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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
In order to investigate the mutagenic activity of Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate was investigated in two bacterial reverse mutation assays (Ames test; test strains used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvr A) performed with the structural analogues (Octyl phosphonic acid and Phosphoric acid, dodecyl ester, potassium salt; for read across justification please refer to IUCLID Chapter 7.6.1), in an in vitro gene mutation study in mammalian cells (Chinese Hamster V79 cells), one intro chromosome aberration in Chinese Hamster V79 cells and in one in vitro chromosome aberration study in human lymphocytes performed with Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate. An increase in chromosome aberrations in Chinese hamster V79 cells was observed in the absence of the S9 mix. However, of Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate did not induce chromosome aberrations up to 3060 µg/mL (10 mM) in the absence and presence of metabolic activation in an in vitro Chromosome Aberration test performed in human lymphocytes which is considered to be the most relevant cell system for it contains the whole human metabolic enzyme set (e.g. V79 are not p53 competent).
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012-04-11 to 2012-07-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment for experiment I (short-term exposure, with and without metabolic activation):
0.010, 0.025, 0.05, 0.10, 0.20, 0.25, 0.50, 1.0, 2.5, 5 µL/mL

Pre-experiments for experiment II (only without metabolic activation, 20 h long-term exposure assay):
0.005, 0.010, 0.025, 0.050, 0.075, 0.10, 1.0, 2.0, 3.0, 4.0 µL/mL and
0.0005, 0.001, 0.025, 0.050, 0.075, 0.10, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 µL/mL

Experiment I
without metabolic activation:
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.12 and 0.14 µL/mL

and with metabolic activation:
0.200, 0.250, 0.300, 0.350, 0.400, 0.450, 0.475 and 0.50 µL/mL

Experiment II
without metabolic activation:
0.050, 0.075, 0.100, 0.125, 0.140, 0.155, 0.170, 0.185, and 0.20 µL/mL

and with metabolic activation:
0.20, 0.24, 0.28, 0.32, 0.36, 0.40, 0.42 and 0.44 µL/mL
Vehicle / solvent:
Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation

Migrated to IUCLID6: 300 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation

Migrated to IUCLID6: 1.0 and 1.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth
Evaluation criteria:
A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
Statistics:
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I without S9: ≥ 0.10 μg/mL; experiment I with S9: ≥ 0.4 μg/mL; Experiment II without S9: ≥ 0.125 μg/mL; Experiment II with S9:≥ 0.36 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Afilan V5760 is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

In a mammalian cell gene mutation assay (HPRT locus),V79 cells cultured in vitro were exposed to Afilan V5760 dissolved in cell culture medium at concentrations of

0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.12 and 0.14 µL/mL (without metabolic activation, Exp. I)

0.200, 0.250, 0.300, 0.350, 0.400, 0.450, 0.475 and 0.50 µL/mL (with metabolic activation, Exp. I)

0.050, 0.075, 0.100, 0.125, 0.140, 0.155, 0.170, 0.185, and 0.20 µL/mL (without metabolic activation, Exp. II)

0.20, 0.24, 0.28, 0.32, 0.36, 0.40, 0.42 and 0.44 µL/mL (with metabolic activation, Exp. II)

Afilan V5760 was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 8.4% for the highest concentration (0.14 µL/mL) evaluated.The highest biologically relevant concentration evaluated with metabolic activation was 0.5 µL/mL with a relative growth of 13.9%. In experiment II without metabolic activation the relative growth was 13.1% for the highest concentration (0.20 µL/mL) evaluated. The highest concentration evaluated with metabolic activation was 0.44 µL/mL with a relative growth of 15.2%.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 2.70 was found at a concentration of 0.14 µL/mL with a relative growth of 8.4%.At this concentration a slight increase in the number of mutant colonies was observed, but due to the observed strong cytotoxic effect, this finding is considered to be not biologically relevant.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 3.75 was found at the lowest concentration tested (0.200 µL/mL) with a relative growth of 106.5%. Only at this concentration an increased number of mutant colonies (63.11 mutants per 106cells) was observed, exceeding the historical data range.

In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 2.32 was found at a concentration of 0.155 µL/mL with a relative growth of 42.1%.

In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.09 was found at a concentration of 0.28 µL/mL with a relative growth of 86.5%.

The observed elevated mutation rate of 3.75 in experiment I (with metabolic activation) at the lowest concentration tested (0.200 µl/mL) could not be reproduced in experiment II with metabolic activation. At the same concentration a mutation factor of only 0.68 was observed. Due to this finding, the absence of a dose-response relationship as well as due to the overall integrity of the data the observed increased mutation factor in experiment I (with metabolic activation) is considered to be not biologically relevant.

The positive controls did induce the appropriate response. 

There was no evidence of a concentration related positive response of induced mutant colonies over background.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The structural analogues (Octyl phosphonic acid and Phosphoric acid, dodecyl ester, potassium salt; for read across justification please refer to IUCLID Chapter 7.6.1) showed negative results in the study for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The study was performed with the test strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvr A. Test concentrations up to the limit concentration of 5000 µg/plate were tested in the experiments. The test compounds proved to be not mutagenic to the bacterial strains.

Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate yielded negative results in an in vitro gene mutation study in mammalian cells in concentration up to cytotoxic in the presence and absence of metabolic activation.

Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate was assessed for its potential to induce chromosome aberrations in Chinese Hamster cells (V79) and in human lymphocytes in vitro. In V79 cells negative results were obtained in the presence of metabolic activation. An increase in chromosome aberrations in Chinese hamster V79 cells was observed in the absence of the S9 mix. However, of reaction mass of potassium ethyl octylphosphonate and diethyl octyl phosphonate did not induce chromosome aberrations up to 3060 µg/mL (10 mM) in the absence and presence of metabolic activation in an in vitro Chromosome Aberration test performed in human lymphocytes which is considered to be the most relevant cell system for it contains the relevant human metabolic enzyme set (e.g. V79 are not p53 competent).


Justification for selection of genetic toxicity endpoint
This study is selected as key study representing the toxicological endpoint "Genetic toxicity" since it was performed using mammalian cells and examines the most sensitive genotoxic mechanism. The study was performed according to the current OECD Guideline 476 and und GLP.

Justification for classification or non-classification

In conclusion, Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate or its structural analogues are not mutagenic in the bacterial reverse mutation assay, the in vitro gene mutation study in human lymphocytes and the in vitro chromosome aberration in the presence and absence of metabolic activation up to the tested concentrations.

Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate does not have to be not classified for mutagenicity since this substance or its structural analogues did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation in concentrations up to 5000 µg/plate (by read across approach), in the in vitro gene mutation assay (up to cytotoxic concentrations) or in the in vitro chromosome aberration study.