Registration Dossier
Registration Dossier
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EC number: 701-298-1 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
1- Ames test (OECD 471, GLP, Kr.1): not mutagenic in Bacteria.
2- In vitro gene mutation study in mammalian cells (OECD 490, GLP, Kr.1): not mutagenic in mammalian cells with and without metabolic activation.
3- in vitro cytogenicity / micronucleus study (OECD 487, GLP, Kr.1): not clastogenic.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 July to 20 October 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Alkenyl phosphonate
- Expiration date of the lot/batch: 09 September 2017
- Purity test date: 08/06/2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%)
- Stability under test conditions: yes - Target gene:
- Histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Dose range finding test: dose range: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate with and without S9-mix (TA98 and TA100)
Experiment 1, with and without metabolic activation: 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate
Experiment 2, with and without metabolic activation: 1.581, 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate. - Vehicle / solvent:
- In the study three vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive control chemicals. The following chemicals were used for vehicle (solvent) control groups:
1- Dimethyl sulfoxide (puriss. p.a., dried, water ≤ 0.02%):
- Supplier: Sigma-Aldrich Co.
- Batch No.: STBG8411
- Expiry date: 29 February 2020
- Purity: >99.99%
2- Distilled water:
- Manufacturer: Hungaro-Gal Kft.
- Batch No.: 802 0117 / 8080617
- Expiry date: 23 July 2017 / 06 December 2017
3- N,N-dimethylformamide (DMF)
- Supplier: VWR
- Batch No.: 17C034009
- Expiry date: 28 February 2022 - Untreated negative controls:
- yes
- Remarks:
- Dimethyl sulfoxide, Distilled water and N,N-dimethylformamide (DMF).
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulfoxide, Distilled water and N,N-dimethylformamide (DMF).
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-1,2-phenylenediamine (NDP) and 2-aminoanthracene (2AA)
- Remarks:
- with and without metabolic activation.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation method
DURATION
- Preincubation period: 20 minutes at 37°C
NUMBER OF REPLICATIONS: 3 plates/dose/strain
DETERMINATION OF CYTOTOXICITY
- Method: background lawn of microcolonies observation - Evaluation criteria:
- The test article was considered to be positive for mutagenicity if the mean number of revertant colonies in the test article group was no less than 2 times that of the negative control group, and if the number of revertant colonies increased with increasing dose of the test article.
- Statistics:
- Statistical methods were not used in the determination of test results.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was on the plates in the Preliminary Concentration Range Finding Test. Precipitate/slight precipitate/ microdrops was/were detected on the plates in the main tests at 5000 μg/plate concentration without metabolic activation.
RANGE-FINDING: yes
Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 15.81 and 5 μg/plate, in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.
COMPARISON WITH HISTORICAL CONTROL DATA: yes - Remarks on result:
- other:
- Remarks:
- Slightly reduced background lawn was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA100 and TA1537 strains without metabolic activation on the plates at 5000 and 1581 µg/plate.
- Conclusions:
- Under the test conditions of this study, Alkenyl phosphonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
- Executive summary:
In a reverse gene mutation assay in bacteria, performed according to the OECD No.471 guideline, Alkenyl phosphonate was tested in S. typhimurium TA1535, TA1537, TA100 and TA98 and in E. coli WP2 uvr A in the presence and the absence of mammalian metabolic activation (S9) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats,using the preincubation method.
Based on the results of the Compatibility Test, the test item was dissolved in N,N-Dimethylformamide (DMF) at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 15.81 and 5 μg/plate, in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.
No precipitate was on the plates in the Preliminary Concentration Range Finding Test. Precipitate/slight precipitate/ microdrops was/were detected on the plates in the main tests at 5000 μg/plate concentration without metabolic activation. Inhibitory, cytotoxic effect of the test item was not detected in thePreliminary Concentration Range Finding Test and in theInitial Mutation Test. Slightly reduced background lawn was observed in the Confirmatory Mutation Test inSalmonella typhimuriumTA100 and TA1537 strains without metabolic activation on the plates at 5000 and 1581 µg/plate. In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.
The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid.
Under the test conditions of this study, Alkenyl phosphonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP-compliant OECD guideline study.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Alkenyl phosphonate
- Expiration date of the lot/batch: 09 September 2017
- Purity test date: 08/06/2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%)
- Stability under test conditions: yes - Target gene:
- Not applicable.
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France).
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL). This medium was supplemented by heat inactivated horse serum at 10% (v/v).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route.
- Test concentrations with justification for top dose:
- The dose-levels selected for treatments were as follows:
- 4.1, 12.3, 37, 111.1, 333.3 and 1000 µg/mL for the 3-hour treatments, with and without S9 mix,
- 7.81, 15.6, 31.3, 62.5, 125, 187.5, 250 and 500 µg/mL for the 24-hour treatment without S9 mix.
The dose-levels selected for micronucleus analysis were as follows:
- 12.3, 37 and 111.1 µg/mL for the 3-hour treatment with and without S9 mix, the latter being the lowest dose-level showing precipitate in the culture medium at the end of the treatment period,
- 15.6, 62.5 and 125 µg/mL for the 24-hour treatment without S9 mix, the latter being the lowest dose level showing precipitate in the culture medium at the end of the treatment period and inducing the recommended level of cytotoxicity (i.e. a 54% decrease in the PD). - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: according to available solubility data, the vehicle used for the preparation of test item dose formulations
and the treatment of vehicle control cultures was ethanol. - Untreated negative controls:
- yes
- Remarks:
- Sovent control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Colchicine (without S9 mix).
- Details on test system and experimental conditions:
- The assay using duplicate cultures was performed both with and without S9. A single sampling time was used: 24 hours, i.e. 1.5 cell cycle times from the beginning of treatment.
For each culture, approximately 5 x 10E5 cells were seeded in 3 mL DMEM (Eagle medium modified by Dulbecco) medium containing 10% fetal calf serum, 1% L. glutamine, penicillin, streptomycin and fungizon in flasks of 25 cm2. The flasks were then placed at 37°C in a humidified atmosphere of 5% CO2 in 95% air. After 24 hours, the cultures were exposed to the test subtance:
- for 3 hours at 37°C in culture medium without fetal calf serum for the assay without S9,
- for 3 hours at 37°C in the culture medium without fetal calf serum containing 15% S9, for the assay with S9.
After treatment, the cells were observed under a microscope to assess any morphological alterations, the treatment medium removed, the cells rinsed and fresh medium added. The cultures were then incubated for 21 hours at 37°C.
Two hours before cell collection, the cells were treated with a colcemid solution in order to stop mitosis at the metaphase-stage.
The cells were then rinsed, trypsinated, harvested and centrifuged. After a hypotonic treatment, they were fixed in a methanol/acetic acid mixture, spread on slides and stained with Giemsa. Two slides/culture were prepared. - Evaluation criteria:
- 1- Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
- or at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.
2- Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met. - Statistics:
- The incidence of cells wtih aberrations (excluding gaps) in treated cultures was compared to that of the solvent cultures using the "Chi-square" test.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At the highest dose-level of 2500 µg/mL, the pH of the culture medium was approximately 7.4 (as for the vehicle control) Therefore, none of the selected dose-levels was considered to produce extreme culture conditions.
- Effects of osmolality: At the highest dose-level of 2500 µg/mL, the osmolality was 348 mOsm/kg H2O (395 mOsm/kg for the vehicle control). Therefore, none of the selected dose-levels was considered to produce extreme culture conditions.
- Evaporation from medium: none
- Water solubility: the test substance is poorly soluble in water.
- Precipitation:
A precipitate was observed in the culture medium at dose-levels >= 250 µg/mL at the end of both treatment periods (preliminary cytotoxicity test).
A precipitate was observed in the culture medium at dose-levels >= 111.1 µg/mL at the end of the 3-hour treatment period, and >= 125 µg/mL at the end of the 24-hour treatment period (Main test).
RANGE-FINDING/SCREENING STUDIES:
Based on available solubility data, the highest selected dose-level to be used in the preliminary cytotoxicity test was 2500 µg/mL, since it was expected to produce precipitation in the culture medium. This dose-level was obtained using a test item stock solution at the concentration of 500 mg/mL and a treatment volume of 0.5% (v/v) in the culture medium.
The dose-levels selected for the treatment of the preliminary test were 5, 50, 250, 500, 1250 and 2500 µg/mL.
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used:- Remarks on result:
- other:
- Remarks:
- Following the 3-hour treatment without S9 mix, a severe cytotoxicity was induced at the dose-level of 1000 µg/mL, as shown by a 100% decrease in the PD. Following the 24-hour treatment without S9 mix, a moderate to severe cytotoxicity was induced at dose levels ≥ 31.3 µg/mL, as shown by a 40 to 100% decrease in the PD. Following the 3-hour treatment with S9 mix, no cytotoxicity was induced at any dose-levels, as shown by the absence of any decrease in the PD.
- Conclusions:
- Under the experimental conditions of the study, the test item Alkenyl phosphonate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using
L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system. - Executive summary:
In an in vitro Micronucleus Test (K. Chevallier, 2017), performed according to the OECD guideline N° 487 and in compliance with good laboratory practice, L5178Y TK+/- mouse lymphoma
cells were treated with Alkenyl phosphonate diluted in ethanol at three dose levels and in each treatment condition, in duplicate, together with negative and positive controls. The dose range was selected on the basis of the results of a preliminary toxicity test and was12.3, 37 and 111.1 µg/mL for the 3-hour treatment with and without S9 mix, the latter being the lowest dose-level showing precipitate in the culture medium at the end of the treatment period,15.6, 62.5 and 125 µg/mL for the 24-hour treatment without S9 mix, the latter being the lowest dose‑level showing precipitate in the culture medium at the end of the treatment period and inducing the recommended level of cytotoxicity (i.e.a 54% decrease in the PD).
Following the 3-hour treatments with and without S9 mix or the 24-hour treatment without S9 mix, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the analyzed dose levels in comparison to the corresponding vehicle control. Moreover, none of the analyzed dose-levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle historical range. Thus, these results met the criteria of a negative response.
Under the experimental conditions of the study, the test item Alkenyl phosphonate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27/02/2017 - 13/06/2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: Mammalian Cell Gene Mutation Test with L5178Y Mouse Lymphoma Cells
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Alkenyl phosphonate
- Expiration date of the lot/batch: 09 September 2017
- Purity test date: 08/06/2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%)
- Stability under test conditions: yes - Target gene:
- thymidine kinase (TK)
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA).
- Suitability of cells: Recommended test system in OECD guidelines.
- Methods for maintenance in cell culture: Cell density was kept below 1 x 106 cells/ml.
- The cultures were checked for Mycoplasma contamination.
- Stock cultures of the cells were stored in a cryoprotective medium [10% horse serum and 10% dimethylsulfoxide (DMSO)] at -80°C.
- Cleansing: The cells were maintained in flasks as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 10% v/v, in a 37°C, 5% CO2 humidified incubator.
MEDIA USED
The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL).
This medium (RPMI 0) was supplemented by heat inactivated horse serum at 10% v/v (RPMI 10) or 20% v/v (RPMI 20).
RPMI 10 was diluted with RPMI 0 (1:1, v/v) in order to obtain RPMI 5 which was used for the 3-hour treatment.
ENVIRONMENTAL CONDITIONS
- All incubations were carried out in a humid atmosphere (80 - 100%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C
- Temperature and humidity were continuously monitored throughout the experiment.
- The CO2 percentage was monitored once on each working day. - Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver microsomal fraction (S9 fraction). S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.
- Test concentrations with justification for top dose:
- 1- Preliminary cytotoxicity test (with and without metabolic activation, 3 hours of exposure): 0, 5, 50, 250, 500, 1250 and 2500 µg/mL
2- Mutagenicity test (first experiment with and without metabolic activation, 3 hours of exposure): 0, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL
3- Cytotoxicity test (second experiment with metabolic activation, 3 hours of exposure): 0, 7.8, 15.6, 31.3, 62.5, 125 and 250 µg/mL
4- Mytotoxicity test (second experiment with metabolic activation, 3 hours of exposure): 0, 7.8, 15.6, 31.3, 62.5, 125 and 250 µg/mL - Vehicle / solvent:
- The test item was dissolved in ethanol.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- The negative control was ethanol, the vehicle of the test item.
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 5 x 105 cells/mL
DURATION
- Exposure duration:
* First experiment: 3 hours with and without metabolic activation
* Second experiment: 3 hours with metabolic activation.
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 2000 cells/well
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
OTHER: The following parameters were used for the scoring of colonies in mutant plates:
* well containing mutant colony (small or large),
* well not containing mutant colony,
* when both small and large colonies are present in the same well, two mutant colonies were counted (one small and one large). - Evaluation criteria:
- A positive result, which should fulfill the following criteria:
- at least at one dose level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10-6,
- a dose-response relationship is demonstrated by a statistically significant trend test.
Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.
Noteworthy increases in the mutation frequency observed only at high-levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose levels with Adj. RTG between 10 and 20%, are not considered as positive results.
A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if:
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG. - Statistics:
- To assess the dose-response relationship, a linear regression was performed between dose levels and individual mutation frequencies obtained at dose levels showing a mean Adj. RTG ≥ 10%. This statistical analysis was performed using SAS Enterprise Guide software.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- A severe cytotoxicity was induced at the dose level of 500 µg/mL, as shown by a 98% decrease in the Adj. RTG.
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At the highest tested dose level, i.e. 2500 µg/mL (preliminary cytotoxicity test), the pH of the culture medium was approximately 7.4 (as for the vehicle control)
- Effects of osmolality: The osmolarity at a concentration of 2500 μg/ml was 340 mOsm/kg H2O (393 mOsm/kg H2O for the vehicle control).
- Precipitation:
1- Main experiment (With S9):
A precipitate, which did not prevent any scoring, was noted in the culture medium at the end of the treatment period, at dose levels ≥ 62.5 µg/mL in both experiments.
2- Main experiment (Without S9):
A precipitate, which did not prevent any scoring, was noted in the culture medium at the end of the treatment period at dose levels ≥ 62.5 µg/mL.
RANGE-FINDING/SCREENING STUDIES:
Based on available solubility data, the highest selected dose level was 2500 µg/mL since it was expected to produce precipitation in the culture medium. This dose level was obtained using a test item concentration of 500 mg/mL and a treatment volume of 0.5% (v/v) in the culture medium (i.e. 100 µL/20 mL culture medium). The dose levels selected for the treatment of the preliminary test were 5, 50, 250, 500, 1250 and 2500 µg/mL.
At the end of the treatment period, a precipitate was observed in the culture medium at dose levels ≥ 250 µg/mL.
Following the 3-hour treatment without S9 mix, a moderate to severe cytotoxicity was induced at dose levels ≥ 500 µg/mL, as shown by a 48 to 92% decrease in the Adj. RTG.
Following the 3-hour treatment with S9 mix, no noteworthy cytotoxicity was induced at any dose levels, as shown by the absence of any noteworthy decrease in the Adj. RTG
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (95%)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes - Remarks on result:
- other:
- Remarks:
- A precipitate, which did not prevent any scoring, was noted in the culture medium at the end of the treatment period, at dose levels ≥ 62.5 µg/mL in both experiments.
- Conclusions:
- Under the experimental conditions of this study, the test item, Alkenyl phosphonate, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.
- Executive summary:
The mutagenic potential of Alkenyl phosphonate was evaluated by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells. This study was conducted according to the OECD 490 guideline (Kr.1, GLP).
After a preliminary cytotoxicity test, the test item Alkenyl phosphonate, diluted in ethanol, was tested in two independent experiments with or without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.
In the first experiment, the test item was tested up to concentrations of 500 µg/mL both in the absence and the presence of S9-mix. A precipitate, which did not prevent any scoring, was noted in the culture medium at the end of the treatment period at dose levels ≥ 62.5 µg/ml. A severe cytotoxicity was induced at the dose level of 500 µg/mL, as shown by a 98% decrease in the Adj. RTG. No noteworthy increase in the mutation frequency was noted at any of the tested dose levels, relative to the corresponding vehicle control and no dose-response relationship was demonstrated by the linear regression.
In the second experiment, the test item was tested up to concentrations of 500 µg/mL in the absence of S9-mix and 250 µg/ml in the presence of S9 mix. A precipitate, which did not prevent any scoring, was noted in the culture medium at the end of the treatment period, at dose levels ≥ 62.5 µg/mL in both experiments. No noteworthy cytotoxicity was observed at any of the tested dose levels, as shown by the absence of any noteworthy decrease in the Adj. RTG, in either experiment. No noteworthy increase in the mutation frequency was noted at any of the tested dose levels in either experiment, relative to the corresponding vehicle control, and no dose-response relationship was demonstrated by the linear regression. These results did not meet the criteria of a positive response. Considering the negative results obtained in both independent experiments performed, the overall results were considered as suitable to allow a reliable interpretation.
The mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.
For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid.
Under the experimental conditions of this study, the test item, Alkenyl phosphonate, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.
Referenceopen allclose all
Table 1: Summary Table of the Preliminary Concentration Range Finding Test
Concentrations
|
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimuriumtester strains |
||||
TA 98 |
TA 100 |
|||||
-S9 |
+S9 |
-S9 |
+S9 |
|||
Untreated control |
Mean |
24.0 |
27.3 |
95.0 |
100.3 |
|
MF |
1.09 |
1.17 |
1.04 |
1.03 |
||
Distilled water control |
Mean |
-- |
-- |
90.7 |
-- |
|
MF |
-- |
-- |
0.99 |
-- |
||
DMSO control |
Mean |
21.0 |
27.0 |
-- |
104.3 |
|
MF |
0.95 |
1.16 |
-- |
1.08 |
||
DMF control |
Mean |
22.0 |
23.3 |
91.7 |
97.0 |
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
||
5000 |
Mean |
24.0 |
27.0 |
90.0 |
108.7 |
|
MF |
1.09 |
1.16 |
0.98 |
1.12 |
||
2500 |
Mean |
18.0 |
25.0 |
99.3 |
102.0 |
|
MF |
0.82 |
1.07 |
1.08 |
1.05 |
||
1000 |
Mean |
27.7 |
25.0 |
108.0 |
114.3 |
|
MF |
1.26 |
1.07 |
1.18 |
1.18 |
||
316 |
Mean |
25.0 |
27.7 |
107.0 |
105.0 |
|
MF |
1.14 |
1.19 |
1.17 |
1.08 |
||
100 |
Mean |
22.7 |
26.3 |
110.0 |
113.0 |
|
MF |
1.03 |
1.13 |
1.20 |
1.16 |
||
31.6 |
Mean |
21.3 |
24.0 |
104.0 |
124.0 |
|
MF |
0.97 |
1.03 |
1.13 |
1.28 |
||
10 |
Mean |
21.3 |
22.3 |
107.7 |
148.3 |
|
MF |
0.97 |
0.96 |
1.17 |
1.53 |
||
NPD (4mg) |
Mean |
396.7 |
-- |
-- |
-- |
|
MF |
18.89 |
-- |
-- |
-- |
||
2AA (2mg) |
Mean |
-- |
2401.3 |
-- |
2412.7 |
|
MF |
-- |
88.94 |
-- |
23.12 |
||
SAZ (2mg) |
Mean |
-- |
-- |
1212.7 |
-- |
|
MF |
-- |
-- |
13.38 |
-- |
||
Table 2: Summary Table of the Initial Mutation Test
Concentrations
|
Mean values of revertants / Mutation factor (MF) |
Salmonella thyphimuriumtester strains |
Escherichia coli |
|||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||
Untreated control |
Mean |
20.7 |
24.3 |
92.7 |
95.7 |
12.3 |
11.0 |
12.3 |
14.7 |
50.7 |
56.0 |
|
MF |
1.02 |
1.16 |
1.07 |
1.01 |
1.19 |
0.97 |
0.93 |
1.05 |
0.92 |
1.06 |
||
Distilled water control |
Mean |
-- |
-- |
92.0 |
-- |
11.3 |
-- |
-- |
-- |
56.3 |
-- |
|
MF |
-- |
-- |
1.07 |
-- |
1.10 |
-- |
-- |
-- |
1.02 |
-- |
||
DMSO control |
Mean |
19.0 |
26.7 |
-- |
90.7 |
-- |
9.0 |
13.7 |
14.7 |
-- |
56.0 |
|
MF |
0.93 |
1.27 |
-- |
0.96 |
-- |
0.79 |
1.03 |
1.05 |
-- |
1.06 |
||
DMF control |
Mean |
20.3 |
21.0 |
86.3 |
94.3 |
10.3 |
11.3 |
13.3 |
14.0 |
55.3 |
53.0 |
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
||
5000 |
Mean |
21.3 |
25.0 |
38.7 |
74.0 |
10.7 |
10.7 |
19.7 |
15.7 |
44.0 |
55.3 |
|
MF |
1.05 |
1.19 |
0.45 |
0.78 |
1.03 |
0.94 |
1.48 |
1.12 |
0.80 |
1.04 |
||
1581 |
Mean |
21.0 |
22.7 |
54.0 |
79.0 |
16.0 |
11.7 |
17.0 |
19.3 |
47.0 |
51.0 |
|
MF |
1.03 |
1.08 |
0.63 |
0.84 |
1.55 |
1.03 |
1.28 |
1.38 |
0.85 |
0.96 |
||
500 |
Mean |
19.0 |
21.0 |
65.3 |
78.7 |
12.3 |
10.7 |
16.7 |
20.0 |
43.5 |
46.7 |
|
MF |
0.93 |
1.00 |
0.76 |
0.83 |
1.19 |
0.94 |
1.25 |
1.43 |
0.79 |
0.88 |
||
158.1 |
Mean |
20.7 |
21.0 |
72.0 |
80.0 |
9.0 |
12.0 |
16.7 |
17.0 |
47.7 |
50.0 |
|
MF |
1.02 |
1.00 |
0.83 |
0.85 |
0.87 |
1.06 |
1.25 |
1.21 |
0.86 |
0.94 |
||
50 |
Mean |
21.0 |
22.0 |
70.7 |
79.7 |
11.0 |
8.7 |
18.3 |
21.7 |
50.0 |
52.0 |
|
MF |
1.03 |
1.05 |
0.82 |
0.84 |
1.06 |
0.76 |
1.38 |
1.55 |
0.90 |
0.98 |
||
15.81 |
Mean |
20.7 |
22.7 |
81.3 |
88.0 |
16.0 |
12.7 |
21.0 |
17.0 |
47.7 |
48.0 |
|
MF |
1.02 |
1.08 |
0.94 |
0.93 |
1.55 |
1.12 |
1.58 |
1.21 |
0.86 |
0.91 |
||
5 |
Mean |
19.7 |
23.3 |
83.7 |
82.3 |
10.0 |
7.0 |
16.7 |
21.0 |
40.7 |
46.3 |
|
MF |
0.97 |
1.11 |
0.97 |
0.87 |
0.97 |
0.62 |
1.25 |
1.50 |
0.73 |
0.87 |
||
NPD (4mg) |
Mean |
429.3 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
MF |
22.60 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
||
2AA (2mg) |
Mean |
-- |
2481.3 |
-- |
2482.7 |
-- |
201.3 |
-- |
200.7 |
-- |
-- |
|
MF |
-- |
93.05 |
-- |
27.38 |
-- |
22.37 |
-- |
13.68 |
-- |
-- |
||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
282.7 |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.05 |
||
SAZ (2mg) |
Mean |
-- |
-- |
1158.7 |
-- |
1156.0 |
-- |
-- |
-- |
-- |
-- |
|
MF |
-- |
-- |
12.59 |
-- |
102.00 |
-- |
-- |
-- |
-- |
-- |
||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
404.0 |
-- |
-- |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
29.56 |
-- |
-- |
-- |
||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
986.7 |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
17.51 |
-- |
||
Table 3: Summary Table of the Confirmatory Mutation Test
Concentrations
|
Mean values of revertants / Mutation factor (MF) |
Salmonella thyphimuriumtester strains |
Escherichia coli |
|||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2 uvrA |
||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||
Untreated control |
Mean |
24.7 |
32.3 |
99.0 |
99.0 |
12.3 |
14.7 |
12.7 |
14.3 |
53.0 |
57.7 |
|
MF |
0.84 |
1.03 |
1.03 |
0.99 |
0.82 |
1.26 |
0.86 |
0.98 |
0.95 |
1.09 |
||
Distilled water control |
Mean |
-- |
-- |
97.0 |
-- |
15.0 |
-- |
-- |
-- |
55.3 |
-- |
|
MF |
-- |
-- |
1.01 |
-- |
1.00 |
-- |
-- |
-- |
0.99 |
-- |
||
DMSO control |
Mean |
28.3 |
29.7 |
-- |
99.3 |
-- |
14.7 |
13.3 |
12.0 |
-- |
53.7 |
|
MF |
0.97 |
0.95 |
-- |
1.00 |
-- |
1.26 |
0.91 |
0.82 |
-- |
1.02 |
||
DMFcontrol |
Mean |
29.3 |
31.3 |
95.7 |
99.7 |
15.0 |
11.7 |
14.7 |
14.7 |
56.0 |
52.7 |
|
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
||
5000 |
Mean |
20.0 |
31.3 |
56.7 |
97.7 |
12.7 |
14.7 |
11.7 |
16.0 |
47.7 |
57.7 |
|
MF |
0.68 |
1.00 |
0.59 |
0.98 |
0.84 |
1.26 |
0.80 |
1.09 |
0.85 |
1.09 |
||
1581 |
Mean |
18.0 |
29.3 |
57.3 |
92.7 |
16.0 |
16.0 |
11.3 |
14.3 |
48.0 |
55.0 |
|
MF |
0.61 |
0.94 |
0.60 |
0.93 |
1.07 |
1.37 |
0.77 |
0.98 |
0.86 |
1.04 |
||
500 |
Mean |
18.3 |
27.0 |
60.0 |
92.3 |
16.3 |
15.0 |
11.0 |
13.7 |
44.3 |
56.0 |
|
MF |
0.63 |
0.86 |
0.63 |
0.93 |
1.09 |
1.29 |
0.75 |
0.93 |
0.79 |
1.06 |
||
158.1 |
Mean |
23.7 |
27.0 |
72.3 |
92.0 |
16.3 |
15.3 |
12.3 |
14.7 |
48.3 |
55.7 |
|
MF |
0.81 |
0.86 |
0.76 |
0.92 |
1.09 |
1.31 |
0.84 |
1.00 |
0.86 |
1.06 |
||
50 |
Mean |
20.7 |
30.0 |
76.3 |
93.0 |
18.0 |
16.3 |
11.7 |
15.0 |
48.3 |
59.0 |
|
MF |
0.70 |
0.96 |
0.80 |
0.93 |
1.20 |
1.40 |
0.80 |
1.02 |
0.86 |
1.12 |
||
15.81 |
Mean |
18.0 |
27.7 |
74.7 |
95.7 |
16.3 |
18.7 |
13.3 |
13.0 |
51.7 |
58.7 |
|
MF |
0.61 |
0.88 |
0.78 |
0.96 |
1.09 |
1.60 |
0.91 |
0.89 |
0.92 |
1.11 |
||
5 |
Mean |
20.7 |
26.3 |
70.0 |
98.0 |
16.7 |
16.7 |
14.0 |
14.3 |
52.3 |
63.0 |
|
MF |
0.70 |
0.84 |
0.73 |
0.98 |
1.11 |
1.43 |
0.95 |
0.98 |
0.93 |
1.20 |
||
1.581 |
Mean |
21.3 |
24.3 |
88.3 |
87.7 |
18.0 |
16.3 |
11.3 |
14.3 |
56.3 |
59.0 |
|
MF |
0.73 |
0.78 |
0.92 |
0.88 |
1.20 |
1.40 |
0.77 |
0.98 |
1.01 |
1.12 |
||
NPD (4mg) |
Mean |
400.7 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
MF |
14.14 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
||
2AA (2mg) |
Mean |
-- |
2432.0 |
-- |
2352.0 |
-- |
202.7 |
-- |
204.0 |
-- |
-- |
|
MF |
-- |
81.98 |
-- |
23.68 |
-- |
13.82 |
-- |
17.00 |
-- |
-- |
||
2AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
318.7 |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.94 |
||
SAZ (2mg) |
Mean |
-- |
-- |
1202.7 |
-- |
1172.0 |
-- |
-- |
-- |
-- |
-- |
|
MF |
-- |
-- |
13.36 |
-- |
78.13 |
-- |
-- |
-- |
-- |
-- |
||
9AA (50mg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
452.0 |
-- |
-- |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
33.90 |
-- |
-- |
-- |
||
MMS (2mL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1038.7 |
-- |
|
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
18.77 |
-- |
||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro, the following genotoxic endpoints were assessed :
1- Gene mutation potential:
1 -1 One reverse bacterial gene mutation (Ames) test (OECD 471, Kr.1) was available and considered as the key study. In this study, Alkenyl phosphonate was tested in S. typhimurium TA1535, TA1537, TA100 and TA98 and in E. coli WP2 uvr A in the presence and the absence of mammalian metabolic activation (S9) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats,using the preincubation method. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 15.81 and 5μg/plate, in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581μg/plate. Slightly reduced background lawn was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA100 and TA1537 strains without metabolic activation on the plates at 5000 and 1581 µg/plate. In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect. The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. Under the test conditions of this study, Alkenyl phosphonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
1 -2 The mutagenic potential of Alkenyl phosphonate was evaluated by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells. This study was conducted according to the OECD 490 guideline.
The test was performed with a 3 hour treatment period, first in the absence and in the presence of metabolic activation (S9 -mix) with a single culture (first experiment) and then, only in the presence of S9-mix with duplicate cultures (second experiment).
In the first experiment, the test item was tested up to concentrations of 500 µg/mL both in the absence and the presence of S9-mix. A precipitate, which did not prevent any scoring, was noted in the culture medium at the end of the treatment period at dose levels ≥ 62.5 µg/ml. A severe cytotoxicity was induced at the dose level of 500 µg/mL, as shown by a 98% decrease in the Adj. RTG.No noteworthy increase in the mutation frequency was noted at any of the tested dose levels, relative to the corresponding vehicle control and no dose-response relationship was demonstrated by the linear regression.
In the second experiment, the test item was tested up to concentrations of 500 µg/mL in the absence of S9-mix and 250 µg/ml in the presence of S9 mix. A precipitate, which did not prevent any scoring, was noted in the culture medium at the end of the treatment period, at dose levels ≥ 62.5 µg/mL in both experiments. No noteworthy cytotoxicity was observed at any of the tested dose levels, as shown by the absence of any noteworthy decrease in the Adj. RTG, in either experiment.No noteworthy increase in the mutation frequency was noted at any of the tested dose levels in either experiment, relative to the corresponding vehicle control, and no dose-response relationship was demonstrated by the linear regression. These results did not meet the criteria of a positive response.Considering the negative results obtained in both independent experiments performed, the overall results were considered as suitable to allow a reliable interpretation.
Under the experimental conditions of this study, the test item, Alkenyl phosphonate, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.
2 - Chromosomal aberration potential:
In an in vitro Micronucleus Test, performed according to the OECD guideline N° 487 and in compliance with good laboratory practice,L5178Y TK+/- mouse lymphoma. Cells were treated with Alkenyl phosphonate diluted in ethanol at three dose levels and in each treatment condition, in duplicate, together with negative and positive controls. The dose range was selected on the basis of the results of a preliminary toxicity test and was12.3, 37 and 111.1 µg/mL for the 3-hour treatment with and without S9 mix, the latter being the lowest dose-level showing precipitate in the culture medium at the end of the treatment period,15.6, 62.5 and 125 µg/mL for the 24-hour treatment without S9 mix, the latter being the lowest dose‑level showing precipitate in the culture medium at the end of the treatment period and inducing the recommended level of cytotoxicity (i.e.a 54% decrease in the PD).
Following the 3-hour treatments with and without S9 mix or the 24-hour treatment without S9 mix, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the analyzed dose levels in comparison to the corresponding vehicle control. Moreover, none of the analyzed dose-levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle historical range. Thus, these results met the criteria of a negative response.
Under the experimental conditions of the study, the test item Alkenyl phosphonate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
Justification for classification or non-classification
Harmonized classification:
No harmonized classification is available according to the Regulation (EC) No 1272/2008.
Self-classification:
Based on available data (OECD 471, OECD 490 and OECD 487, Kr.1, GLP), Alkenyl phosphonate is considered as not genotoxic. Therefore, no classification is required according to EU criteria.
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