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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (Ames test according to OECD471): negative with and without metabolic activation Cytogenicity in mammalian cells (In vitro Mammalian Cell Micronucleus Test according to OECD487): negative with and without metabolic activation Gene mutation in mammalian cells (Mouse Lymphoma Assay according to OECD476): negative with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-Sep-2012 to 06-Nov-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
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: 10, 33, 100, 333, 820 µg/mL
Without S9-mix, 24 hours treatment: 10, 33, 100, 333, 820 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 0.3, 1, 3, 10, 33, 100, 333 and 820 µg/mL
With S9-mix, 3 hours treatment: 100, 300, 400, 500, 600, 700, 750 and 800 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 33, 100, 400, 500, 600, 700, 750 and 820 µg/mL
With S9-mix, 3 hours treatment: 33, 100, 300, 500, 600, 700, 750 and 820 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Exposure medium (RPMI 1640 Hepes buffered medium (Dutch modification) )
- Justification for choice of solvent/vehicle:

Test compound was soluble in exposure medium and exposure 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)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: cyclophosphamide 10 µg/mL
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

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: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10^6 survivors, and for CP not below 700 per 10^6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than 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.
Statistics:
The global evaluation factor (GEF) has been defined by the IWTGP 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.

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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH:
Solvent control: 7.4
820 µg/ml: 6.2
333 µg/ml: 6.9
- Effects of osmolality:
Solvent control: 0.284 mOsm/kg
820 µg/ml: 0.295 mOsm/kg
333 µg/ml: 0.291 mOsm/kg
- Precipitation: No precipitation was observed up to and including the top dose of 820 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed in the absence of S9, 3 hours treatment; Toxicity was observed at the dose level of 820 µg/mL in the presence of S9, 3 hours treatment and at dose level of 820 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:
Appropriate toxicity was observed up to and including the highest tested dose level in both experiments in the absence and presence of S9-mix.
Remarks on result:
other: strain/cell type: Test system L5178Y/TK+/-3.7.2C
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Phosphonic acid (Phosphorous acid) is considered not mutagenic in the absence and presence of S9-mix in the mouse lymphoma L5178Y test system
Executive summary:

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. Except the response of the solvent control cultures in the first experiment in the presence of S9-mix. Although these responses were below the lower limit of the range, clear negative results were obtained in this part of the study, the validity of the test was considered to be not affected.

 

Mutation frequencies in cultures treated with positive control chemicals were increased 8.6 and

10-fold for in the absence of S9-mix, and 14- and 17-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 after a 3 hours treatment period, Phosphonic acid (Phosphorous acid) did not induce a significant increase in the mutation frequency. However Phosphonic acid (Phosphorous acid) induced a 3.4-fold increase in the mutation frequency at the prolonged treatment period of 24 hours. The mutation frequency (213 x 10-6mutant colonies) was above the global evaluation factor plus mutation frequency of the negative controls (GEF + MF(controls): 190 x 10-6) and outside the historical control data range. This increase was only observed at the highest tested dose level of 820 µg/ml with severe toxicity, a relative total growth of 7%.

 

In the presence of S9-mix, Phosphonic acid (Phosphorous acid) did not induce a significant increase in the mutation frequency in the first experiment. However Phosphonic acid (Phosphorous acid) induced a 5.1-fold increase in the mutation frequency in the repeat experiment. The mutation frequency (323 x 10-6mutant colonies) was above the global evaluation factor plus mutation frequency of the negative controls (GEF + MF(controls): 190 x 10-6) and outside the historical control data range. This increase was only observed at the highest tested dose level of 820 µg/ml with severe toxicity, a relative total growth of 4%.

  

Since the RTG is below 10% both in the absence and presence of S9-mix, these concentrations are too toxic according to the guidelines and therefore the mutagenic responses are not biologically relevant. Furthermore, the next dose level of 750 µg/ml with a RTG of 17 and 32% in the absence and presence of S9-mix, respectively, did not even show a two-fold increase in the mutation frequency.

The dose- response was not seen as the dose levels of 700 and 750 µg/ml showed no increase in the mutation frequency more than the MF(controls) + the GEF and were within the limit of the historical control data.Therefore, Phosphonic acid (Phosphorous acid) is considered not mutagenic in the absence and presence of S9-mix in the mouse lymphoma L5178Y test system.

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:
April 22, 2010 - May 11, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test was conducted according to OECD Test Guideline No. 471, 1997, under GLP Standards, and QA.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
His-gene: Amino acid histidine and Trp-gene: Amino acid tryptophan
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: All strains: rfa, uvrB; TA98 and TA100: plasmid pKM101
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
Male Wistar rat liver S9 induced by Phenobarbital/B-Naphthoflavone
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: Strain TA 100 and TA 1535: sodium-azide; Strain TA 1537 and TA 98: 4-nitro-o-phenylene-diamine; WP2 uvrA: methyl methane sulfonate; With metabolic activation: All strains: 2-aminoanthracene.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

- Preincubation period: 60 minutes
- Selection time (if incubation with a selection agent): at least 48 hours

SELECTION AGENT (mutation assays): agar containing Histidine or Tryptophan

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: 10*9

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
Mean and Standard Deviation
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The experimental conditions in the pre-experiment were the same as described for experiment I (plate incorporation test). The pre-experiment is reported as experiment I, since the following criteria were met: evaluable plates (>0 colonies) at 5 concentrations or more in all strains used.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Conclusions:
During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Phosphonic acid (EC 237-066-7; CAS RN 13598-36-2) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay, and does not need to be classified for genetic toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

This study was performed according to OECD Test Guideline No. 471 to investigate the potential of Phosphonic acid (EC 237-066-7; CAS RN 13598-36-2) to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate. Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate. 

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five test strains was observed following treatment with Phosphonic acid at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct in-crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Phosphonic acid (EC 237-066-7; CAS RN 13598-36-2) is considered to be non-mutagenic in this Salmonella typhimurium and 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
Study period:
05-Nov-2012 to 07-Feb-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
other: 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
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
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, 3hr exposure; 27 hr fixation: 33, 100, 333, 500 and 820 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 33, 100, 333, 500 and 820 µg/mL
First cytogenetic test:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 100, 333 and 820 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 100, 700 and 820 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle:
Test compound was stable in water and soluble in culture medium. Culture medium has been accepted and approved by authorities and international guidelines

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 Migrated to IUCLID6: MMC-C 0.25 µg/mL for a 3 hours exposure period and 0.15 µg/mL for a 24 hours exposure period
Positive control substance:
other: colchicine: 0.1 µg/mL
Remarks:
without S9
Positive control substance:
cyclophosphamide
Remarks:
with S9 Migrated to IUCLID6: 15 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)
Evaluation criteria:
A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.
Statistics:
The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics:
Key result
Species / strain:
lymphocytes: human peripheral blood
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:
Solvent control: 7.7
820 µg/ml: 6.1
333 µg/ml: 6.8
- Effects of osmolality:
Solvent control: 287 mOsm/kg
820 µg/ml: 291 mOsm/kg

- Precipitation: No precipitation was observed up to and including the top dose of 820 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was only observed after the prolonged treatment period.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- T oxicity was only observed after the prolonged treatment period
Conclusions:
Phosphonic acid (Phosphorous acid) is not clastogenic or aneugenic in human lymphocytes
Executive summary:

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures in the presence of S9-mix and in the absence of S9-mix at the 24 hours exposure time was within the historical control data range per 2000 mono- or binucleated cells. In the absence of S9-mix at the 3 hours exposure time the number of binucleated cells with micronuclei of the solvent control were outside the historical control data range (8 and 9 per 1000 binucleated cells). However, the results of the test substance treated cultures were clearly negative. In addition, the positive control chemicals mitomycin C showed a statistically significant increase in the number of binucleated cells with micronuclei and colchicine showed a statistically significant increase in the number of mono- and binucleated cells with micronuclei. 

 

The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

Phosphonic acid (Phosphorous acid) did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The mutagenic potential of phosphonic acid were investigated in three in vitro studies.

The Ames test was performed in accordance with OECD Guidance 471 to investigate the potential of Phosphonic acid to induce gene mutations. In the plate incorporation test (experiment I) and the pre-incubation test (experiment II) Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA were used. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. In both experiments, the test item was tested up to and including 5000μg/plate. 

It was concluded that the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Phosphonic acid (EC 237 -066-7; CAS RN 13598-36-2) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

The clastogenicity of phosphonic acid was investigated in an in vitro micronucleus assay with Phosphonic acid in cultured peripheral human lymphocytes (with independent repeat), which was performed in accordance with OECD Guidance 487.

In the first cytogenetic assay, Phosphonic acid was tested up to 820 µg/ml (= 0.01 M) for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. In the second cytogenetic assay, Phosphonic acid was also tested up to 820 µg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9 -mix. Since the positive control substances acted as expected, the test conditions were considered adequate and that the metabolic activation system (S9 -mix) functioned properly.

Phosphonic acid did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments. It is concluded that this test is valid and that Phosphonic acid is not clastogenic or aneugenic in this vitro micronucleus assay in human lymphocytes.

The potential of phosphonic acid to induce gene mutations in mammalian cells was investigated in an in vitro Mouse Lymphoma Assay (MLA), which was performed according to OECD Guidance 476. The MLA test was considered valid, the test conditions were appropriate and the metabolic activation system (S9-mix) worked properly.

In the absence of S9-mix after a 3 hours treatment period, Phosphonic acid did not induce a significant increase in the mutation frequency. However, Phosphonic acid induced a 3.4-fold increase in the mutation frequency at the prolonged treatment period of 24 hours. This increase was only observed at the highest tested dose level of 820 µg/ml with severe toxicity, a relative total growth (RTG) of 7%.

In the presence of S9-mix, Phosphonic acid did not induce a significant increase in the mutation frequency in the first experiment. However, Phosphonic acid induced a 5.1-fold increase in the mutation frequency in the repeat experiment. This increase was only observed at the highest tested dose level of 820 µg/ml with severe toxicity, a RTG of 4%.

Since the RTG is below 10% both in the absence and presence of S9-mix, these concentrations are too toxic for a reliable measurement according to the guidelines. Therefore, these mutagenic responses are not biologically relevant. Furthermore, the next dose level of 750 µg/ml with a RTG of 17 and 32% in the absence and presence of S9-mix, respectively, did not even show a two-fold increase in the mutation frequency. A dose- response was not seen as the dose levels of 700 and 750 µg/ml showed no significant increase in the mutation frequency. Therefore, Phosphonic acid is considered not mutagenic in the absence and presence of S9-mix in the mouse lymphoma L5178Y test system.

Justification for selection of genetic toxicity endpoint
No mutagenic effects were observed in three in vitro tests.

Justification for classification or non-classification

Based on the available information, Phosphonic acid does not need to be classified as mutagenic in accordance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2002/EC.