Tetrakis(hydroxymethyl)phosphonium chloride, oligomeric reaction products with urea

Genetic toxicity: in vitro

Administrative data

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 April 2014 - 10 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Remarks:

2013-01-10

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Details on test material:

- Name of test material: Tetrakis(hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea
- Physical state: colorless to pale yellow liquid
- Lot/batch No.: PCCP28G1
- Active ingredient content: 66.7% w/w
- Water content: 31.1% w/w
- Correction factor applied: 1.5
- Expiry date: 31 July 2014
- Storage condition: at room temperature.

Method

Target gene:

Histidine operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Experiments without S9 mix
The selected treatment-levels were:
- 3.91, 7.81, 15.6, 31.3, 62.5 and 125 µg/plate for the TA 1535 and TA 100 strains in the first and second experiments as well as for the TA 1537 strain in the second experiment,
- 7.81, 15.6, 31.3, 62.5, 125 and 250 µg/plate for the TA 102 strain in the first and second experiments as well as for the TA 98 strain in the first experiment,
- 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 1537 strain in the first experiment,
- 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/plate for the TA 98 strain in the second experiment, as well as for the TA 1535, TA 98, TA 100 and TA 102 strains in the third experiment.

Experiments with S9 mix
The selected treatment-levels were:
- 3.91, 7.81, 15.6, 31.3, 62.5 and 125 µg/plate for the TA 1535 in the first and second experiments as well as for the TA 1537 strain in the second experiment,
- 7.81, 15.6, 31.3, 62.5, 125 and 250 µg/plate for the TA 98, TA 100 and TA 102 strains in the first experiment,
-7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 100 strain in the second experiment,
- 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 1537 strain in the first experiment,
- 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/plate for the TA 98 and TA 102 strains in the second experiment, as well as for the TA 98 strain in the third experiment using the direct incorporation method.
- 7.81, 15.6, 31.3, 62.5, 93.75, 125 and 250 µg/plate for the TA 98 strain in the third experiment using the pre-incubation method.

Vehicle / solvent:

- Vehicle used: water for injections
- Justification for choice: Since the test item was found to be cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn.

Evaluation criteria:

In all case, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item is considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose-level,
- and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose-levels,
- nor any evidence of a dose-response relationship is noted.

Results and discussion

Test results

Test results 1

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

 

Methods

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

Since cytotoxicity was not shown with the selected dose-levels in each of the tested conditions of the main experiments, a third experiment was undertaken for some strains and conditions, using higher ranges of dose-levels. In order to check the reliability of slight increases in the number of revertants noted in the TA 98 strain in the second experiment with S9 mix, a third experiment was also undertaken with this strain under the same experimental conditions but using a narrower range of dose-levels.

 

All experiments were performed according to the direct plate incorporation method, except for the second experiment with S9 mix as well as a part of the third experiment with S9 mix, which were performed according to the pre-incubation method (60 minutes, 37°C).

 

The test item was dissolved in water for injections.

 

Results

 

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

 

Since the test item was found to be cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines.

 

Experiments without S9 mix

The selected treatment-levels were ranged from 3.91 to 1000 µg/plate.

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

 

A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels >= 62.5 µg/plate in the TA 1537 strain, >= 125 µg/plate in the TA 1535 and TA 100 strains and >= 250 µg/plate in the TA 98 and TA 102 strains.

 

Slight increases in the number of revertants were noted in the TA 98 strain in the first experiment. These increases seemed dose-related and exceeded the positive threshold of 2-fold the vehicle control value. Nevertheless the mean number of revertants remained within the vehicle control historical range (up to 39 revertants/plate versus [12-39] for the vehicle control historical data). Furthermore, no similar results were noted either in the second or in the third experiment. The slight increases noted in the first experiment were therefore considered to be not biologically relevant.

 

The test item did not induce any other noteworthy increase in the number of revertants, in any other strains or test conditions.

 

Experiments with S9 mix

The selected treatment-levels were ranged from 3.91 to 1000 µg/plate.

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

 

A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels >= 125 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and >= 250 µg/plate in the TA 102 strain.

Slight increases in the number of revertants were noted in the TA 98 strain in the second experiment (pre‑incubation method). These increases seemed dose-related, they exceeded the positive threshold of 2-fold the vehicle control value and the mean number of revertants exceeded the vehicle control historical range (up to 67 revertants/plate versus [16-44] for the vehicle control historical data). Nevertheless, no similar effect was observed either in the third experiment using the pre-incubation method, despite the use of a narrower range of dose-levels, or in the first and third experiments using the direct plate incorporation method. Since the slight increases noted in the second experiment were not reproducible they were considered to be not biologically relevant.

 

The test item did not induce any other noteworthy increase in the number of revertants, in any other strains or test conditions.

 

Conclusion

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium either in the presence or absence of a rat liver metabolizing system.