Phosphorous ester

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:

2008-04-01 to 2008-04-17

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

esterification products of castor oil and polyphosphoric acids

EC Number:

614-406-6

Cas Number:

68308-61-2

Molecular formula:

C57H107P3O18

IUPAC Name:

esterification products of castor oil and polyphosphoric acids

Constituent 2

Reference substance name:

Polyphosphoric acids, esters with caster oil

IUPAC Name:

Polyphosphoric acids, esters with caster oil

Details on test material:

- Name of test material (as cited in study report): GARDO TP10451
- Physical state: liquid
- Analytical purity: 100%
- Lot/batch No.: 2939R
- Expiration date of the lot/batch: 2010-02-18

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- => his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– => trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 mix.

Test concentrations with justification for top dose:

The test concentratoins were: 5000; 1581.1; 500; 158.1; 50 and 15.81 µg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation; Strain S. typhimurium: TA 100; TA 1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation; Strain S. typhimurium: TA 1537

Migrated to IUCLID6: 9AA

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation; Strain E. coli WP2uvrA

Migrated to IUCLID6: MMS

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Name 4 -Nitro-1,2-phenylenediamine, 4-NOPD (or NPD)

Remarks:

Without metabolic activation; Strain S. typhimurium: TA 98

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

With metabolic activation; Strains S. typhimurium: TA 100; TA 98; TA1535; TA 1537 and E. coli WP2uvrA

Details on test system and experimental conditions:

Preparation of bacteria:
The frozen cultures were thawed at room temperature and a measured amount was used for inoculating the over-night cultures in the assay. 200 μL inoculum was used for each 50 mL of broth. The bacterial strains were grown up in nutrient broth. The cultures were incubated for 10-14 h at 37°C up to the late exponential or early stationary growth phase (approx. 109 cells/mL) in a Gyrotory Water Bath Shaker. The nutrient broth contained 25 g Nutrient Broth No 2. per litre.
Agar Plates:
Ready-to-use minimal agar plates obtained from Merck (Merckoplate) were used in the experiments.
Overlay Agar:
Histidine- Biotin overlay agar (for Salmonella typhimurium strains) contains per litre: Agar Bacteriological 3.6 g; NaCl 4.5 g; D-Biotin (F.W. 244.3) 12.2 mg; L-Histidine·HCl H2O (F.W. 209.63) 10.5 mg. The agar solution was sterilised at 121 °C in an autoclave, and the Histidine – Biotin solution (0.5 mM) was sterilised by filtration through a 0.22 m membrane filter.
Tryptophan overlay agar (for Escherichia coli strain) contains per litre: Agar Bacteriological 3.79 g; NaCl 4.74 g; Nutrient Broth (see "Preparation of Bacteria") 50.0 mL; L-Tryptophan (F.W. 204.23) 5.0 mg. The agar solution and the nutrient broth was sterilised at 121 °C in an autoclave. The Tryptophan solution (2 mg/mL) was sterilised by filtration through a 0.22 m membrane filter.
Experimental Performance:
As an initial mutation test the standard plate incorporation procedure was performed. Bacteria (cultured in Nutrient Broth No. 2.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Counting of Colonies:
Revertant colonies were counted manually.

Evaluation criteria:

The colony numbers on the control, positive control and the test plates will be determined, the mean values and appropriate standard deviations will be calculated.

Statistics:

NA

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

Remarks:

Salmonella typhimurium TA 100 minor increases numbers were detected at the concentration of 2500 μg/plate furthermore in the concentration range of 316.23-10 g/plate with metabolic activation (+S9 Mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of GARDO TP10451 in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). In general, the pre-incubation method is more sensitive than the plate incorporation assay. Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate (positive and negative controls were run concurrently). Following concentrations of GARDO TP10451 were tested in experiment I (the concentrationswere chosen based on results obtained in the pre-experiment for toxicity):
5000; 1581.1; 500; 158.1; 50 and 15.81 g/plate.
The examined concentration levels were 5000; 1581.1; 500; 158.1; 50; 15.81 and 5 μg/plate in the experiment II (the additional concentration level, 5 μg/plate was investigated based on the results of the experiment I.
In experiment I and experiment II most of the observed revertant colony number increases were of minor intensity, not dose related, without any biological significance (i.e. below the respective biological threshold value) and in the historical control range.
The highest revertant rate was observed in the experiment I in case of Salmonella typhimurium TA 1535 at 15.81 μg/plate (MF=1.55), without metabolic activation (–S9 Mix). There were no additional dose-dependent relationship and these values were well below the biological threshold value.
In case of Salmonella typhimurium TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA at different concentration levels sporadically lower revertant colony numbers compared to the revertant colony numbers of the solvent control plates were detected in the performed experiments.
Because of the minor intensity of these variations (increases and decreases in the historical control range), the observed changes should be considered as reflecting the biological variability of the test.
After 48 hours incubation microdrops were observed at the concentration of 5000 g/plate ( S9 Mix), using plate incorporation method, and at the concentrations of 5000 and 1581.1 g/plate ( S9 Mix), using the pre-incubation method.
Positive and negative controls were run concurrently. The revertant colony numbers of solvent control plates without S9 Mix were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

no remarks

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

GARDO TP10451 is classified as non mutagenic

Executive summary:

The reported data of this mutagenicity assay shows, that under the experimental conditions described, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, GARDO TP10451 is considered non-mutagenic in this bacterial reverse mutation assay.