9-Octadecen-1-ol, (Z)-, phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reaction product of oleylalcohol with polyphosphoric acid is considered non-mutagenic in this bacterial reverse mutation assay. In addition reaction product of oleylalcohol with polyphosphoric acid did not induce structural chromosome aberrations in Chinese Hamster lung cells and therefore is considered as not clastogenic in this system. Reaction product of oleylalcohol with polyphosphoric acid was not mutagenic in this in vitro test in Chinese hamster ovary cells .

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-01-08 to 2013-03-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: according to GLP, OECD and EC guidelines

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 21st July, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

of 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

, August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes them suitable for gene toxicity assays with
low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the
high proliferation rate (doubling time 12-14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC. The cell stocks were kept in a freezer at -80 °C.
Checking of mycoplasma infections was carried out before freezing. Trypsin-EDTA (0.25 % Trypsin, 1 mM EDTA x 4 Na) solution was used for cell
detachment to subculture. The laboratory cultures were maintained in 75 cm³ plastic flasks at 37 °C in a humidified atmosphere containing 5 % CO2.The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2 mM) and 1 % of Antibiotic-
antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated foetal bovine serum (final concentration 10 %). During the 3 and 20 hours treatments with test item, solvent (negative control) and positive controls, the serum
content was reduced to 5 %.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

The following concentrations were selected ranging from little to maximum (< 50% survival) toxicity and evaluated in the main studies (Experiment A and B). All concentrations were run in duplicate (incl. negative and positive controls) and at least 200 well-spread metaphases were assessed:

Experiment A with 3/20 h treatment/sampling time
without S9 mix: 20, 30, 40, 45 and 50# μg/mL
with S9 mix: 80, 90, 100, 110 and 120# μg/mL

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 10, 20, 30, 35 and 40# μg/mL

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 10, 20, 30, 35 and 40# μg/mL

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 80, 90, 100, 110 and 120# μg/mL

# These concentrations were tested but not evaluated because the second highest concentrations have already fulfilled the criteria of cyctotoxicity
(< 50% viability).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: Good solubility in DMSO. The solvent was adjusted to the optimal survival of the V79 cells and the activity of the metabolic
activation system (S9 mix).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2 mM) and 1 % of Antibiotic-
antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated foetal bovine serum (final concentration 10 %).

EXPERIMENTAL PRE-TEST ON TOXICITY
During the cytotoxicity assay 1-3 day old cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in DME medium. Cells were seeded into 92 x 17 mm dishes (for tissue cultures in TC sterile quality) at 5 x 105 cells each and were incubated for 24 hours in 10 mL of DME medium containing 10 % foetal bovine serum. After 24 hours the cells were treated using increasing concentrations of test item in the absence or presence of S9 mix (50 μL/mL) and were incubated at 37 °C for 3 hours. After treatment the cultures were washed with DME medium and covered with DME containing 10 % foetal bovine serum. Cell counts were performed at 20 hours (without and with S9 mix) and 28 hours with S9 mix after
treatment start. Additional groups of cells were treated for 20 hours without metabolic activation and cell counts were performed at 20 and 28 hoursafter treatment start. The cell numbers of the treatment groups were given as % cells in relation to the negative control. The results obtained were
used for dose selection of the test item used in the Chromosome Aberration Assays. In addition, pH and osmolality were considered for dose
selection.

CHROMOSOME ABERRATION ASSAY
The Chromosome Aberration Assays were conducted in two independent experiments, each in the presence and in the absence of S9 mix.
Experimental Design Cytogenetic Experiment (Experiment A): 3-hour treatment, harvest 20 hours from the beginning of treatment.
Duplicate cultures were used at each concentration and the solvent control cultures as well as the positive controls for treatment without and with S9 mix. 5 x 105 cells were set up at each group. The culture medium of exponentially growing cell cultures was replaced with medium containing the
test item. The exposure period was 3 hours. The exposure period was followed by washing the cells with DME medium and then growth medium
(containing 10 % foetal bovine serum) was added. Sampling was made at 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment). For concurrent measures of cytotoxicity for all treated and negative control cultures, 5 x 105 cells were set up.

Experimental Design Cytogenetic Experiment (Experiment B)
20-hour treatment, harvest 20 hours from the beginning of treatment.
20-hour (without S9 mix) and 3-hour (with S9 mix) treatment, harvest 28 hours from the beginning of treatment.
In Experiment B, as in Experiment A, included concurrent non-activated and S9-activated positive and negative controls.
For each group 5 x 105 cells/dish were seeded. Sampling was made at 1.5 cell cycles (20 hours, without S9 mix only) and at approximately 2 normal cell cycles (28 hours, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.

DETERMINATION OF TOXICITY:
Toxicity was determined by cell counting and results noted as cell survival in the treatment group (in %) in relation to the negative solvent control.

Evaluation criteria:

INTERPRETATION OF RESULTS
The criteria for determining a positive result are:
– a concentration-related increase or a reproducible increase in the number of cells with aberrations.
– biological relevance of the results should be considered first, however, for the interpretation of the data both biological and statistical significance should be considered together.
– an increase in the number of polyploid cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical
chromosome aberrations.
– an increase in the number of cells with endoreduplicated chromosomes may indicate that the test item has the potential to inhibit cell cycle
progression.
A test item for which the results do not meet the above criteria is considered as non mutagenic in this system.

Statistics:

no information on statistical analysis available

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolaty: No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.
- Precipitation: There was no precipitation of the test item at any dose level tested.
- Other confounding effects: There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either
experiment in the presence or absence of metabolic activation

RANGE-FINDING/SCREENING STUDIES:
The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Chromosome
Aberration Assays (Experiment A and B), both in the absence and in the presence of a metabolic activation system (rodent S9 mix). Toxicity was
determined by cell counting and results noted as cell survival in the treatment group (in %) in relation to the negative solvent control.
These results were used to select concentrations of Reaction product of oleylalcohol with polyphosphoric acid for the Chromosome Aberration
Assays. The following concentrations were selected ranging from little to maximum (< 50% survival) toxicity and evaluated in the main studies
(Experiment A and B). All concentrations were run in duplicate (incl. negative and positive controls) and at least 200 well-spread metaphases were assessed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The observed chromosome aberration rates of the negative and positive controls were within the ranges of historical control data.

Remarks on result:

other: strain/cell type: Chinese hamster lung fibroblasts (V79)

Remarks:

Migrated from field 'Test system'.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations. In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates of the negative and positive controls were within the ranges of historical control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The validity of the test was shown using Ethyl methanesulfonate (0.4 or 1.0 μL/mL) in the absence of metabolic activation and Cyclophosphamide (5.0 μg/mL) in the presence of metabolic activation as concurrent positive controls.

Conclusions:

Interpretation of results (migrated information):
negative

Reaction product of oleylalcohol with polyphosphoric acid tested up to cytotoxic concentrations, both with and without mammalian metabolic
activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Therefore, Reaction product of oleylalcohol with polyphosphoric acid is considered as not clastogenic in this system.

Executive summary:

The test item, Reaction product of oleylalcohol with polyphosphoric acid was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in Dimethyl sulfoxide (DMSO) and the test concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 200 well-spread metaphase cells were analysed.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates of the negative and positive controls were within the ranges of historical control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The validity of the test was shown using Ethyl methanesulfonate (0.4 or 1.0 μL/mL) in the absence of metabolic activation and Cyclophosphamide (5.0 μg/mL) in the presence of metabolic activation as concurrent positive controls.

Reaction product of oleylalcohol with polyphosphoric acid tested up to cytotoxic concentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Therefore, Reaction product of oleylalcohol with polyphosphoric acid is considered as not clastogenic in this system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Key studies:

Ames test

In compliance with the OECD Guideline No. 417 and EU Method B.13/14, five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of Reaction product of oleylalcohol with polyphosphoric acid in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (S9 Mix). Additionally a Complementary Plate Incorporation Test was performed with Escherichia coli WP2 uvrA, without metabolic activation. The concentrations, including the controls in each assay, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. All of the validity criteria, regarding the investigated strains, negative and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Reaction product of oleylalcohol with polyphosphoric acid at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

Sporadic increases in revertant colony numbers (compared to the vehicle control values) within the actual historical control data ranges were observed in the experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

Strong inhibitory effect of the test item was observed in the examined Salmonella typhimurium strains. The inhibition appeared unequivocally down to and including the concentration of 5 μg/plate (Salmonella typhimurium TA100 and TA1537, Confirmatory Mutation Test) in absence; and at 1000 μg/plate (all Salmonella typhimurium strains in both experiments) in presence of exogenous metabolic activation.

Chromosome aberration test (in vitro):

The test item, Reaction product of oleylalcohol with polyphosphoric acid was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in Dimethyl sulfoxide (DMSO) and the test concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 200 well-spread metaphase cells were analysed.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates of the negative and positive controls were within the ranges of historical control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The validity of the test was shown using Ethyl methanesulfonate (0.4 or 1.0 μL/mL) in the absence of metabolic activation and Cyclophosphamide (5.0 μg/mL) in the presence of metabolic activation as concurrent positive controls.

Reaction product of oleylalcohol with polyphosphoric acid tested up to cytotoxic concentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Therefore, Reaction product of oleylalcohol with polyphosphoric acid is considered as not clastogenic in this system.

HPRT test:

The test item, Reaction product of oleylalcohol with polyphosphoric acid was tested in a Mammalian Cell Gene Mutation Test in CHO-K1 cells. The test item was dissolved in Dimethyl sulfoxide (DMSO) and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix).

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no biologically significant differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency.

As in Experiment 1, no statistical differences between treatment and solvent control groups and no dose-response relationships were noted in Experiment 2. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

Reaction product of oleylalcohol with polyphosphoric acid tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells. Thus, Reaction product of oleylalcohol with polyphosphoric acid was not mutagenic under the conditions of this study.

Justification for selection of genetic toxicity endpoint
GLP and guideline compliant study with test item. However there are two more key studies (Key.001.:AMES and Key.003:HPRT) which justify the
conclusion.

Justification for classification or non-classification

Based on the results obtained in the in vitro studies reaction product of oleylalcohol with polyphosphoric acid is considered to be non-genotoxic/non-mutagenic or non-clastogenic and thus is not classified according to Directive 67/548/EEC and Regulation (EC) No 1272/2008.