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EC number: 908-996-7 | CAS number: -
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 15 March 2012 until 04 October 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: according to OECD 476, GLP conform
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guidelineopen allclose all
- 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:
- first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation - GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- dimethoxyphosphinic acid; methoxyphosphonic acid; phosphoric acid
- EC Number:
- 908-996-7
- IUPAC Name:
- dimethoxyphosphinic acid; methoxyphosphonic acid; phosphoric acid
- Test material form:
- other: liquid
Constituent 1
Method
- Target gene:
- HPRT
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
- Test concentrations with justification for top dose:
- Experiment I:
without metabolic activation: 156.3; 312.5; 625.0; 1250; 2500; 5000 µg/mL
with metabolic activation: 156.3; 312.5; 625.0; 1250; 2500; 5000 µg/mL
Experiment II:
without metabolic activation: 156.3; 312.5; 625.0; 1250; 2500; 5000 µg/mL
with metabolic activation. 156.3; 312.5; 625.0; 1250; 2500; 5000 µg/mL
In both main experiments the cultures at the lowest concentration in the presence and absence of metabolic activation were not continued since a minimum of only four analys-able concentrations is required by the guidelines. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility properties
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days
SELECTION AGENT (mutation assays): 6-Thioguanine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1,5x10exp. 6
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
experimental group: p-value:
experiment I, culture I without S9 mix 0.831
experiment I, culture II without S9 mix 0.564
experiment I, culture I with S9 mix 0.553
experiment I, culture II with S9 mix 0.308
experiment II, culture I without S9 mix 0.871
experiment II, culture II without S9 mix 0.773
experiment II, culture I with S9 mix 0.373
experiment II, culture II with S9 mix 0.789
Results and discussion
Test results
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
In the pre-experiment the pH at concentrations of 156.3 µg/mL and above was adjusted to neutral with 2 N sodium hydroxide. In the main experiments the pH at concentrations of 312.5 µg/mL and above was adjusted as described above.
- Effects of osmolality: Not effected
- Evaporation from medium: Not examined
- Water solubility: Not indicated
- Precipitation:
The test medium was checked for precipitation or phase separation at the end of each treatment period prior to removal of the test item. No precipitation occurred up to the maximum concentration in the presence and absence of metabolic activation following 4 and 24 hours treatment.
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four ana-lysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20 % relative survival or cell density at subcultivation and the ana-lysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up or beyond their limit of solubility. Precipitation should be evalu-ated at the end of treatment by the unaided eye.
In the range finding pre-experiment test item concentrations between 39.1 and 5000 µg/mL according to OECD guideline were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.
Relevant cytotoxic effects indicated by a relative suspension growth below 50% were noted at 2500 µg/mL and above without metabolic activation following 4 hours treatment.
The test medium was checked for precipitation or phase separation at the end of each treatment period prior to removal of the test item. No precipitation occurred up to the maximum concentration in the presence and absence of metabolic activation following 4 and 24 hours treatment.
In the pre-experiment the pH at concentrations of 156.3 µg/mL and above was adjusted to neutral with 2 N sodium hydroxide. In the main experiments the pH at concentrations of 312.5 µg/mL and above was adjusted as described above.
Based on the results of the pre-experiment, the maximum concentration of the main ex-periments was again 5000 µg/mL. The individual concentrations were spaced by a factor of 2.
In both main experiments the cultures at the lowest concentration in the presence and absence of metabolic activation were not continued since a minimum of only four analys-able concentrations is required by the guidelines.
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Main experiments:
No relevant cytotoxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Summary Table
relative | relative | relative | mutant | relative | relative | relative | mutant | |||||
conc. | S9 | cloning | cell | cloning | colonies/ | induction | cloning | cell | cloning | colonies/ | induction | |
µg/mL | mix | efficiency I | density | efficiency II | 106cells | factor | efficiency I | density | efficiency II | 106cells | factor | |
% | % | % | % | % | % | |||||||
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
Experiment I / 4 h treatment | culture I | culture II | ||||||||||
Solvent control with water | - | 100.0 | 100.0 | 100.0 | 21.7 | 1.0 | 100.0 | 100.0 | 100.0 | 18.2 | 1.0 | |
Positive control (EMS) | 150.0 | - | 102.2 | 76.5 | 96.9 | 138.6 | 6.4 | 96.3 | 91.0 | 95.7 | 148.6 | 8.2 |
Test item | 156.3 | - | 104.0 | culture was not continued# | 101.6 | culture was not continued# | ||||||
Test item | 312.5 | - | 101.9 | 88.6 | 87.8 | 15.2 | 0.7 | 86.4 | 113.9 | 106.9 | 13.9 | 0.8 |
Test item | 625.0 | - | 106.8 | 79.5 | 100.2 | 19.6 | 0.9 | 103.2 | 86.8 | 99.3 | 16.2 | 0.9 |
Test item | 1250.0 | - | 103.5 | 69.9 | 97.5 | 12.0 | 0.6 | 101.8 | 86.4 | 100.0 | 13.1 | 0.7 |
Test item | 2500.0 | - | 98.9 | 85.0 | 100.8 | 23.6 | 1.1 | 100.6 | 92.1 | 96.6 | 43.3 | 2.4 |
Test item | 5000.0 | - | 97.3 | 81.4 | 91.2 | 18.4 | 0.8 | 103.5 | 79.8 | 99.8 | 18.3 | 1.0 |
Experiment I / 4 h treatment | culture I | culture II | ||||||||||
Solvent control with water | + | 100.0 | 100.0 | 100.0 | 12.3 | 1.0 | 100.0 | 100.0 | 100.0 | 20.3 | 1.0 | |
Positive control (DMBA) | 1.1 | + | 34.8 | 252.7 | 94.4 | 862.0 | 70.2 | 38.2 | 57.6 | 84.5 | 1147.2 | 56.4 |
Test item | 156.3 | + | 101.9 | culture was not continued# | 95.9 | culture was not continued# | ||||||
Test item | 312.5 | + | 100.8 | 86.4 | 108.9 | 18.5 | 1.5 | 92.2 | 154.9 | 111.3 | 26.4 | 1.3 |
Test item | 625.0 | + | 102.3 | 127.7 | 123.2 | 18.6 | 1.5 | 97.3 | 79.3 | 124.2 | 12.8 | 0.6 |
Test item | 1250.0 | + | 97.2 | 162.8 | 110.8 | 11.6 | 0.9 | 92.6 | 233.4 | 95.3 | 24.6 | 1.2 |
Test item | 2500.0 | + | 99.8 | 86.5 | 108.5 | 44.0 | 3.6 | 93.9 | 109.5 | 102.3 | 30.3 | 1.5 |
Test item | 5000.0 | + | 100.5 | 153.2 | 108.4 | 18.3 | 1.5 | 90.2 | 88.4 | 89.8 | 27.0 | 1.3 |
Experiment II / 24 h treatment | culture I | culture II | ||||||||||
Solvent control with water | - | 100.0 | 100.0 | 100.0 | 10.8 | 1.0 | 100.0 | 100.0 | 100.0 | 15.8 | 1.0 | |
Positive control (EMS) | 150.0 | - | 103.3 | 159.8 | 69.4 | 385.4 | 35.5 | 108.4 | 105.0 | 90.3 | 280.5 | 17.8 |
Test item | 156.3 | - | 101.9 | culture was not continued# | 98.5 | culture was not continued# | ||||||
Test item | 312.5 | - | 102.0 | 157.7 | 69.7 | 21.7 | 2.0 | 103.2 | 148.2 | 99.8 | 12.7 | 0.8 |
Test item | 625.0 | - | 105.1 | 158.4 | 85.8 | 19.5 | 1.8 | 99.7 | 131.6 | 100.9 | 17.1 | 1.1 |
Test item | 1250.0 | - | 105.3 | 171.1 | 84.9 | 24.1 | 2.2 | 96.8 | 102.0 | 92.3 | 16.1 | 1.0 |
Test item | 2500.0 | - | 107.3 | 143.8 | 82.6 | 17.3 | 1.6 | 97.4 | 133.4 | 93.6 | 23.6 | 1.5 |
Test item | 5000.0 | - | 105.9 | 134.9 | 72.6 | 16.2 | 1.5 | 74.0 | 90.5 | 86.2 | 14.5 | 0.9 |
Experiment II / 4 h treatment | ||||||||||||
Solvent control with water | + | 100.0 | 100.0 | 100.0 | 17.0 | 1.0 | 100.0 | 100.0 | 100.0 | 16.6 | 1.0 | |
Positive control (DMBA) | 1.1 | + | 94.8 | 83.1 | 66.3 | 771.1 | 45.4 | 65.6 | 63.2 | 77.1 | 789.9 | 47.4 |
Test item | 156.3 | + | 102.4 | culture was not continued# | 101.4 | culture was not continued# | ||||||
Test item | 312.5 | + | 105.2 | 110.6 | 95.8 | 25.3 | 1.5 | 99.9 | 92.2 | 83.2 | 40.4 | 2.4 |
Test item | 625.0 | + | 113.4 | 101.1 | 97.1 | 9.4 | 0.6 | 102.4 | 92.2 | 81.3 | 12.5 | 0.8 |
Test item | 1250.0 | + | 113.1 | 92.8 | 75.9 | 24.2 | 1.4 | 101.1 | 85.9 | 100.9 | 19.7 | 1.2 |
Test item | 2500.0 | + | 104.0 | 124.7 | 56.1 | 18.2 | 1.1 | 89.4 | 82.6 | 94.7 | 12.5 | 0.8 |
Test item | 5000.0 | + | 113.1 | 105.6 | 66.5 | 26.6 | 1.6 | 97.5 | 81.4 | 90.7 | 21.9 | 1.3 |
# culture was not continued since a minimum of only four analysable concentrations is required
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Hordaphos CC MS is considered to be non-mutagenic in this HPRT assay. - Executive summary:
The test item Hordaphos CC MS was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The cell cultures were evaluated at the following concentrations:
exposure
periodS9
mixconcentrations
in µg/mLExperiment I
4 hours
-
312.5
625.0
1250
2500
5000
4 hours
+
312.5
625.0
1250
2500
5000
Experiment II
24 hours
-
312.5
625.0
1250
2500
5000
4 hours
+
312.5
625.0
1250
2500
5000
No relevant cytotoxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration.
In the first culture of experiment I the induction factor exceeded the threshold of three times the mutation frequency of the corresponding solvent control at 2500 µg/mL in the presence of metabolic activation. The historical range of solvent controls was also exceeded at this experimental point (44.0 versus 36.6 mutant colonies per 106cells, see Historical Data, Annex II). However, the effect was not reproduced in the parallel culture under identical experimental conditions, and was therefore, judged as biologically irrelevant.
In the second culture of the second experiment the historical range of solvent controls was also exceeded at 312.5 µg/mL with metabolic activation (40.4 versus 36.6 mutant colonies per 106cells).The increase was not dose dependent and was again not reproduced in the parallel culture under indentical experimental conditions and was therefore, judged as biologically irrelevant.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies.No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 10.8 up to 21.7 mutants per 106cells; the range of the groups treated with the test item was from 9.4 up to 44.0 mutant colonies per 106cells.
EMS(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
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