Slags, phosphorus-manufg.

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:

01 February 2010 - 11 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to OECD guideline 471 and under GLP conditions.

Justification for data waiving:

other:

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:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

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

Remarks:

Statement of Compliance

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Slags, phosphorus-manufg.

EC Number:

273-732-3

EC Name:

Slags, phosphorus-manufg.

Cas Number:

69012-32-4

Molecular formula:

not applicable due to the multi constituent mineral structure of the substance

IUPAC Name:

aluminium(3+) heptacalcium magnesium(2+) λ²-iron(2+) disodium tris([(trioxidosilyl)oxy]silanetris(olate)) oxosilanebis(olate) hydroxysilanoylolate difluoride

Details on test material:

- Name of test material (as cited in study report): Phosphorous slag
- Physical state: powder
- Analytical purity: confidential
- Expiration date of the lot/batch: confidential
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

His-gene: Amino acid histidine and Trp-gene: Amino acid tryptophan

Species / strain

Species / strain 1

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, gal, chl, bio, uvrB; TA98 and TA100: plasmid pKM101

Species / strain 2

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:

Dose range finding test: 3; 10; 33; 100; 333; 1000; 3330; and 5000 µg/plate
Mutation assay: 100; 333; 1000; 3330; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: Strain TA1535: sodium-azide; Strain TA 1537: 9-aminoacridine; Strain TA98: 2-nitrofluorene; Strain TA100: methylmethanesulfonate; WP2uvrA; 4-nitroquinoline N-oxide. With metabolic activation: All strains: 2-aminoanthracene.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Selection time (if incubation with a selection agent): 48 ± 4 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: a reduction of the bacterial background lawn, an increase in the size of the microcolonies or a reduction of the revertant colonies

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in test strain TA100 is not greater than two times the concurrent control, and the total number of revertants in test strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in test strain TA100 is greater than two times the concurrent control, or the total number of revertants in test strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the test strains, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

Mean and Standard Deviation

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of Phosphorous slag on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 ug/plate.

RANGE-FINDING/SCREENING STUDIES: Phosphorous slag was tested in the test strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 ug/plate in the absence and presence of S9-mix. Precipitation of Phosphorous slag on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 ug/plate. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. No increase in the number of revertants was observed upon treatment with Phosphorous slag under all conditions tested. The dose range finding test is reported as a part of the first experiment of the mutation test.

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: The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

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 with metabolic activation
negative without metabolic activation

Phosphorous slag did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the 4 test strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in test strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that Phosphorous slag is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Phosphorous slag was tested in the Salmonella typhimurium reverse mutation assay with 4 strains of Salmonella typhimurium (TAI535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a strain of Escherichia coli (WP2uvrA). The test was performed in 2 independent experiments in the presence and absence of S9-mix. The study procedures described in this report were based on OECD guideline 471.

In the dose range finding test, Phosphorous slag was tested up to concentrations of 5000 ug/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first experiment of the mutation assay. Phosphorous slag precipitated on the plates at the top dose of 5000 ug/plate. Based on the results of the dose range finding test, Phosphorous slag was tested in the first mutation assay at concentrations of 100, 333, 1000, 3330, and 5000 µg/plate in the absence and presence of S9-mix in test strains TA1535, TA1537 and TA98. An independent repeat of the assay was performed in all 5 test strains.

The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Furthermore, the negative and strain-specific positive control values were within the laboratory historical control data ranges in this study. Phosphorous slag did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the 4 test strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in test strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that Phosphorous slag is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.