2-hydroxy-3-phenoxypropyl acrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2016 - 13 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 September 2014

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

A technical error was noted in the first main experiment: the sampling volume of the test item was not taken into account in the calculation of the volume of vehicle to add for the preparation of stock formulation. However, if the density measured in the preliminary test is extrapolated, the volume of test item sampled for the first main experiment was 20 µL. Thus, the corrected concentration of the stock formulation was 15.79 mg/mL (instead of 16.01 mg/mL), corresponding to a difference of -1.37%. Since the difference is < ±10% (criterion based on the formulation galenic, i.e. a solution), this minor deviation was considered not to prejudice the overall GLP status of the study and the scientific reliability of the study conclusions. The historical data used for the validation of long treatment period without S9 mix (24 hours treatment + 0 hour recovery) were generated with non-audited data from non-GLP studies. These data were performed in compliance with CiToxLAB France’s standard operating procedures. Since CiToxLAB France is a Test facility certified by the French National Authorities for Good Laboratory Practice, and the procedures undertaken are the same, this deviation is considered not to prejudice the overall GLP status of the study and the scientific reliability of the study conclusions. Moreover, the corresponding mean frequency of micronucleated cells in the vehicle control was 3‰ in the first experiment or 2‰ in the second experiment, therefore = 5‰ as specified in the acceptance criteria.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-3-phenoxypropyl acrylate

EC Number:

241-045-8

EC Name:

2-hydroxy-3-phenoxypropyl acrylate

Cas Number:

16969-10-1

Molecular formula:

C12H14O4

IUPAC Name:

2-hydroxy-3-phenoxypropyl acrylate

Test material form:

liquid

Method

Target gene:

Not applicable (not a gene mutation assay).

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10%(v/v) heat inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

L5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei. The average cell cycle time is approximately 10-12 hours.
L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France).

The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C and each batch of frozen cells was checked for the absence of mycoplasma.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected for treatments were as follows:
- 1.25, 2.5, 5, 10, 20, 30, 40 and 80 µg/mL, for the 3-hour treatment in the first experiment,
- 1.25, 2.5, 5, 10, 20, 40, 60 and 80 µg/mL, for the 24-hour treatment in the first experiment,
- 1.25, 2.5, 5, 6.25, 7.5, 8.75, 10 and 15 µg/mL, both for the 3- and 24-hour treatments in the second experiment.

Experiments with S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected for treatments were as follows:
- 1.25, 2.5, 5, 10, 20, 40, 60 and 80 µg/mL in the first experiment,
- 7.5, 15, 30, 40, 45, 50, 55 and 60 µg/mL in the second experiment.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: the test item was found to be soluble in DMSO and the highest recommended dose-level could be reached using a solution of the test item at a concentration of 400 mg/mL and a treatment volume of 0.5% (v/v) in culture medium.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION

For the preliminary cytotoxicity test and both cytogenetic experiments:
Without S9 mix: 3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix: 3 h treatment + 24 h recovery

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells / dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling

Evaluation criteria:

The biological relevance of the results was always taken into account when evaluating results.

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.

Statistics:

no

Results and discussion

Test results

Key result

Species / strain:

other: mouse lymphoma L5178Y TK+/- cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Emulsion: >= 1000 µg/mL at the end of the short treatment period; no emulsion at the end of the long treatment period
- Definition of acceptable cells for analysis: Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. 1995, according to the following criteria:
* micronuclei should be clearly surrounded by a nuclear membrane,
* the micronucleus area should be less than one-third of the area of the main nucleus,
* non-refractility of the micronuclei,
* micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
* micronuclei should be located within the cytoplasma of the cell,
* only mononucleated cells with a number of micronuclei <= 5 should be scored to exclude apoptosis and nuclear fragmentation.

- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
Using a solution of the test item in the vehicle at a concentration of 400 mg/mL and a treatment volume of 0.5 % (v/v) in culture medium, the highest recommended dose-level of 2000 µg/mL was achievable. Thus, the dose levels selected for the treatment of the preliminary test were 4, 40, 200, 400, 1000 and 2000 µg/mL.

At the highest dose-level of 2000 µg/mL, the pH of the culture medium was approximately 7.7 (as for the vehicle control) and the osmolality was 350 mOsm/kg H2O (394 mOsm/kg for the vehicle control). Therefore, none of the selected dose-levels was considered to produce extreme culture conditions and the highest recommended dose-level of 2000 µg/mL could be selected as the highest dose-level for the main experiments.

An emulsion was observed in the culture medium at the dose-levels = 1000 µg/mL, at the end of the short treatment period. No emulsion was observed in the culture medium at any of the tested dose-levels at the end of the long treatment period.

Following the 3-hour treatments with and without S9 mix, a slight to severe cytotoxicity was observed from the lowest tested dose-level (i.e. 4 µg/mL), as shown by a 28 to 100% decrease in the PD.
Following the 24-hour treatment without S9 mix, a moderate to severe cytotoxicity was observed at dose levels = 40 µg/mL as shown by a 48 to 100% decrease in the PD.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see attached document.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached document.
- Negative (solvent/vehicle) historical control data: see attached document.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: population doubling.

Applicant's summary and conclusion

Conclusions:

2-Hydroxy-3-phenoxypropyl acrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
 

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK^+/-.

 

After a preliminary toxicity test, the test item, diluted in dimethylsulfoxide (DMSO), was tested in two independent experiments with (3h treatment) and without (3hr and 24h treatment) a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

Then, after the final cell counting, the cells were washed and fixed. Then, cells from at least three dose-levels of the test item-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines.

 

The mean Population Doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Experiments without S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected for treatments were as follows:

- 1.25, 2.5, 5, 10, 20, 30, 40 and 80 µg/mL, for the 3-hour treatment in the first experiment,

- 1.25, 2.5, 5, 10, 20, 40, 60 and 80 µg/mL, for the 24-hour treatment in the first experiment,

- 1.25, 2.5, 5, 6.25, 7.5, 8.75, 10 and 15 µg/mL, both for the 3- and 24-hour treatments in the second experiment.

 

No emulsion was observed in the culture medium at any dose-levels, at the end of the treatment periods.

 

Following the 3-hour treatments, a cytotoxicity was induced at dose-levels = 10 µg/mL in the first experiment, and at dose-levels = 5 µg/mL in the second experiment, as shown by a 30 to 100% decrease in the PD.

Following the 24-hour treatments, a cytotoxicity was induced at dose-levels = 10 µg/mL in the first experiment, and at dose-levels = 8.75 µg/mL in the second experiment, as shown by a 29 to 100% decrease in the PD.

 

The dose-levels selected for micronucleus analysis were as follows:

- 1.25, 2.5 and 5 µg/mL for the 3-hour treatment in the first experiment, the latter inducing no decrease in the PD but higher dose-levels being too cytotoxic,

- 2.5, 5 and 6.25 µg/mL for the 3-hour treatment in the second experiment, the latter inducing a 46% decrease in the PD and higher dose-levels being too cytotoxic,

- 1.25, 2.5 and 5 µg/mL for the 24-hour treatment in the first experiment, the latter inducing only a 7% decrease in the PD but higher dose-levels being too cytotoxic,

- 7.5, 8.75 and 10 µg/mL for the 24-hour treatment in the second experiment, the latter inducing a 58% decrease in the PD.

 

In the first experiment, no noteworthy increase in the frequency of micronucleated cells was noted at any of the analyzed dose-levels, either following the 3- or 24-hour treatments. Furthermore, no dose-response relationship in the frequency of micronucleated cells was demonstrated by the linear regression, whatever the treatment period.

Since none of the dose-levels analyzed in the first experiment reached the recommended level of cytotoxicity (i.e. 55±5% cytotoxicity), a second experiment was undertaken using a narrower range of dose-levels.

 

In the second experiment, no noteworthy increase in the frequency of micronucleated cells was noted at any of the analyzed dose-levels,either following the 3- or 24-hour treatments. Furthermore, no dose-response relationship in the frequency of micronucleated cells was evidenced, whatever the treatment period.

Despite the use of a narrower range of dose-levels, none of the dose-levels selected for the 3-hour treatment induced the recommended level of cytotoxicity. Considering the narrow dose-levels spacing used in this experiment and the negative results obtained in both independent experiments performed, the overall available results were considered as suitable to allow a reliable interpretation.

 

The overall results without S9 mix were considered to meet the criteria of a negative response.

 

Experiments with S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected for treatments were as follows:

- 1.25, 2.5, 5, 10, 20, 40, 60 and 80 µg/mL in the first experiment,

- 7.5, 15, 30, 40, 45, 50, 55 and 60 µg/mL in the second experiment.

 

No emulsion was observed in the culture medium at any dose-levels, at the end of the treatment periods.

 

In the first experiment, a slight to severe cytotoxicity was induced at dose-levels = 40 µg/mL, as shown by a 31 to 100% decrease in the PD.

In the second experiment, a moderate to severe cytotoxicity was induced at dose-levels = 30 µg/mL, as shown by a 45 to 92% decrease in the PD.

The dose-levels selected for micronucleus analysis were as follows:

- 10, 20 and 40 µg/mL in the first experiment, the latter inducing only a 31% decrease in the PD but higher dose-levels being too cytotoxic,

- 15, 30 and 40 µg/mL in the second experiment, the latter inducing a 52% decrease in the PD.

 

In the first experiment,a statistically significant increase in the frequency of micronucleated cells was noted at the dose-level of 40 µg/mL (p < 0.05). However, in the absence of any dose-response relationship, the criteria of a positive response were only partially met. Thus, these results remained equivocal.

Since none of the dose-levels of the first experiment induced the recommended level of cytotoxicity (i.e. 55 ± 5% cytotoxicity), and since the results remained equivocal, a second experiment was undertaken using a narrower range of dose-levels.

 

In the second experiment, no noteworthy increase in the frequency of micronucleated cells was noted at any of the analyzed dose-levels and no dose-response relationship was evidenced. Since the slight increase observed in the first experiment was not reproduced in the second experiment, using a narrower range of dose-levels and reaching the recommended level of cytotoxicity, it was considered to be non biologically relevant. Thus the overall results with S9 mix met the criteria of a negative response.

 

Under the experimental conditions of the study, the test item 2-Hydroxy-3-phenoxypropyl acrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.