Aluminium tridiethylphosphinate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registration substance is not mutagenic in the bacterial reverse mutation assay, not clastogenic in chromosome aberration test and not mutagenic in reverse mutation test in mammalian cells.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

HPRT

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Performed using four bacterial strains.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

liver extract, S9-mix

Test concentrations with justification for top dose:

4 to 5000 µg/ plate

Vehicle / solvent:

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

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation; for strain TA100 and TA 1535

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation; for strain TA 1537

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation; for strain TA 98

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation; for all strains

Evaluation criteria:

the solvent control data are within the range of spontaneous mutant frequency; the positive controls induce increases in the mutation frequency

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Not mutagenic in the bacterial reverse mutation assay.

Executive summary:

The registration substance was investigated for its mutagenicity according to the OECD Guideline 471 (Bacterial reverse mutation assay). No mutagenicity was found.

Comments on the study:

The study was performed using four bacterial strains, thus missing the investigation of mutation in E-coli. But it can be reasonably assumed that no increase of mutation will occur, because the registration substance is of limited water solubility (1.6 g/l) and not likely soluble in incubation media of Ames test. Due to the low bioavailability for the bacterial cells, no biological interaction is possible, thus no mutation is likely.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

trypsin

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix, rat liver

Test concentrations with justification for top dose:

7.8, 25.0, 78.0, 250.0 and 780.0 µg/ml

Vehicle / solvent:

0.2M disodium hydrogenephosphate and 0.2 M sodium dihydrogenphosphate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

exposure period without activation: 3 h, 20 h and with metabolic activation: 3h ; fixation time: 20 h (with and without activation);

Evaluation criteria:

-number of induced structural chromosome aberrations
-concentration-related increase of the number of chromosome aberrations;

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

strong precipitation at >78 µg/ml;

Conclusions:

Not clastogenic in in-vitro chromosome aberration test

Executive summary:

The registration substance was investigated for its clastogenicity according to the OECD Guideline 473 (Chromosome aberration test). V79 cells were treated up to the concentration with visible precipitation.

In the first experiment the incubation time was 3h with and without metabolic activation. In the second experiment the incubation time was 3h with and 20h without metbolic activation. No increase of aberrant cells were found in both experiments. The registration substance is not clastogenic in in-vitro chromosome aberration test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The registration substance did not induce an increase of micronuclei in bone marrow in mouse.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Route of administration:

oral: unspecified

Vehicle:

PEG 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: formulated in PEG 400

Duration of treatment / exposure:

24 h; 48 h

Frequency of treatment:

orally once

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: orally
- Doses / concentrations: 40 mg/kg b.w.

Tissues and cell types examined:

polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The highest dose (2000 mg/kg) was estimated by a pre-experiment to be suitable; The animals treated with 2000 mg/kg b.w. expressed toxic reactions (ruffled fur);

Evaluation criteria:

the negative and positive controls are in the range of historical control data

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The registration substance was investigated for its clastogenicity in mouse according to the OECD Guideline 474. The registration substance did not induce increase of micronuclei in bone marrow cells in mouse.

Executive summary:

The registration substance was investigated for its clastogenicity in mouse according to the OECD Guideline 474. The registration substance was suspended in PEG and given to mouse per gavage at dose of 0, 500, 1000 and 2000 mg/kg. 24 or 48h after the treatment the bone marrow was collected and the polychromatic erythrocytes were investigated for the micronuclei. The treatment of up to the dose of 2000 mg/kg did not alter the number of polychromatic erythrocytes and the number of cells with microcleis. The registration substance did not induce increase of micronuclei in bone marrow cells in mouse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Comments on the bacterial reverse mutation assay:

The study was performed using four bacterial strains, thus missing the investigation of mutation in E-coli. But it can be reasonably assumed that no increase of mutation will occur, because the registration substance is of limited water solubility and not likely soluble in incubation media of Ames test. Due to the low bioavailability for the bacterial cells, no biological interaction is possible, thus no mutation is likely.

Justification for classification or non-classification

Negative results were obtained in three in-vitro assays and one in-vivo assay, indicating absence of genotoxicity of the registration substance. No classification is justified.