Aluminium tridiethylphosphinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept - Nov 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

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:

first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Details on test material:

Batch No.: DEH2029007
Purity: 96.9%
Expiry Date: 13.6.2016

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without metabolic activation: 19.8; 59.3 (p); 177.8 (p); 533.3 (p); 1600 (p) µg/mL
with metabolic activation: 19.8; 59.3 (p); 177.8 (p); 533.3 (p); 1600 (p) µg/mL
Experiment II:
without metabolic activation: 6.6; 19.8; 59.3; 177.8 (p); 533.3 (p) µg/mL
with metabolic activation: 6.6; 19.8; 59.3 (p); 177.8 (p); 533.3 (p) µg/mL

(p) = precipitation visible to the unaided eye

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
On the day of the experiment (immediately before treatment), the test item was suspended in DMSO (purity 99.9 %). The final concentration of DMSO in the culture medium was 0.5 % (v/v).

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.

A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the corresponding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.33 in the solvent control versus pH 7.31 at 3200 µg/mL) measured in the pre-experiment
- Effects of osmolality: No relevant increase (358 in the solvent control versus 376 at 3200 µg/mL) measured in the pre-experiment
- Precipitation: Precipitation of the test item visible to the naked eye at the end of treatment was noted in the first experiment at 59.3 µg/mL and above with and without metabolic activation. In the second experiment precipitation as described above occurred at 59.3 µg/mL and above with and at 177.8 µg/mL and above without metabolic activation.

- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:

The highest concentration used in the pre-test was 3200 µg/mL limited by the solubility of the test item in DMSO and aqueous medium. Test item concentrations between 28.0 µg/mL and 3200 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant toxic effect occurred up to 1600 µg/mL with and without metabolic activation following 4 and 24 hours treatment.

The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred after 4 hours treatment with and without metabolic activation at 25 µg/mL and above.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. The individual concentrations were generally spaced by a factor of 2.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effect defined as a reduction of the relative cloning efficiency I and/or relative cell density to values below 50% was observed at 1600 µg/mL without metabolic activation following 4 hours of treatment.

Applicant's summary and conclusion

Conclusions:

Not mutagenic in HPRT.

Executive summary:

The registration substance was investigated for its mutagenicity in mammalian cells according to the OECD Guideline 476 (HPRT). V79 cells were treated with the registration substance up to the concentrations causing visible precipitations. In the first experiment the incubation time of 4 hours with and without metabolic activation and in the seconed experiment 4 hours with metabolic activation and 24 hours without metabolic activation. No increase of mutation rate was found in two independent experiments. The registration substance in not mutagenic in HPRT test.