Poly[oxy(methyl-1,2-ethanediyl)], .alapha.,α'-(2,2-dimethyl-1,3-propanediyl)bis[ω-[(1-oxo-2-propenyl)oxy

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 April 2018 - 06 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

1

Reference substance name:

Esterification products of acrylic acid with reaction products of 2,2-dimethylpropane-1,3-diol and methyloxirane

EC Number:

701-440-2

Molecular formula:

(C3H6O)x (C3H6O)y C11H16O4

IUPAC Name:

Esterification products of acrylic acid with reaction products of 2,2-dimethylpropane-1,3-diol and methyloxirane

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% 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

Cytokinesis block (if used):

n/a

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 to be severely cytotoxic in the preliminary tests, the highest dose levels selected for the main experiments were based on the level of cytotoxicity, according to the criteria specified in the international regulations.

Experiment without S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the 3- and 24-hour treatments were 0.63, 1.3, 2.5, 3.8, 5, 7.5, 10 and 15 µg/mL.

Experiments with S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the treatments were as follows:
- 6.3, 12.5, 25, 37.5, 50, 75, 100 and 150 µg/mL in the first experiment,
- 0.781, 1.17, 1.56, 2.34, 3.13, 4.69, 6.25, 9.38, 12.5, 18.8, 25, 37.5, 50, 75, 100 and 150 µg/mL in the second experiment.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice:
Using a test item concentration of 500 mg/mL in the vehicle (DMSO) and a treatment volume of 1% (v/v) in culture medium, the highest recommended dose level of 5000 µg/mL was achievable. Thus, the dose levels selected for the treatment of the first preliminary test were 20.58, 61.73, 185.2, 555.6, 1667 and 5000 µg/mL.

Using a test item concentration of 10 mg/mL in DMSO and a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the treatment of the second preliminary test were 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5 and 25 µg/mL in the absence of S9 mix and 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL in the presence of S9 mix.

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:
Preliminary 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 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/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:

For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the Khi2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.

To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

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:

not applicable

True 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
- Precipitation: none

RANGE-FINDING STUDIES:
Using a test item concentration of 500 mg/mL in the vehicle (DMSO) and a treatment volume of 1% (v/v) in culture medium, the highest recommended dose level of 5000 µg/mL was achievable. Thus, the dose levels selected for the treatment of the first preliminary test were 20.58, 61.73, 185.2, 555.6, 1667 and 5000 µg/mL.
At the highest dose level of 5000 µg/mL, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 361 mOsm/kg H2O (447 mOsm/kg H2O for the vehicle control). Therefore, none of the selected dose levels was considered to produce extreme culture conditions.
An emulsion was observed in the culture medium at dose levels >= 555.6 µg/mL, at the end of both treatment periods.
Following the 3- and 24-hour treatments without S9 mix, a severe cytotoxicity was observed at all from the lowest dose levels (i.e. from 20.58 µg/mL), as shown by a 100% decrease in the PD (Table 1).
Following the 3-hour treatment with S9 mix, a moderate to severe cytotoxicity was observed from the lowest dose levels of 20.58 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 Tables enclosed.
- Indication whether binucleate or mononucleate where appropriate: mononucleates cells 'L5178Y
cell line).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see document attached.

RESULTS OF CYTOTOXICITY:
Since the test item was found to be severely cytotoxic in the preliminary tests, the highest dose levels selected for the main experiments were based on the level of cytotoxicity, according to the criteria specified in the international regulations.

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.

First of all, no emulsion was observed in the culture medium at any dose levels, at the end of both treatment periods in either main experiment.


Experiment without S9 mix:
With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the 3- and 24-hour treatments were 0.63, 1.3, 2.5, 3.8, 5, 7.5, 10 and 15 µg/mL.

Cytotoxicity:
Following the 3- and 24-hour treatments, a 40 to 100% decrease in the PD was induced at dose levels = 2.5 µg/mL (Tables 3 and 5).

Micronucleus analysis:
The dose levels selected for the micronucleus analysis were as follows:
- 0.63, 1.3 and 2.5 µg/mL for the 3-hour treatment, the latter inducing a 50% decrease in the PD (i.e. the recommended level of cytotoxicity of 55 ± 5% cytotoxicity),
- 0.63, 1.3 and 2.5 µg/mL for the 24-hour treatment, the latter inducing only a 40% decrease in the PD but higher dose levels being too cytotoxic.

Following the 3- or 24-hour treatments without S9 mix, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the analyzed dose levels in comparison to the corresponding vehicle control (Tables 4 and 6; p>0.05). Moreover, none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle control historical range.
Following the 24-hour treatment, none of the selected dose levels induced the recommended level of cytotoxicity. Nevertheless, considering the narrow dose levels spacing used in this experimental condition, the available results were considered as suitable to allow a reliable interpretation. These results without S9 mix are thus considered to meet the criteria of a negative response.

Experiments with S9 mix:
With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the treatments were as follows:
- 6.3, 12.5, 25, 37.5, 50, 75, 100 and 150 µg/mL in the first experiment,
- 0.781, 1.17, 1.56, 2.34, 3.13, 4.69, 6.25, 9.38, 12.5, 18.8, 25, 37.5, 50, 75, 100 and 150 µg/mL in the second experiment.

Cytotoxicity:
A 68 to 100% decrease in the PD was induced at dose levels = 12.5 µg/mL in the first experiment (Table 7).
A 46 to 100% decrease in the PD was induced at dose levels = 12.5 µg/mL in the second experiment (Table 8).

Micronucleus analysis:
Since not enough analyzable dose levels were obtained in the first experiment with S9 mix due to the high cytotoxicity of the test item, the corresponding micronucleus analysis was not performed.
In the second experiment, the dose levels selected for the micronucleus analysis were 4.69, 9.38 and 25 µg/mL, the latter inducing a 49% decrease in the PD and higher dose levels being too cytotoxic.

Following the 3-hour treatment with S9 mix, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the analyzed dose levels in comparison to the vehicle control (Table 9; p>0.05). Moreover, none of the analyzed dose levels showed frequency of micronucleated cells above the corresponding vehicle control historical range.
None of the selected dose levels induced the recommended level of cytotoxicity, however the values are near to the recommended level of cytotoxicity (49 vs 55 ± 5%). Nevertheless, the values are close to the recommended level of cytotoxicity (up to 49% versus 55 ± 5%) and considering the narrow dose levels spacing used in this experimental condition, the available results were considered as suitable to allow a reliable interpretation. These results with S9 mix are thus considered to meet the criteria of a negative response.

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^+/-.

The study was performed according to the international guidelines and in compliance with the principles of Good Laboratory Practice.

 

Methods

After two preliminary cytotoxicity tests, the test item, diluted in dimethylsulfoxide (DMSO) was tested in two independent experiments with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

 

	First experiment	Second experiment
Without S9 mix	3 h treatment + 24 h recovery	-
	24 h treatment + 0 h recovery	-
With S9 mix	3 h treatment + 24 h recovery	3 h treatment + 24 h recovery

 

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.

After the final cell counting, the cells were washed and fixed. Then, when applicable (i.e. when enough analyzable dose levels were obtained), 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). When the slide analysis was undertaken, micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in a 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 to be severely cytotoxic in the preliminary tests, the highest dose levels selected for the main experiments were based on the level of cytotoxicity, according to the criteria specified in the international regulations.

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.

 

First of all, noemulsionwas observed in the culture medium at any dose levels, at the end of both treatment periods in either main experiment.

Experiment without S9 mix

With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the 3- and 24-hour treatments were 0.63, 1.3, 2.5, 3.8, 5, 7.5, 10 and 15 µg/mL.

 

Cytotoxicity

Following the 3- and 24-hour treatments, a 40 to 100% decrease in the PD was induced at dose levels = 2.5 µg/mL.

 

Micronucleus analysis

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

. 0.63, 1.3 and 2.5 µg/mL for the 3-hour treatment, the latter inducing a 50% decrease in the PD (i.e. the recommended level of cytotoxicity of 55 ± 5% cytotoxicity),

. 0.63, 1.3 and 2.5 µg/mLfor the 24-hour treatment,the latter inducing only a 40% decrease in the PD but higher dose levels being too cytotoxic.

 

Following the 3- or 24-hour treatments without S9 mix, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the analyzed dose levels in comparison to the corresponding vehicle control. Moreover, none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle control historical range.

Following the 24-hour treatment, none of the selected dose levels induced the recommended level of cytotoxicity. Nevertheless, considering the narrow dose levels spacing used in this experimental condition, the available results were considered as suitable to allow a reliable interpretation. These results without S9 mix are thus considered to meet the criteria of a negative response.

 

Experiments with S9 mix

With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the treatments were as follows:

. 6.3, 12.5, 25, 37.5, 50, 75, 100 and 150 µg/mL in the first experiment,

. 0.781, 1.17, 1.56, 2.34, 3.13, 4.69, 6.25, 9.38, 12.5, 18.8, 25, 37.5, 50, 75, 100 and 150 µg/mL in the second experiment.

 

Cytotoxicity

A 68 to 100% decrease in the PD was induced at dose levels = 12.5 µg/mL in the first experiment.

A 46 to 100% decrease in the PD was induced at dose levels = 12.5 µg/mL in the second experiment.

Micronucleus analysis

Since not enough analyzable dose levels were obtained in the first experiment with S9 mix due to the high cytotoxicity of the test item, the corresponding micronucleus analysis was not performed.

In the second experiment, the dose levels selected for the micronucleus analysis were 4.69, 9.38 and 25 µg/mL, the latter inducing a 49% decrease in the PD and higher dose levels being too cytotoxic.

Following the 3-hour treatment with S9 mix, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the analyzed dose levels in comparison to the vehicle control. Moreover, none of the analyzed dose levels showed frequency of micronucleated cells above the corresponding vehicle control historical range.

None of the selected dose levels induced the recommended level of cytotoxicity. Nevertheless, the values are close to the recommended level of cytotoxicity (up to 49% versus 55 ± 5%) and considering the narrow dose levels spacing used in this experimental condition, the available results were considered as suitable to allow a reliable interpretation. These results with S9 mix are thus 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.