1000, 2000 µg/mL (first trial without S-9 mix); 0, 600, 1000, 1400, 1800, 2200, 2600 µg/mL (second trial, without S-9 mix); 0, 1700, 1900, 2100, 2300, 2500 µg/mL (first and second trial with S-9 mix) in two independent experiments with and without metabolic activation (S9 mix). Dimethyl phosphonate induced gene mutations in L5178Y mouse lymphoma cells with metabolic activation at concentration > or = 2100 µg/mL in the presence of metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Comparable to guideline with acceptable restrictions (No GLP; determination of cytotoxicity not reported; number of replicate not reported; longer exposure times). However, according to OECD SIDS or other official EU regulatory a reliability of 1 was given.
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- No GLP; determination of cytotoxicity not reported; number of replicate not reported; longer exposure times.
- Principles of method if other than guideline:
- NTP standard protocol (Galloway S.M. et al, 1985).
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: CHO cells were maintained in McCoy’s 5 A medium (modified) supplemented with L-glutamine (2 mM), antibiotics, and 10% foetal bovine serum (FBS).
- Periodically checked for Mycoplasma contamination: yes. - Metabolic activation:
- with and without
- Metabolic activation system:
- Liver fraction (S9) prepared from Aroclor 1254-induced male Sprague Dawley rat.
- Test concentrations with justification for top dose:
- without S-9 mix: 0, 50, 160, 500, 1600 µg/mL (second trial: 0, 500, 1000, 1600 µg/mL); with S-9 mix: 0, 16, 50, 160, 500, 1600 µg/mL (second trial: 0, 1600, 3000, 4000, 5000 µg/mL).
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: serum-free culture medium.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- serum-free culture medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Migrated to IUCLID6: -S9, 2.5 and 0.5 µg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- serum-free culture medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- Migrated to IUCLID6: +S9, 50 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
PROTOCOL FOR CHROMOSOMAL ABERRATION ASSAY
Approximately 24 hours prior cell treatment, 1.2 X 10*6 cells were seeded per 75 cm² flask. A culture was established for each dose both with and without metabolic activation. For assays without metabolic activation, the medium was replaced with fresh medium immediately before the chemical treatment.
Cells were treated with test control or control substances for about 10 hours. Colcemid was added 2-3 hours prior to cell harvest by mitotic shake-off.
For the assay with metabolic activation, the cells were rinsed twice with phosphate buffered saline (PBS), after which culture medium without FBS was added. Cells were incubated for 2 hours in the presence of the test or control substance and the S9 mixture. After 2 hours exposure period, cells were washed twice with PBS, and then complete medium containing 10% FBS was added. Cells were incubated for an additional 26 hours. Cells were harvested 11 hours after removal of the S9 fraction. Colcemid was added 2-3 hour prior to cell harvest by mitotic shake off.
Slides were stained in 6% Giemsa for 5-10 min. One hundred cells were scored for each dose in early studies and 200 cells in later studies. All slides except high-dose positive controls were coded. Only metaphase cells in which the chromosome number was between 19 and 23 were scored.
The chromosome number was recorded for each cell and chromosome or chromatid type aberrations were classified into three categories: simple (breaks, fragments, double minutes), complex (interchanges, rearrangements), and other (pulverized, more than ten aberrations/cell).
REPEAT TEST
Positive results in initial tests were confirmed by additional tests. If both -S9 and + S9 studies gave a positive response and required confirmation, they were done sequentially (-S9 first). If the -S9 repeat was positive, the repeat +S9 study was not always performed.
CELL CYCLE DELAY
For chemicals that caused cell cycle delay, harvest times were extended, generally in 5 hours increments, with colcemid present for the last 2 hours.
pH DURING CHEMICAL TREATMENT
In instances when a change in the pH of the culture medium was noticed after addition of the test chemical and the overall response was negative, the test was considered negative, the test was considered sufficient. If however, the overall response was positive, the experiment was repeated with the pH adjusted to 7.4.
PRECIPITATION OF THE TEST COMPOUND
If the test chemical was not soluble at 5 mg/mL, it was tested up to doses at which precipitate was visible. - Evaluation criteria:
- Not reported
- Statistics:
- The percentage of cells with aberrations was analyzed. Dose-response curve and individual dose points were statistically analyzed. A statistically significant (P< 0.003) trend test or a significantly elevated dose point (P<0.05) was sufficient to indicate a chemical effect.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- other: >= 5000 µg/ml
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- Dimethyl phosphonate clearly induced chromosomal aberrations in the presence of S-9 mix and was weakly positive in the absence of S-9 mix at concentrations of > or = 1600 µg/mL each.
- Conclusions:
- Interpretation of results: positive.
- Executive summary:
An in vitro chromosome aberration test was performed with dimethyl phosphonate in Chinese hamster Ovary (CHO) cells performed according to a method equivalent to OECD guideline 473 (No GLP; determination of cytotoxicity not reported; number of replicate not reported; longer exposure times). The occurrence of chromosome aberrations was investigated in the presence and absence of metabolic activation (rat liver S9-mix). Dimethyl phosphonate clearly induced chromosomal aberrations in the presence of S-9 mix and was weakly positive in the absence of S-9 mix at concentrations of > or = 1600 µg/mL each. Relevant cytotoxic effect (reduction of mitotic index) was observed at concentration >= 5000 µg/mL of the compound. Positive controls significantly increased the rate of chromosome aberrations indicating the sensitivity of the assay.
In conclusion, dimethyl phosphonate induced chromosome aberrations in CHO, in the presence and in the absence of a metabolic activation system, under these experimental conditions.
Referenceopen allclose all
Table 1. Mutagenicity of dimethyl phosphonate (Case Western University)
TA 100 | TA 1535 | TA 1537 | TA 98 | |||||||||||||
Dose (µg/plate) | NA |
NA |
10% HLI |
10% HLI |
10% HLI |
10% RLI |
10% RLI | NA | 10% HLI | 10% HLI | NA | 10% HLI | 10% RLI | NA | 10% HLI | 10% RLI |
0 | 145±10.4 | 149±5.4 | 223±3.5 | 209±3.5 | 110±6.1 | 188±1.9 | 197±8.4 | 26±3.0 | 13±2.2 | 11±1.7 | 16±8.9 | 22±3.2 | 19±1.5 | 31±3.2 | 43±4.2 | 39±2.7 |
100 | 144±13.8 | 152±11.5 | 177 ±17.9 | 200 ±7.8 | 92±3.3 | 188 ±21.9 | 170 ±4.4 | 34±3.3 | 13 ±2.0 | 10±3.0 | 14±2.2 | 27±4.3 | 27±1.9 | 35±2.6 | 36 ±8.5 | 35±2.1 |
333 | 192±12.0 | 156±9.8 | 192 ±15.3 | 225 ±1.5 | 112±6.2 | 239 ±11.9 | 186±17.7 | 33±1.9 | 11±1.2 | 13±1.5 | 13±0.7 | 19±2.9 | 18±2.4 | 33±1.8 | 39±4.3 | 36±2.5 |
1000 | 161±16.2 | 151 ±6.5 | 204± 5.8 | 248±8.3 | 110±4.3 | 225±22.2 | 189±13.3 | 32±2.8 | 12 ±0.3 | 12±2.0 | 18±2.7 | 25±2.4 | 19±4.2 | 37±4.4 | 31±3.5 | 26±6.0 |
3333 | 283 ±6.4 | 179 ±9.6 | 197±18.2 | 242 ±11.3 | 84 ±13.8 | 268±2.4 | 199±5.9 | 32 ±1.9 | 13 ±2.4 | 14±4.1 | 14±0.6 | 24 ±1.8 | 19 ±2.6 | 37±3.2 | 29±6.1 | 34±1.9 |
10000 | 204 ±13.5 | 168 ±9.1 | 206 ±9.3 | 234 ±10.1 | t | 269±22.9 | 224±3.6 | 26 ±1.5 | t | t | 11±1.2 | 17±4.2 | 15±0.9 | 42±5.9 | t | t |
POS | 1882±197.2 | 1505 ±23.3 | 3362 ±176.8 | 1014±68.0 | 1919 ±139.9 | 1405 ±77.4 | 2073±71.3 | 816 ±93.2 | 367 ±45.1 | 447±23.4 | 275 ±84.8 | 281 ±20.8 | 142±21.1 | 228 ±8.6 | 1213±142.9 | 1125±80.5 |
RLI = Rat liver S-9, Aroclor 1254-induced; HLI = Hamster liver S-9, Aroclor 1254-induced
Table 2. Mutagenicity of dimethyl phosphonate (EGG Mason Research Institute, later Microbiological Associates)
TA 100 | TA 1535 | TA 1537 | TA 98 | |||||||||||||
Dose (µg/plate) | NA | NA | 10% HLI | 10% HLI | 30% HLI | 10% RLI | 10% RLI | NA | 10% HLI | 10% HLI | NA | 10% HLI | 10% RLI | NA | 10% HLI | 10% RLI |
0 | 144± 5.5 | 134±1.7 | 136±2.4 | 142±6.7 | 165±7.8 | 126±4.3 | 148± 4.0 | 37±0.4 | 12±2.2 | 13±2.3 | 5±1.0 | 8±1.8 | 6±1.3 | 19±1.9 | 30±1.2 | 31±1.7 |
100 | 139±6.6 | 149±2.6 | 143 ±9.9 | |||||||||||||
333 | 145±3.7 | 153 ±6.0 | 148 ±8.7 | |||||||||||||
1000 | 156±2.0 | 162 ±2.8 | 139± 2.6 | 147±3.5 | 155±5.8 | 143±9.1 | 133±5.8 | 31±5.8 | 19 ±2.3 | 13±1.3 | 4±0.6 | 10±1.2 | 7±2.0 | 18±1.2 | 29±3.2 | 20±1.8 |
3333 | 157 ±1.8 | 173 ±1.2 | 170 ±8.3 | 174 ±13.9 | 171 ±11.1 | 146±12.1 | 166±5.5 | 31±2.8 | 18 ±2.1 | 19±3.8 | 3±0.6 | 6 ±2.1 | 7 ±0.9 | 19±2.2 | 27±4.2 | 25±3.5 |
5000 | 175 ±6.0 | 187 ±11.7 | 177 ±15.6 | 170±4.4 | 33 ±2.0 | 17±2.4 | 16±2.7 | 7±2.0 | 7±1.2 | 7±2.2 | 18±1.8 | 26±4.3 | 28±1.5 | |||
7500 | 193 ±2.3 | 200 ±6.4 | 189 ±12.9 | 162 ±7.4 | 34±1.7 | 21 ±2.6 | 13±1.2 | 6±1.5 | 9±0.7 | 6±0.6 | 17± 4.8 | 26±4.1 | 25±1.5 | |||
10000 | 172±9.8 | 190 ±2.9 | 190 ±1.0 | 193±10.0 | 196 ±9.7 | 156 ±6.6 | 161±7.8 | 33 ±1.0 | 18±2.7 | 19±2.2 | 3 ±0.4 | 9±2.3 | 5±1.2 | 18 ±1.2 | 32±4.9 | 34±2.6 |
POS | 1210±14.0 | 1000±3.2 | 804±24.5 | 1004±168 | 1105 ±37.3 | 563±13.5 | 1450±43.1 | 920±38.9 | 237±28.5 | 73 ±4.2 | 341±49.1 | 136±16.5 | 200 ±14.5 | 1433±41.3 | 1056±75.2 | 2011 ± 47.5 |
RLI = Rat liver S-9, Aroclor 1254-induced; HLI = Hamster liver S-9, Aroclor 1254-induced.
Table 1
Without S9 trial 1 (i) | Without S9 trial 2 (-) | Induced S9 Trial 1 (+) | Induced S9 trial 2 (+) | ||||||||||||||||||||
Concµg/mL | CE | RTG | MC | MF | AVGMF | Conc µg /mL | CE | RTG | MC | MF | AVGMF | Conc µg/ mL | CE | RTG | MC | MF | AVGMF | Conc µg/mL | CE | RTG | MC | MF | AVGMF |
FOP | 143 | 136 | 74 | 17 | F0P | 112 | 106 | 226 | 67 | F0P | 51 | 100 | 57 | 37 | F0P | 130 | 101 | 133 | 34 | ||||
0 | 81 | 75 | 68 | 28 | 0 | 93 | 105 | 205 | 73 | 0 | 45 | 85 | 54 | 40 | 0 | 120 | 107 | 139 | 38 | ||||
84 | 92 | 77 | 30 | 90 | 98 | 180 | 67 | 69 | 107 | 96 | 46 | 87 | 97 | 100 | 38 | ||||||||
85 | 97 | 77 | 30 | 26 | 74 | 91 | 184 | 83 | 73 | 42 | 108 | 69 | 54 | 45 | 92 | 95 | 110 | 40 | 38 | ||||
125 | 75 | 98 | 78 | 35 | 600 | 78 | 84 | 184 | 79 | 1700 | 49 | 76 | 101 | 68 | 1700 | 148 | 91 | 227 | 51 | ||||
91 | 96 | 107 | 39 | 37 | 88 | 79 | 172 | 65 | 72 | 36 | 64 | 77 | 72 | 70 | 124 | 71 | 222 | 60 | 55 | ||||
250 | 92 | 101 | 58 | 21 | 1000 | 70 | 71 | 160 | 76 | 1900 | 40 | 58 | 54 | 45 | 1900 | 103 | 73 | 189 | 61 | ||||
102 | 108 | 78 | 25 | 23 | 90 | 85 | 147 | 55 | 65 | 34 | 56 | 54 | 52 | 49 | 100 | 71 | 188 | 62 | 62 | ||||
500 | 113 | 109 | 53 | 16 | 1400 | 75 | 75 | 203 | 90 | 2100 | 26 | 50 | 75 | 97 | 2100 | 100 | 69 | 235 | 78 | ||||
59 | 80 | 49 | 27 | 22 | 82 | 70 | 202 | 82 | 86 | 28 | 43 | 97 | 115 | 106 | 101 | 74 | 245 | 81 | 79 | ||||
1000 | 83 | 70 | 73 | 29 | 1800 | 57 | 39 | 174 | 101 | 2300 | 10 | 14 | 61 | 194 | 2300 | 112 | 66 | 243 | 72 | ||||
99 | 74 | 97 | 33 | 31 | 79 | 57 | 176 | 75 | 88 | 3R | 4 | 18 | 225 | 209 | 103 | 65 | 217 | 70 | 71 | ||||
2000 | 92 | 55 | 110 | 40 | 2200 | 70 | 20 | 233 | 111 | 2500 | 1 | 1 | 9 | 450 | 2500 | 134 | 42 | 509 | 126 | ||||
88 | 59 | 108 | 41 | 40 | 95 | 28 | 295 | 104 | 107 | 6R | 8 | 23 | 118 | 284 | 89 | 46 | 378 | 141 | 134 | ||||
2600 | Tox | ||||||||||||||||||||||
Tox | |||||||||||||||||||||||
MMS | 54 | 32 | 323 | 200 | MMS | 20 | 18 | 289 | 474 | 3-MC | 32 | 34 | 419 | 432 | 3-MC | 84 | 42 | 736 | 293 | ||||
15 µg/mL | 41 | 30 | 238 | 193 | 197 | 15 µg/mL | 29 | 19 | 356 | 414 | 444 | 2.5 µg/mL | 49 | 52 | 463 | 314 | 373 | 2.5 µg/mL | 91 | 38 | 775 | 282 | 288 |
CE=cloning efficiency
RTG= relative total growth
MC= mutant clones
MF=mutant fraction
AVG MF=average mutant fraction
Table 1: Induction of Chromosomal Aberrations in chinese hamster ovary cells by dimethyl phosphonate (without S9 activation)
Without S9 |
|||||
Compound |
Dose (µg/mL) |
Total cells |
% cell with aberrations (Total) |
% cell with aberrations (Simple) |
% cell with aberrations (Complex) |
Solvent |
0 |
100 |
1.00 |
1.00 |
0.00 |
Mitomycin C |
0.25 |
100 |
24.00 |
14.00 |
17.00 |
Dimethyl phosphonate |
50 |
100 |
0.00 |
0.00 |
0.00 |
160 |
100 |
0.00 |
0.00 |
0.00 |
|
500 |
100 |
0.00 |
0.00 |
0.00 |
|
Dimethyl phosphonate |
1600 | 100 | 10.00* | 4.00 | 6.00 |
Table 2: Induction of Chromosomal Aberrations In Chinese Hamster Ovary Cells by dimethyl phosphonate (without S9 activation)
Without S9 |
|||||
Compound |
Dose (µg/mL) |
Total cells |
% cell with aberrations (Total) |
% cell with aberrations (Simple) |
% cell with aberrations (Complex) |
Solvent |
0.00 |
100 |
0.00 |
0.00 |
0.00 |
Mitomycin C |
0.5 |
100 |
37.00 |
23.00 |
19.00 |
Dimethyl phosphonate |
500 |
100 |
2.00 |
2.00 |
0.00 |
1000 |
100 |
3.00 |
2.00 |
1.00 |
|
1600 |
50 |
10.00* |
4.00 |
6.00 |
Table3: Induction of Chromosomal Aberrations in chinese hamster ovary cells by dimethyl phosphonate (with S9 activation)
With S9 |
|||||
Compound |
Dose (µg/mL) |
Total cells |
% cell with aberrations (Total) |
% cell with aberrations (Simple) |
% cell with aberrations (Complex) |
Solvent |
0 |
100 |
0.00 |
0.00 |
0.00 |
cyclophosphamide |
50 |
100 |
38.00 |
22.00 |
22.00 |
Dimethyl phosphonate |
16 |
100 |
1.00 |
0.00 |
1.00 |
50 |
100 |
1.00 |
1.00 |
0.00 |
|
160 |
100 |
0.00 |
0.00 |
0.00 |
|
Dimethyl phosphonate |
500 | 100 | 0.00 | 0.00 | 0.00 |
Dimethyl phosphonate |
1600 | 100 | 0.00 | 0.00 | 0.00 |
Table 4 : Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by dimethyl phosphonate (with S9 activation)
With S9 |
|||||
Compound |
Dose (µg/mL) |
Total cells |
% cell with aberrations (Total) |
% cell with aberrations (Simple) |
% cell with aberrations (Complex) |
Solvent |
0.00 |
100 |
1.00 |
1.00 |
0.00 |
cyclophosphamide | 50 |
100 |
53.00 |
36.00 |
43.00 |
Dimethyl phosphonate |
1600 |
100 |
0.00 |
0.00 |
0.00 |
3000 |
100 |
3.00 |
2.00 |
1.00 |
|
4000 |
100 |
3.00 |
2.00 |
1.00 |
|
5000 | 100 | 44.00** | 26.00 | 20.00 |
* significant dose
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
IN VIVO STUDIES
2 micronucleus assays: one positive (equivalent to OECD 474) and one
negative (GLP, OECD 474).
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Comparable to guideline study with restrictions (No information about environmental conditions and housing, only male mice were tested, misssing reproducibility). Adopted according to OECD SIDS (public available peer reviewed source). The original source is available and has been reviewed.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- No information about environmental conditions and housing, only male mice were tested, missing reproducibility
- GLP compliance:
- not specified
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: the animals were obtained from the National Toxicology Program production facility at Taconic Farms, USA.
- Age at study initiation: between 9 and 14 weeks old.
- Weight at study initiation: within a 2 g range of a mean weight between 25 and 33 g. - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: PBS
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test chemical was prepared in the appropriate solvent and mixed using a S/P vortex Mixer. The test chemical was administered within 30 minutes of preparation. - Duration of treatment / exposure:
- The animals were sacrified 24 hour after last injection.
- Frequency of treatment:
- Three consecutive days (one injection/day).
- Post exposure period:
- 24 hours after the last exposure
- Remarks:
- Doses / Concentrations:
0, 250, 500 mg/kg bw/d
Basis:
nominal conc. - No. of animals per sex per dose:
- 5 (only male mice were tested).
- Control animals:
- other: negative control animals (concurrent vehicle); positive control animals.
- Positive control(s):
- 7,12-dimethylbenzanthracene (DMBA); mitomycin C (MMC)
- Route of administration: intraperitoneal injection
- Doses / concentrations: 7,12-dimethylbenzanthracene - DMBA (12.5 mg/kg), mitomycin C - MMC (0.2 mg/kg) - Tissues and cell types examined:
- Bone marrow.
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
The selection of the maximum dose to be tested for micronucleus induction was based on either mortality, administration characteristics (ability to be administered as a homogeneous suspension in corn oil or dissolved in PBS), depression in the percentage of bone marrow PCE (no less than 15% of the erythrocytes), or on the arbitrary maximum dose of 2000 mg/kg/day.
Generally, unless LD50 data were available to suggest an appropriate dose range to test, the initial doses tested were 200, 1000, and 2000 mg/kg.
Groups of 5 mice were administered the test chemical by i.p. injection on the three consecutive days. Animals were monitored twice daily, and 48 hours after the third treatment, the surviving mice were euthanized by CO2 asphyxiation. Bone marrow smears (two/slides/tissue/mouse) were prepared by a direct technique.
Air-dried smears were fixed using absolute methanol and stained with acridine orange. Bone marrow from each animal were evaluated at 1000x magnification using epi-illuminated fluorescence microscopy (450-490 nm excitation, 520 nm emission) for determination of the percentage of PCE among 200 erythrocytes. Based on the results obtained, the maximum administered dose was estimated or additional dose determination experiments were conducted to more accurately estimate the maximum dose to be tested in the primary micronucleus test.
TREATMENT AND SAMPLING TIMES
For the initial micronucleus test, groups of 5 animals were injected i.p. on the three consecutive days with either the test chemical, a weakly active dose of the positive control chemical (DMBA in corn oil, MMC in PBS), or the appropriate solvent. Mice were euthanized with CO2 24 hours after the thirds treatment.
DETAILS OF SLIDE PREPARATION
Bone marrow smears (two slides mouse) were prepared, fixed in absolute methanol, and stained with acridine orange. For each animal, slides were evaluated at 1000x magnification for number for the number of MN-PCE among 2000 PCE and for the percentage of PCE among 200 erythrocytes.
- Statistics:
- The data were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.4), which was designed specifically for in vivo micronucleus data [ILS, 1990. Integrated Laboratory Systems. P.O Box 13501, Research Triangle Park, NC 27709]. The level of significance was set at an alpha level of 0.05. To determine whether a specific treatment resulted in a significant increase in MN-PCE, the number of MN-PCE were pooled within each dose group and analyzed by one-tailed trend test. In the software package used, the trend test incorporates a variance inflation factor to account for excess animal variability. In the event that the increase in the dose response curve is nonmonotonic, the software program allows for the data to be analyzed for a significant positive trend after data at the highest dose only has been excluded. However, in this event, the alpha level is adjusted to 0.01 to protect against false positive. The % PCE data were analyzed by an analysis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each groups and the concurrent solvent control group was by an unadjusted one-tailed Pearson chi-squared test which incorporated the calculated variance inflation factor for the study.
- Sex:
- male
- Genotoxicity:
- positive
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The initial test gave a positive trend from 2.1 in the control to 6.1 at 500 mg/kg (trend P-value <0.001); the trend analysis of the repeat test gave p= 0.078 with the MNC-PCE frequency ranging from a control of 2.7 to 4.2 in high dose. Although not showing a reproducible, statistically significant increase in MN-PCE, the data were judged to be adequate evidence of an effect.
- Conclusions:
- Interpretation of results: positive
- Executive summary:
Dimethyl phosphonate was tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection (i.p.). Bone marrow samples were obtained 24 hours following the final exposure.
For the initial micronucleus test, groups of 5 animals were injected i.p.during three consecutive days with either the test chemical (0, 250, 500 mg/kg bw), a weakly active dose of the positive control chemical (DMBA in corn oil, MMC in PBS), or the appropriate solvent. Mice were euthanized with CO2 24 hours after the third treatment.
The initial test gave a positive trend from 2.1 in the control to 6.1 at 500 mg/kg (trend P-value < 0.001); the trend analysis of the repeat test gave p= 0.078 with the MNC-PCE frequency ranging from a control of 2.7 to 4.2 in high dose. Although not showing a reproducible, statistically significant increase in MN-PCE, the data were judged to be adequate evidence of an effect.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP-guideline study with restrictions (positive controls outside laboratory historical range). Adopted according to OECD SIDS (public available peer reviewed source). The original source is available and has been reviewed
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- (positive controls outside laboratory historical control range)
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6-12 weeks of age
- Weight at study initiation: 28-45 g
- Assigned to test groups randomly: yes
- Housing: the females were kept in groups of a maximum of three mice in Makrolon type I cages. Males were kept singly in type I cages.
- Diet (e.g. ad libitum): Altromin 1324 Standard Diet ad libitum
- Water (e.g. ad libitum): tape water ad libitum
- Acclimation period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1.5
- Humidity (%): 40-45
- Air changes (per hr): ten times per hour
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: physiol. saline (dimethylphosphite); water (cyclophosphamide).
- Duration of treatment / exposure:
- 16, 24 and 48 hours after single administration.
- Frequency of treatment:
- Single exposure.
- Post exposure period:
- No data.
- Dose / conc.:
- 2 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- other: negative control animals (concurrent vehicle); positive control animals.
- Positive control(s):
- Cyclophosphamide, in the form of Endoxan 100 mg injection vials of dry substance, (ASTA Medica AG), Batch 082525
- Route of administration: intraperitoneal injection
- Doses / concentrations: 20 mg/kg bw
-Justification for choice of positive control(s): cyclophosphamide is a proven cytostatic agent and known clastogen with bifunctional alkylation action. - Tissues and cell types examined:
- Femur bone-marrow cells.
- Details of tissue and slide preparation:
- Smears were prepared according to Schmid's method and were stained with an Ames Hema-Tek Slide Stainer.
At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor).
CRITERIA FOR DOSE SELECTION:
The selection of the dimethylphophite dose was based on a pilot test in which groups of five animals, including both males and females, were intraperitoneally administered 1000 mg/kg, 1500 mg/kg, 2000 mg/kg, 2250 mg/kg and 2500 mg/kg dimethylphosphite.
The following symptoms were recorded for up to 48 hours, starting at 1000 mg/kg: apathy, roughened fur, staggering gait, sternal recumbency, spasm and difficulty in breathing. In addition, 2 of 5 animals died in the 2250 mg/kg group and 5 of 5 animals died in the 2500 mg/kg group.
Based on these results, 2000 mg/kg dimethylphosphite was chosen as MTD for this test.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The femur was separated from muscular tissue.
The femur was completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off.
The contents were then flushed several times and the bone marrow was passed into the serum as a fine suspension.
Finally, the flushing might be repeated from the other end, after it had been opened.
The tube containing the serum and the bone marrow was centrifuged at approximately 1000 rpm for five minutes.
The supernatant was removed with a suitable pipette (e.g. Pasteur pipette), leaving only a small remainder.
The sediment was mixed to produce a homogenous suspension.
DETAILS OF SLIDE PREPARATION:
One drop of the viscous suspension was placed on a well-cleaned slide and spread with a suitable object, to allow proper evaluation of the smear.
The labelled slides were dried overnight. If fresh smears needed to be stained, the needed to be dried with heat for a short period.
METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes.
Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.
The number of normochromatic erythrocytes per 1000 polychromatic ones was noted.
A relevant, treatment related alteration of the ratio polychromatic to normochromatic erythrocytes can only be concluded if it is clearly lower for a majority of the animals in the treated group than in the negative control.
In addition to the number of normochromatic erythrocytes per 1000 polychromatic ones, the number of normochromatic erythrocytes showing micronuclei was also established. - Evaluation criteria:
- A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in the rate which, according to the laboratory’s experience was within the range of the negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. In this case, a second test had to be performed at the most sensitive interval. - Statistics:
- The dimethylphosphite groups with the highest mean and the positive control were checked by Wilcoxon´s non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi²-test. A variation was considered statistically significant if the error probability was below 5% and the treatment group figure was higher than that of the negative control. In addition, standard deviations were calculated for all means. - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- other: Although statistically significant, the values for the positive control group were unusually low. It is therefore not certain, whether this test was sufficiently sensitive.
- Additional information on results:
- TOLERATION BY THE ANIMALS
Compound-related symptoms: apathy, roughened fur, staggering gait, difficulty in breathing (all symptoms were noted until sacrifice); all animals survived until the end of the study. No symptoms were recorded in the control group. No animals died in these groups.
MICROSCOPIC EVALUATION
Concerning the assessment of the clastogenic potential of dimethylphosphite, there were no relevant variations in results between males and females. Therefore they were evaluated jointly.
The ratio of polychromatic to normochromatic erythrocytes was time-dependently altered by the treatment with dimethylphosphite, being 1000: 842 (1s*=240) in the negative control, 1000: 1066 (1s=495) in the 16 hours group, 1000: 1250 (1s=573) in the 24 hours group and 1000: 1559 (1=397) in the 48 hours group. Relevant variations were thus noted.
No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 2000 mg/kg dimethylphosphite, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 1.3/1000 (1s=1.1) in the negative control, and 0.8 /1000 (1s=1.1), 1.8/1000 (1s=1.5) and 2.7/1000 (1s=3.1) in the dimethylphosphite groups.
Similarly, there could be no biologically significant variation between the negative control and dimethylphosphite groups in the number of micronucleated normochoromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed.
The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 7.3/ 1000 (1s=5.5), which represents a biologically relevant increase in comparison to the negative control. There could not have been biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones.
No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary to a biologically relevant degree [1000:963 (1s=421)], as, against 1000:842 in the negative control]. This clearly demonstrates that an alteration of the ratio of the polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.
* Standard deviations (1s ranges). - Conclusions:
- Interpretation of results: negative.
Referenceopen allclose all
Table1. Micronucleus Data
Chemical |
Tissue |
Trend P-value |
Dose (mg/kg) | MN-PCE/1000 (No. animals) |
Pair-wise |
Survivals |
% PCE |
First trial | |||||||
Dimethyl hydrogen phosphite | bone marrow | < 0.001 |
0 | 2.10 ± 0.64 (5) | 5/5 | 29.5 |
|
250 | 1.10 ± 0.37 (5) | 0.9616 | 5/5 | 42.8 |
|||
500 | 6.10 ± 0.94 (5) | <0.001 | 5/5 | 30.6 |
|||
Second trial | |||||||
Dimethyl hydrogen phosphite | bone marrow | 0.078 |
0 | 2.70 ± 0.56 (5) | 5/5 | 50.3 | |
250 | 2.20 ± 0.26 (5) | 0.7627 | 5/5 | 41.7 | |||
500 | 4.17 ± 0.44 (5) | 0.0573 | 3/5 | 19.7 |
Positive Control DMBA / mean mn PCE: 6.93 +/- 2.59
Positive Control MMC / mean mn PCE: 6.82 +/- 1.24
*: statistically significant
PCE - polychromatic erythrocytes
According to the study author, there was no biologically relevant or statistically significant variation between negative controls and dimethylphosphite treated groups. There was, however, a statistically non-significant doubling of micro-nucleated PCEs after 48 hours (negative controls: 1.3 ± 1.1; 16h: 0.8 ± 1.1; 24h: 1.8 ± 1.5, 48 h: 2.7 ± 3.1). Although statistically significant, the values for the positive control group were unusually low (7.3 ± 5.5 as compared to the laboratory’s historical positive control range of 10.2 - 25.1). It is therefore not certain, whether this test was sufficiently sensitive.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Additional information
Bacterial tests
Ames tests performed with dimethyl phosphonate were primarily negative. In one NTP assay the results of the Salmonella typhimurium strains TA 98, 100, 1535, 1537 in concentrations up to 10000 µg/plate were judged negative with and without metabolic activation. Cytotoxicity was reached at 10000 µg dimethyl phosphonate/plate (NTP, 1985). A further assay equivalent to OECD TG 471 and conducted with GLP conditions was judged negative: 775 to 12400 µg dimethyl phosphonate/plate were tested with the strains TA 98, 100, 1535 and 1537 in duplicates (Herbold, 1988). In the first experiment the mutant counts of TA 100 with S-9 mix were significantly increased. This result could not be reproduced in the replicate (Herbold, 1988). At 6200 µg/plate bacteriotoxic effects were observed but the test could be evaluated (Herbold, 1988). In a reliable and good quality study Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were treated with dimethyl phosphonate diluted in H2O using the Ames preincubation method equivalent to OECD Guideline No. 471 with deviations (No GLP. Only 4 strain tested), both with and without the addition of a rat and hamster liver homogenate metabolising system . The dose range was determined in a preliminary toxicity assay with upper dose 10 mg/plate. The mutagenicity of dimethyl phosphonate was tested in two laboratories. In one laboratory (Case Western University) the mutagenic effect of dimethyl phosphonate was judged equivocal; in the other laboratory (EGG Mason Research Institute, later Microbiological Associates) dimethyl phosphonate resulted to be mutagenic in Salmonella typhimurium strain TA 100 (Mortelmanns et al., 1986). Dimethyl phosphonate was therefore evaluated to be positive in TA 100 strain, and negative with TA 98, 1535, 1537 or 97 strains. The data from the study by Tennant et al. (1987a) were re-evaluated by Prival and Dunkel (1989) using more stringent criteria for a positive result. The positive results with TA100 were made negative by disregarding the positive results at a dose of 10000 µg/plate (Prival and Dunkel, 1989). It is noted that this concentration also exceeds the limit dose of 5000 µg/plate which is recommended in current guidelines.
In vitro Studies in Mammalian cells
In a cytogenetic assay, performed according to NTP standard protocol, with L5178Y mouse lymphoma cells, dimethyl phosphonate showed positive results with metabolic activation at concentrations of > or = 1700 µg/mL (Tennant et al., 1987a). A further mouse lymphoma assay showed also mutagenic activity of dimethyl phosphonate in concentrations of > or = 2100 µg/mL in the presence of S-9 mix (McGregor et al., 1988). Concentrations > 2200 µg/mL (without S-9 mix) and > 2500 µg/mL (with S-9 mix) resulted to be cytotoxic in this assay.
In chromosomal aberration tests with Chinese hamster ovary cells performed after NTP standard protocol dimethyl phosphonate clearly induced chromosomal aberrations in the presence of S-9 mix and was weakly positive in the absence of S-9 mix at concentrations of > or = 1600 µg/mL each (Gulati et al. 1989).
Dimethyl phosphonate was positive in a DNA damage and repair assay with primary rat hepatocytes pretreated with Aroclor-1254 (Aro) and 3-methylcholanthrene (3-MC). The netto nuclear grains (NNG) and the percentage of cells with three NNGs above the solvent control (% IR) respectively were evaluated.
The % IR was clearly elevated in the rat hepatocytes pretreated with Aro (in concentrations of > or = 0.01 µg/mL) and 3-MC (in concentrations of > or = 0.025 µg/mL) representing unscheduled DNA synthesis and indicating DNA mutations of dimethyl phosphonate (Shaddock et al., 1990). Dimethyl phosphonate was negative in untreated primary rat hepatocytes (Shaddock et al. 1990). A further negative result was obtained in an unscheduled DNA synthesis assay with primary rat hepatocytes and limited documentation (Tennant et al., 1987b).
In a sister chromatid exchange (SCE) assay with Chinese hamster ovary cells dimethyl phosphonate caused increased total SCE numbers in cells and increased numbers of SCE/cell with and without metabolic activation at concentrations of > or = 250 µg/mL. The concentration range tested was 5 - 1600 µg/mL without S-9 mix and 16 - 4000 µg/mL with S-9 mix and fifty second-division metaphase cells were scored per dose (Gulati et al., 1989).
In vivo Studies
In a micronucleus assay in bone marrow cells of B6C3F1 mice, which received daily i.p. injections of 250 and 500 mg/kg bw/d dimethyl phosphonate for three days, the number of micro-nucleated polychromatic erythrocytes (PCEs) per 1000 PCEs scored was significantly elevated in the first trial at 500 mg/kg bw/d. This result could not be clearly reproduced in the second trial. The trend analysis of the repeat test gave P=0.078. The authors judged the data as “adequate evidence of an effect”, though not conclusive: “... additional tests would be needed to provide conclusive evidence of MN-inducing ability” of dimethyl phosphonate (Shelby et al., 1993).
In a separate micronucleus assay with NMRI mice, no clastogenic effect was observed according to the study authors after a single i.p. administration of 2000 mg/kg bw dimethyl phosphonate (Bayer AG, 1994). The incidences of micro-nucleated polychromatic erythrocytes (PCEs) per 1000 PCEs scored were measured 16, 24 and 48 hours after i.p. injection of dimethyl phosphonate. There was a statistically non-significant doubling of micro-nucleated PCEs after 48 hours (negative controls 1.3 ± 1.1, 16h 0.8 ± 1.1, 24h 1.8 ± 1.5, 48 h 2.7 ± 3.1). Although statistically significant, the values for the positive control group (cyclophosphamide, 20 mg/kg bw i.p.) were unusually low (7.3 ± 5.5 as compared to the laboratory`s historical positive control range of 10.2 – 25.1). It is therefore not certain, whether this test was sufficiently sensitive.
Conclusion
In vitro data indicate that dimethyl phosphonate has mutagenic and clastogenic potential. The available in vivo data are limited to the bone marrow and the results are conflicting with one study indicating clastogenicity. Dimethyl phosphonate should be regarded as having genotoxic potential in vivo.
Justification for classification or non-classification
According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification as Muta. 2 (H341: Suspected of causing genetic defects) is justified.
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