Calcium phosphinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 23 to May 21, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

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)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Calcium phosphinate

EC Number:

232-190-8

EC Name:

Calcium phosphinate

Cas Number:

7789-79-9

Molecular formula:

Ca.2H3O2P

IUPAC Name:

calcium phosphinate

Test material form:

solid: particulate/powder

Method

Target gene:

Salmonella typhimurium
The tester strains TA 1535, TA 1537, TA 98 and TA 100 selected by Ames and co-workers are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.
The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens.
These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98.
The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 (4) and, in addition to having genes resistant to antibiotics, they have a modified post replication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.
Escherichia coli
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions. The rate of induced back mutations from tryptophan auxotrophy (trp ) to tryptophan independence (trp+) is determined.

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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0; 20 ; 100 ; 500; 2,500 and 5,000 µg/plate

Vehicle / solvent:

Water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene: with S9 mix 4-nitro-o-phenylendiamine: without S9 mix

Details on test system and experimental conditions:

Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.6 % agar + 0.6 % NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants : 0.5 mM histidine + 0 .5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
- 0.1 ml test solution or vehicle
- 0.1 ml fresh bacterial culture
- 0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation )
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Evaluation criteria:

Mutagenicity:
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, five doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st experiment.

Titer:
The titer is generally determined only in the experimental parts with S-9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Toxicity:
Toxicity is recorded for all test groups both with and without S-9 mix in all experiments and it is detected by:
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his or trp" background growth)
- reduction in the titer
The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i .e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
A test substance is generally considered no mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

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:

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A slight decrease in the number of revertants was occasionally observed in the standard plate test.
In the preincubation assay a slight decrease in the number of revertants and/or a slight reduction in the titer was occasionally observed.
No test substance precipitation was found.

Applicant's summary and conclusion

Conclusions:

Calcium phosphinate is not a mutagenic agent in a bacterial reverse mutation test.

Executive summary:

The substance calcium phosphinate was tested for ots mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA.

DOSE RANGE: 20 µg - 5000 µg/plate.

TEST CONDITIONS: with and without metabolic activation (Aroclor-induced rat liver S-9 mix).

SOLUBILITY: no precipitation of the test substance was found.

TOXICITY: a weak bacteriotoxic effect was occasionally observed.

MUTAGENICITY: an increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of metabolizing system.

According to the results of the present study, the test substance calcium phosphinate is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay.