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EC number: 231-764-5 | CAS number: 7722-76-1
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
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:
- 27 April 2001 -17 May 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:
- Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, published by OECD, Paris, February 1998.
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (1997).
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Salmonella tester strain cultures: deep rough mutation (ifa) and the deletion in the uvrB gene.
Cultures of tester strains TA98 and TA100: presence of the pKM101 plasmid R-factor.
All WP2 uvrA cultures: deletion in the uvrA gene - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- other: TA98 + TA1537: reverted from histidine dependence to histidine independence by frameshift mutagens. TA1535: reverted by mutagens that cause basepair substitutions. TA100: reverted by mutagens that cause both frameshift and basepair substitution mutations
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- not applicable
- Additional strain / cell type characteristics:
- other: Specificity of the reversion mechanism in E. coli is sensitive to base-pair substitution mutations, rather than frameshift mutations
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9-mix from male Sprague-Dawley rats
- Test concentrations with justification for top dose:
- Initial toxicity-mutation assay: 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 ug per plate.
Confirmatory mutagenicity assay: 50, 150,500, 1500 and 5000 ug per plate. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: sterile distilled water (CAS# 7732-18-5), obtained from Life Technologies, Inc.
- Justification for choice of solvent/vehicle: Based on compatibility with the target cells and workability of the test article in water. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene: 1.0 and 10 ug/plate
- Remarks:
- All tester stains with S9 activation mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- TA 98 without S9 activation mix: 1.0 ug/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- TA 100 and TA 1535 without S9 activation mix: 1.0 ug/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA 1537 without S9 activation mix: 75 ug/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- WP2 uvrA without S9 activation mix: 1000 ug/plate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in top agar (plate incorporation)
DURATION
- Preincubation period: overnight (To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored)
- Exposure duration: approximately 48 to 72 hours at 37±2°C
NUMBER OF REPLICATIONS:
Initial Toxicity-Mutation Assay in duplicate
Confirmatory Mutagenicity Assay in triplicate
DETERMINATION OF CYTOTOXICITY
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope.
OTHER EXAMINATIONS:
- Other: Precipitate was evaluated by visual examination without magnification.
OTHER:
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. - Evaluation criteria:
- For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for tester strains TA98, TA1OO and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.
- Statistics:
- For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated.
Since no positive responses were observed, no statistical analysis of the responses was performed. - Key result
- 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 applicable
- Positive controls validity:
- valid
- Key result
- 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 applicable
- Positive controls validity:
- valid
- Key result
- 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 applicable
- Positive controls validity:
- valid
- Key result
- 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 applicable
- Positive controls validity:
- valid
- Key result
- 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 applicable
- Positive controls validity:
- valid
- Additional information on results:
- Precipitation conc: Initial Toxicity Mutation Assay: 1,800 μg/plate
Confirmatory Mutagenicity Assay: 5,000 µg/plate - Conclusions:
- All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Diammonium Phosphate (DAP) did not cause a positive response in the presence and absence of Aroclor-induced rat liver S9.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
-RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
- Test concentrations with justification for top dose:
- Dose range finding test:
Without and with S9-mix, 3 hours treatment: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24 hours treatment: 3, 10, 33, 100 and 333 µg/ml and 0.003, 0.01, 0.03, 0.1, 0.3, 1 and 3 µg/mL
Experiment 1:
Without S9-mix, 3 hours treatment: 0.003, 0.03, 0.1, 0.25, 0.5, 1, 1.4 and 2 µg/mL
With S9-mix, 3 hours treatment: 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 12 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 0.01, 0.03, 0.1, 0.25, 0.5, 1, 1.4 and 1.8 µg/mL
With S9-mix, 3 hours treatment: 0.01, 0.1, 1, 10, 12, 14, 16 and 17 μg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: RPMI 1640 medium
- Justification for choice of solvent/vehicle:Test compound was soluble in RPMI 1640 medium and RPMI 1640 medium has been accepted and approved by authorities and international guidelines - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without S9: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
- Positive control substance:
- cyclophosphamide
- Remarks:
- with S9: 7.5 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
TEST SYSTEM
-cells: L5178Y/TK+/-3.7.2C
DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days
SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)
NUMBER OF REPLICATIONS:
- Solvent controls: Duplo cultures
- Treatment groups and positive control: Single cultures
NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells/concentration
DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments) - Evaluation criteria:
- The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more then MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test. - Key result
- 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
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation was observed up to and including the top dose of 850 µg/mL (= 0.01 M)
RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 3 µg/mL in the absence of S9, 3 hours treatment; at dose levels of 33 µg/mL in the presence of S9, 3 hours treatment; at dose levels of 1 µg/mL in the absence of S9, 24 hours treatment
COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 79 and 83% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.
In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level in both experiments. - Conclusions:
- The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.
Mutation frequencies in cultures treated with positive control chemicals were increased by 11- and 16-fold for MMS in the absence of S9-mix, and by 19- and 11-fold for CP in the presence of
S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.
In the absence of S9-mix, Ammonium dihydrogenorthophosphate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.
In the presence of S9-mix, Ammonium dihydrogenorthophosphate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.
It is concluded that Ammonium dihydrogenorthophosphate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report. - Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01 May 2001 - 12 June 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Well-documented, GLP-compliant, according to OECD 473 in Chinese hamster ovary cells.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- (Ninth Addendum; February 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (1996 and 1997)
- Deviations:
- no
- GLP compliance:
- yes
- 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: McCoy's 5A medium supplemented with 10% fetal bovine serum, 100 units penicillin and 100 ug streptomycin/mL and 2 mM L-glutamine for the non-activated study. S9 reaction mixture (3.5 mL serum-free McCoy's 5A medium (supplemented with 100 units penicillin and 100 ug streptomycin/mL and 2 mM L-glutamine) plus 1 mL of S9/cofactor pool) for the activated study. S9/cofactor pool = 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 uL S9 per milliliter serum-free McCoy's 5A medium supplemented with 100 units penicillin and 100 ug streptomycin/mL, and 2 mM L-glutamine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9: from male Sprague-Dawley rats induced with a single ip injection of Aroclor 1254, 500 mglkg, five days prior to sacrifice. S9 was batch prepared and stored frozen at approximately -70C until used.
- Test concentrations with justification for top dose:
- 165, 330, 660 and 1320 μg/mL (2 replicates per concentration)
- Vehicle / solvent:
- Water
- Vehicle(s)/solvent(s) used: sterile water (CAS No.: 7732-18-5) obtained from Life Technologies Company.
- Justification for choice of solvent/vehicle: A solubility test was conducted to select the solvent. The test was conducted using purified water. The test article was tested to determine the solvent, selected in order of preference, that permitted preparation of the highest soluble stock concentration, up to 50 mg/mL for aqueous vehicles. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile water at the same concentrations as that found in the test article-treated groups
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- non-activated test system: 0.1 and 0.2 ug/ml final concentrations
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Sterile water at the same concentrations as that found in the test article-treated groups
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- S9 activated test system: 10 and 20 ug/ml final concentrations
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 16-24 hours.
- Exposure duration: In the non-activated study, the cells were exposed to the test article for 4 hours or continuously for 20 hour. In the S9 activated study, the cells were exposed for 4 hours.
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF REPLICATIONS: 2 replicates per test substance concentration
NUMBER OF CELLS EVALUATED: A minimum of 200 metaphase spreads (100 per duplicate flask) were examined
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: viability of cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Mitotic index = number mitotic figures x 100/500 cells counted.
%Aberrant Cells: numerical cells include polyploid and endoreduplicated cells; structural cells exclude cells with only gaps.
Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures include quadriradials, triradials and complex rearrangements.
Chromosome breaks include breaks and acentric fragments; Die, dicentric chromosome.
Severely damaged cells includes cells with one or more pulverized chromosome and cells with 10 or more aberrations. - Evaluation criteria:
- The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control. The number and types of aberration
found, the percentage ofstructurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell were calculated.
All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test article was considered to induce a
positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations
being statistically significant (p≤0.05). However, values that are statistically significant but do not exceed the range of historic solvent controls may
be judged as not biologically significant. Test articles not demonstrating a statistically significant increase in aberrations will be
concluded to be negative. - Statistics:
- Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent
aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level,
the Cochran-Armitage test was used to measure dose-responsiveness. - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- see Additional information on results
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest concentration of test article in treatment medium was
approximately 7.5
- Effects of osmolality: treatment medium of the highest concentration tested, 1320 ug/mL, was 292 mmol/kg. The osmolality in treatment of the highest soluble dose, 660 ug/mL, was 268 mmol/kg. The osmolality of the solvent (water) in treatment medium was 276 mmol/kg.
- Water solubility: The test article was soluble in treatment medium at dose levels ≤660 ug/mL.
- Precipitation: Visible precipitate was observed in treatment medium at dose level 1320 ug/mL.
- Other confounding effects:
RANGE-FINDING/SCREENING STUDIES:
CHO cells were exposed to solvent alone and to nine concentrations of test article ranging from 0.132 ug/mL to 1320 ug/mL in the absence and
presence of an S9 reaction mixture. The test article was soluble in treatment medium at dose levels 996 ug/mL. Visible precipitate was observed in treatment medium at dose level 1320 ug/mL.
Cell growth inhibition relative to the solvent control was 25% and 17% at 1320 ug/mL, the highest concentration tested in both the non-activated
and S9 activated 4 hour exposure groups, respectively. Cell growth inhibition relative to the solvent control was 37% at 1320 ug/mL, the highest
concentration tested in the non-activated 20 hour continuous exposure group.
COMPARISON WITH HISTORICAL CONTROL DATA:
Both structural and numerical abberations of the cultures treated with the test articel were within ranges of the historical control data.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxicity of Diammonium Phosphate (DAP) (cell growth inhibition relative to the solvent control) in CHO cells when treated for 4 hours in the
absence of S9 activation was 56% at 660 ug/mL, the highest test concentration evaluated for chromosome aberrations.
Toxicity of Diammonium Phosphate (DAP) (cell growth inhibition relative to the solvent control) in CHO cells when treated for 4 hours in the
presence ofS9 activation was 5% at 1320 ug/mL, the highest test concentration evaluated for chromosome aberrations.
Toxicity of Diammonium Phosphate (DAP) (cell growth inhibition relative to the solvent control) in CHO cells when treated for 20 hours in the
absence of S9 activation was 63% at 660 ug/mL, the highest test concentration evaluated for chromosome aberrations. - Conclusions:
- Interpretation of results: negative
The positive and solvent controls fulfilled the requirements for a valid test.
Under the conditions of the assay described in this report, Diammonimn Phosphate (DAP) was concluded to be negative for the induction of
structural and numerical chromosome aberrations in CHO cells.
Referenceopen allclose all
Initial Toxicity-Mutation Assay
With (+) or Without (-) S9-mix |
Test substance concentration (μg/plate) |
Number of colonies/platea) |
|||||
Base-pair substitution type |
Frame shift type |
||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||
-S9 mix |
Solvent control |
130 |
18 |
13 |
12 |
7 |
|
2.5 |
101 |
12 |
12 |
10 |
4 |
||
7.5 |
118 |
16 |
14 |
14 |
4 |
||
25 |
103 |
17 |
11 |
19 |
6 |
||
75 |
102 |
16 |
16 |
14 |
5 |
||
200 |
128 |
14 |
11 |
13 |
7 |
||
600 |
91 |
14 |
10 |
16 |
6 |
||
1800 |
100 * |
14 * |
12 * |
12 * |
5 * |
||
5000 |
83 * |
16 * |
10 * |
12 * |
5 * |
||
+S9 mix |
Solvent control |
145 |
21 |
14 |
15 |
9 |
|
2.5 |
119 |
9 |
18 |
16 |
9 |
||
7.5 |
124 |
11 |
14 |
18 |
6 |
||
25 |
117 |
12 |
17 |
19 |
7 |
||
75 |
99 |
11 |
14 |
13 |
6 |
||
200 |
110 |
14 |
13 |
21 |
6 |
||
600 |
92 |
12 |
17 |
15 |
7 |
||
1800 |
90 * |
13 * |
19 * |
17 * |
9 * |
||
5000 |
95 * |
15 * |
9 * |
13 * |
6 * |
||
Positive control without S9 mix |
Name |
NaN3 |
NaN3 |
MMS |
2-NF |
9-AA |
|
Number of colonies/plate |
396 |
176 |
94 |
97 |
343 |
||
Positive control with S9 mix |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
|
Number of colonies/plate |
486 |
87 |
167 |
459 |
70 |
a) The average number of revertant colonies from two plates
*)Precipitate
NaN3= sodium azide; 2-NF= 2-nitrofluorene; 9-AA = 9-aminoacridine; MMS = methyl methanesulfonate; 2 -AA = 2 -aminoanthracene
Solvent control = water
Confirmatory Mutagenicity Assay
With (+) or Without (-) S9-mix |
Test substance concentration (μg/plate) |
Number of colonies/platea) |
|||||
Base-pair substitution type |
Frame shift type |
||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||
-S9 mix |
Solvent control |
119 |
20 |
16 |
12 |
6 |
|
50 |
101 |
17 |
12 |
12 |
5 |
||
150 |
136 |
17 |
15 |
9 |
5 |
||
500 |
105 |
17 |
15 |
10 |
9 |
||
1500 |
121 |
20 |
13 |
11 |
7 |
||
5000 |
125 * |
19 * |
10 * |
11 * |
5 * |
||
+S9 mix |
Solvent control |
148 |
13 |
19 |
20 |
8 |
|
50 |
144 |
15 |
17 |
17 |
7 |
||
150 |
125 |
15 |
12 |
18 |
8 |
||
500 |
117 |
17 |
13 |
20 |
8 |
||
1500 |
128 |
14 |
16 |
16 |
7 |
||
5000 |
117 * |
15 * |
10 * |
21 * |
5 * |
||
Positive control without S9 mix |
Name |
NaN3 |
NaN3 |
MMS |
2-NF |
9-AA |
|
Number of colonies/plate |
475 |
244 |
100 |
113 |
475 |
||
Positive control with S9 mix |
Name |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
|
Number of colonies/plate |
1094 |
143 |
239 |
977 |
97 |
a) The average number of revertant colonies from three plates
*)Precipitate
NaN3= sodium azide; 2-NF= 2-nitrofluorene; 9-AA = 9-aminoacridine; MMS = methyl methanesulfonate; 2 -AA = 2 -aminoanthracene
Solvent control = water
Treatment |
S9 activation |
Treatment time |
Mean mitotic index |
Cells scored |
Aberrations per cell (Mean +/- SD) |
Cells with Aberrations |
|
Numerical (%) |
Structural (%) |
||||||
Water |
- |
4 |
16.0 |
200 |
0.000±0.000 |
0.5 |
0.0 |
DAP |
|
|
|
|
|
|
|
165 µg/ml |
- |
4 |
14.1 |
200 |
0.005±0.071 |
0.0 |
0.5 |
330 µg/ml |
- |
4 |
11.6 |
200 |
0.005±0.071 |
2.0 |
0.5 |
660 µg/ml |
- |
4 |
6.7 |
200 |
0.015±0.158 |
3.0 |
1.0 |
MMC 0.2 µg/ml |
- |
4 |
15.0 |
200 |
0.105±0.380 |
0.5 |
8.5** |
|
|
|
|
|
|
|
|
Water |
+ |
4 |
17.1 |
200 |
0.025±0.186 |
1.5 |
2.0 |
DAP |
|
|
|
|
|
|
|
330 µg/ml |
+ |
4 |
15.9 |
200 |
0.005±0.071 |
2.5 |
0.5 |
660µg/ml |
+ |
4 |
15.6 |
200 |
0.045±0.231 |
2.5 |
4.0 |
1320 µg/ml |
+ |
4 |
16.4 |
200 |
0.015±0.122 |
1.5 |
1.5 |
CP 10 µg/ml |
+ |
4 |
14.1 |
100 |
0.770±0.962 |
1.5 |
48.0** |
|
|
|
|
|
|
|
|
Water |
- |
20 |
11.8 |
200 |
0.000±0.000 |
1.5 |
0.0 |
DAP |
|
|
|
|
|
|
|
165 µg/ml |
- |
20 |
10.1 |
200 |
0.005±0.071 |
1.5 |
0.5 |
330 µg/ml |
- |
20 |
7.3 |
200 |
0.025±0.157 |
4.5 |
2.5* |
660 µg/ml |
- |
20 |
6.3 |
200 |
0.005±0.071 |
1.0 |
0.5 |
MMC 0.1 µg/ml |
- |
20 |
11.4 |
200 |
0.200±0.521 |
0.5 |
15.5** |
Treatment: Cells from all treatment conditions were harvested at 20 hours after the initiation of the treatments.
Aberrations per cell: Severely damaged cells were counted as 10 aberrations.
Percent aberrant cells: *, p≤0.05; **, p≤0.01; using Fisher's exact test
DAP = Diammonium Phosphate; CP = cyclophosphamide; MMC = mitomycin C
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
With ammonium dihydrogenorthophosphate no in vitro studies were present for the Ames and the chromosome aberration tests, however with diammonium hydrogenorthophosphate studies are present. A reliable TK-assay with this substance itself was present.
In an Ames test according to OECD 471 guideline, Salmonella Typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and E. Coli WP2 uvr A showed no genotoxicity with and without metabolic activation, tested up to the limit concentration. In an in vitro chromosome aberration test with CHO cells performed according to OECD 473 guideline, also no genotoxicity was seen with and without metabolic activation, while cytotoxicity was present. With ammonium dihydrogenorthophosphate a reliable TK-assay was present. In a Thymidine kinase (TK) assay in L5178Y mouse lymphoma cells performed according to OECD 476 and EC B.17 guidelines, the substance did not induce a significant increase in the mutation frequency. Based on these negative results for genotoxicity in in vitro studies, no in vivo studies are necessary.
Justification for selection of genetic toxicity endpoint
An Ames test and a chromosome aberration study with the read-across
substance diammonium hydrogenorthophosphate are available, showing no
adverse effects. A reliable Mouse lymphoma study with the substance is
available, showing no adverse effects.
Short description of key information:
No reliable Ames and chromosome aberration studies with ammonium
dihydrogenorthophoshate are present. Based on reliable in vitro studies
with diammonium hydrogenorthophosphate, the Ames test and the chromosome
aberration study were negative in the presence and absence of metabolic
activation. For the in vitro TK assay a reliable study with ammonium
dihydrogenorthophosphate is present showing negative results in the
presence and absence of metabolic activation. The read-across rationale
can be found in the category approach document attached in IUCLID
Section 13 and fully included in the CSR.
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
Based on the available data, ammonium dihydrogenorthophosphate does not have to be classified according to regulation (EC) No.1272/2008 for genetic toxicity.
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