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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 August 2016 - 30 September 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:
2014
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
reaction mass of decyl acrylate and octyl acrylate
EC Number:
911-295-9
Molecular formula:
C11H20O2 and C13H24O2
IUPAC Name:
reaction mass of decyl acrylate and octyl acrylate
Test material form:
liquid

Method

Target gene:
Not applicable (not a gene mutation assay).
Species / strain
Species / strain / cell type:
other: mouse lymphoma L5178Y TK+/- cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing 10% (v/v) 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
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 and non-freely soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiment was based on the level of cytotoxicity and/or on the presence of emulsion in the culture medium, according to the criteria specified in the international guidelines.

Experiment without S9 mix
With a treatment volume of 0.5% (v/v) in culture medium (24-hour treatment) or 1% (v/v) in culture medium (3-hour treatment), the dose-levels used for treatment were as follows:
- 5, 10, 20, 40, 60, 80, 100 and 120 µg/mL for the 3-hour treatment,
- 3.13, 6.25, 12.5, 25, 50, 100 and 300 µg/mL for the 24-hour treatment.

Experiment with S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatment were 25, 50, 100, 200, 300, 400 and 500 µg/mL.
Vehicle / solvent:
- Vehicle used: ethanol
- Justification for choice: based on available solubility data, the test item was prepared as a solution in ethanol at the concentration of 500 mg/mL, which was expected to produce an emulsion in the 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
Premiminary cytotoxicity test:
Without S9 mix:
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery

With S9 mix: 3 h treatment + 24 h recovery

Main cytogenetic test
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:
yes

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:
* Experiment without S9 mix
An emulsion was observed in the culture medium at the end of the treatment period at dose-levels >= 80 µg/mL following the 3-hour treatment, and at the dose-level of 300 µg/mL following the 24-hour treatment.

* Experiment with S9 mix
An emulsion was observed in the culture medium at the end of the treatment period at dose-levels >= 100 µg/mL. It is to be noted that this emulsion did not prevent any scoring.

- 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:
Based on available solubility data, the test item was prepared as a solution in ethanol at the concentration of 500 mg/mL, which was expected to produce an emulsion in the culture medium.
Using this stock solution and a treatment volume of 1% (v/v) in culture medium, the highest recommended dose-level of 5000 µg/mL was achievable for the 3-hour treatment with and without S9 mix.
Taking into account the maximum practicable volume of 0.5% (v/v) of ethanol in culture medium for a continuous treatment, the highest achievable dose-level of 2500 µg/mL was reached for the 24-hour treatment without S9 mix.

The dose levels selected for the treatment of the preliminary test were the following:
- 0.1, 1, 10, 100, 500, 1000, 2500 and 5000 µg/mL for the 3-hour treatment with and without S9 mix,
- 0.01, 0.1, 1, 10, 100, 500, 1000 and 2500 µg/mL for the 24-hour treatment without S9 mix.

At the highest dose-levels of 2500 or 5000 µg/mL (depending on the treatment volume used in the culture medium), the pH of the culture media was approximately 7.1, as for the corresponding vehicle controls, whatever the treatment volume applied.
Using a treatment volume of 1% (v/v) in the culture medium, the osmolality at the highest dose-level of 5000 µg/mL was equal to 375 mOsm/kg H2O (482 mOsm/kg for the corresponding vehicle control).
Using a treatment volume of 0.5% (v/v) in the culture medium, the osmolality at the highest dose-level of 2500 µg/mL was equal to 334 mOsm/kg H2O (383 mOsm/kg for the corresponding vehicle control).

Therefore, none of the selected dose-levels was considered to produce extreme culture conditions. Thus, the highest recommended dose-level of 5000 µg/mL could be selected as the highest dose-level for the main experiment following the 3-hour treatments, and the dose-level of 2500 µg/mL could be selected as the highest dose-level for the main experiment following the 24-hour treatment.

An emulsion was observed in the culture medium at the end of the treatment periods at dose-levels >= 500 µg/mL.

Following the 3-hour treatment without S9 mix, a severe cytotoxicity was observed at dose-levels >= 100 µg/mL, as shown by a 100% decrease in the PD.
Following the 24-hour treatment without S9 mix, a slight cytotoxicity was noted at 0.1 µg/mL, then a severe cytotoxicity was noted at dose-levels >= 100 µg/mL, as shown by a 33 to 100% decrease in the PD.
Following the 3-hour treatment with S9 mix, a moderate to severe cytotoxicity was observed at dose-levels >= 500 µg/mL, as shown by a 47 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:
Under the experimental conditions of the study, the test item 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+/-.

This study was conducted in compliance with OECD Guideline No. 487 and the principles of Good Laboratory Practices.

 

Methods

After a preliminary cytotoxicity test, the test item, diluted in ethanol, was tested in a single experiment, with a metabolic activation system (3 h treatment + 24 h recovery) and without a metabolic activation system (  3 h treatment + 24 h recovery, and,  24 h treatment + 0 h recovery), 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.

 

Results

Since the test item was found cytotoxic and non-freely soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiment was based on the level of cytotoxicity and/or on the presence of emulsion in the culture medium, 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.

 

Experiment without S9 mix

With a treatment volume of 0.5% (v/v) in culture medium (24-hour treatment) or 1% (v/v) in culture medium (3-hour treatment), the dose-levels used for treatment were as follows:

.            5, 10, 20, 40, 60, 80, 100 and 120 µg/mL for the 3-hour treatment,

.            3.13, 6.25, 12.5, 25, 50, 100 and 300 µg/mL for the 24-hour treatment.

 

An emulsion was observed in the culture medium at the end of the treatment period at dose-levels superior or equal to 80 µg/mL following the 3-hour treatment, and at the dose-level of 300 µg/mL following the 24-hour treatment.


Cytotoxicity

Following the 3-hour treatment, a moderate to severe cytotoxicity was induced at dose-levels superior or equal to 60 µg/mL, as shown by a 49 to 100% decrease in the PD.

Following the 24-hour treatment, a moderate to severe cytotoxicity was induced at dose-levels superior or equal to 100 µg/mL, as shown by a 53 to 100% decrease in the PD.

 

Micronucleus analysis

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

.            20, 40 and 60 µg/mL for the 3-hour treatment, the latter inducing a 49% decrease in the PD (close to the recommended level of cytotoxicity (i.e. 55 ± 5% cytotoxicity)) and higher dose-levels being too cytotoxic,

.            25, 50 and 100 µg/mL for the 24-hour treatment, the latter inducing the recommended level of cytotoxicity (with 53% decrease in the PD).

 

Increases in the frequency of micronucleated cells were noted at 40 and 60 µg/mL after the 3-hour treatment. However, no dose-response relationship was evidenced, and none of these increases were statistically significant when compared to the corresponding vehicle control. Moreover, the frequencies of micronucleated cells of each replicate culture remained within the vehicle historical range.

Despite that the recommended level of cytotoxicity was not reached, considering the narrow dose-levels spacing used in this experiment, the available results were considered as suitable to allow a reliable interpretation. These results are thus considered to meet the criteria of a negative response.

 

No increase in the frequency of micronucleated cells were noted after the 24-hour treatment. Furthermore, no dose-response relationship was evidenced, and the frequencies of micronucleated cells of each replicate culture remained within the vehicle historical range. These results met the criteria of a negative response.

Experiment with S9 mix

With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatment were 25, 50, 100, 200, 300, 400 and 500 µg/mL.

 

An emulsion was observed in the culture medium at the end of the treatment period, at dose-levels superior or equal to 100 µg/mL. It is to be noted that this emulsion did not prevent any scoring.

Cytotoxicity

A severe cytotoxicity was induced at dose-levels superior or equal to 200 µg/mL, as shown by a 100% decrease in the PD.

Micronucleus analysis

The dose-levels selected for micronucleus analysis were 25, 50 and 100 µg/mL, the latter corresponding to the lowest dose-level which induced an emulsion in the culture medium.

 

An increase in the frequency of micronucleated cells was noted at 25 µg/mL. At this dose-level, the frequencies of micronucleated cells of each replicate culture were slightly above the vehicle historical range. However, since this increase was neither statistically significant nor dose-related, the criteria of a positive response were only partially met and the overall results with S9 mix were considered to meet the criteria of a negative response.

Conclusion

Under the experimental conditions of the study, the test item 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.