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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All the three in vitro tests performed on Propoxylated neopentylglycol diacrylate (Ames test, HPRT test, In vitro micronucleus test) showed an absence of genotoxicity effects.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

Not applicable (not a gene mutation assay).

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.

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.

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.

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. 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 August 2012 to 24 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Mouse lymphoma L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5±1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S 9), prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Positive controls
4-nitroquinoline 1 oxide (NQO), Stock concentration; 0.015 and 0.020 mg/mL and final concentrations; 0.15 and 0.20 mg/mL without metabolic activation
Benzo[a]pyrene (B[a]P), Stock concentration; 0.200 and 0.300 mg/mL and final concentrations; 2.00 and 3.00 mg/mL with metabolic activation

Range finding - concentrations ranging from 5 to 100 µg/mL

Main study :
In Experiment 1 twelve concentrations, ranging from 2.5 to 50 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 25 to 300 µg/mL, were tested in the presence of S-9.
In Experiment 2 eleven concentrations, ranging from 2.5 to 25.0 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 25 to 200 µg/mL, were tested in the presence of S-9.

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Untreated negative controls:

yes

Remarks:

dimethyl sulphoxide (DMSO)

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5±1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p < 0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p < 0.05).
3. The effects described above were reproducible.

Statistics:

Not reported

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 46.88 to 1500 µg/mL (limited by solubility in culture medium). The highest concentration to provide >10% relative survival (RS) was 93.75 µg/mL in the presence of S-9, which gave 38% RS. In the absence of S-9 all concentrations analysed gave <10% RS, with the lowest concentration tested (46.88 µg/mL) giving 7% RS.

In Experiment 1 (main study), twelve concentrations, ranging from 2.5 to 50 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 25 to 300 µg/mL, were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 12.5 µg/mL in the absence of S-9 and 150 µg/mL in the presence of S-9, which gave 26% and 9% RS, respectively. In the absence of S-9, excessive heterogeneity for mutation between replicate cultures was observed at 15 µg/mL (giving 10% RS), therefore this concentration was excluded from analysis. In the presence of S-9, steep concentration-related toxicity was observed between 125 and 150 µg/mL (30% and 9% RS, respectively), therefore both concentrations were analysed. In both cases, the highest concentrations analysed were sufficiently close to the target toxicity range of 10-20% RS to be considered acceptable.
In Experiment 2 (main study) eleven concentrations, ranging from 2.5 to 25.0 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 25 to 200 µg/mL, were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed were 17.5 µg/mL in the absence of S-9 and 135 µg/mL in the presence of S-9, which gave 14% and 15% RS, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with Propoxylated neopentylglycol diacrylate (CAS Number 84170-74-1) at any concentration tested in the absence and presence of S-9 and there were no significant linear trends.

Conclusions:

It is concluded that Propoxylated neopentylglycol diacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

Propoxylated neopentylglycol diacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 46.88 to 1500 µg/mL (limited by solubility in culture medium). The highest concentration to provide >10% relative survival (RS) was 93.75 µg/mL in the presence of S-9, which gave 38% RS. In the absence of S-9 all concentrations analysed gave <10% RS, with the lowest concentration tested (46.88 µg/mL) giving 7% RS.

In Experiment 1 twelve concentrations, ranging from 2.5 to 50 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 25 to 300 µg/mL, were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 12.5 µg/mL in the absence of S-9 and 150 µg/mL in the presence of S-9, which gave 26% and 9% RS, respectively. In the absence of S-9, excessive heterogeneity for mutation between replicate cultures was observed at 15 µg/mL (giving 10% RS), therefore this concentration was excluded from analysis. In the presence of S-9, steep concentration-related toxicity was observed between 125 and 150 µg/mL (30% and 9% RS, respectively), therefore both concentrations were analysed. In both cases, the highest concentrations analysed were sufficiently close to the target toxicity range of 10-20% RS to be considered acceptable.

In Experiment 2 eleven concentrations, ranging from 2.5 to 25.0 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 25 to 200 µg/mL, were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed were 17.5 µg/mL in the absence of S-9 and 135 µg/mL in the presence of S-9, which gave 14% and 15% RS, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with Propoxylated neopentylglycol diacrylate (CAS Number 84170-74-1) at any concentration tested in the absence and presence of S-9 and there were no significant linear trends.

It is concluded that Propoxylated neopentylglycol diacrylate (CAS Number 84170-74-1) did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in the absence and presence of a rat liver metabolic activation system (S-9). 

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June 2012 - 12 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

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:

312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments with and without S9 mix.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: test item was soluble in the vehicle at 100 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar
Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.

Statistics:

no

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in strain TA 1537

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate in both mutagenicity experiments with and without S9 mix.
A decrease in the number of revertants was observed in the TA 1537 strain when scoring the revertants at dose-levels = 312.5 µg/plate in the first experiment with S9 mix, and at 5000 µg/plate in the second experiment with S9 mix. No noteworthy toxicity was noted in the other tester strains, either with or without S9 mix.
The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains.

Conclusions:

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the 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 reverse mutation in Salmonella typhimurium.

 The study was performed according to the international guidelines (OECD No. 471 and Council Regulation No. 440/2008 of 30 May 2008, Part B13/14) and in compliance with the principles of Good Laboratory Practice.

 

Methods

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item (dissolved in dimethylsulfoxide) was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

 

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

Results

The test item was dissolved in dimethylsulfoxide (DMSO).

The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered to be valid.

Since the test item was found soluble and non-cytotoxic in the preliminary test, the highest dose-level selected for the mutagenicity experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.

The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments with and without S9 mix.

 

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate in both mutagenicity experiments with and without S9 mix.

 

A decrease in the number of revertants was observed in the TA 1537 strain when scoring the revertants at dose-levels = 312.5 µg/plate in the first experiment with S9 mix, and at 5000 µg/plate in the second experiment with S9 mix.

No noteworthy toxicity was noted in the other tester strains, either with or without S9 mix.

 

The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains.

Conclusion

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence of a rat liver metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation test / Ames test (Sarlang 2012):

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium (OECD 471).

In the key study, performed according to the international guidelines and in compliance with the principles of Good Laboratory Practice, the test item (dissolved in dimethylsulfoxide) was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used.

The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered to be valid.

The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments with and without S9 mix.A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate in both mutagenicity experiments with and without S9 mix. A decrease in the number of revertants was observed in the TA 1537 strain when scoring the revertants at dose-levels = 312.5 µg/plate in the first experiment with S9 mix, and at 5000 µg/plate in the second experiment with S9 mix. No noteworthy toxicity was noted in the other tester strains, either with or without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains. The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence of a rat liver metabolizing system.

Mammalian cell gene mutation assay / HPRT test (Lloyd 2013):

Propoxylated neopentylglycol diacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol (OECD 476). The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for all experiments.

In Experiment 1 twelve concentrations, ranging from 2.5 to 50 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 25 to 300 µg/mL, were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 12.5 µg/mL in the absence of S-9 and 150 µg/mL in the presence of S-9, which gave 26% and 9% RS, respectively. In the absence of S-9, excessive heterogeneity for mutation between replicate cultures was observed at 15 µg/mL (giving 10% RS), therefore this concentration was excluded from analysis. In the presence of S-9, steep concentration-related toxicity was observed between 125 and 150 µg/mL (30% and 9% RS, respectively), therefore both concentrations were analysed. In both cases, the highest concentrations analysed were sufficiently close to the target toxicity range of 10-20% RS to be considered acceptable.

In Experiment 2 eleven concentrations, ranging from 2.5 to 25.0 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 25 to 200 µg/mL, were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed were 17.5 µg/mL in the absence of S-9 and 135 µg/mL in the presence of S-9, which gave 14% and 15% RS, respectively.

In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with Propoxylated neopentylglycol diacrylate (CAS Number 84170-74-1) at any concentration tested in the absence and presence of S-9 and there were no significant linear trends.

It is concluded that Propoxylated neopentylglycol diacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in the absence and presence of a rat liver metabolic activation system (S-9). 

In vitro micronucleus assay (Chevallier 2018) :

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+/- (OECD 487).

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: 3h traitement and 24h treatment without S9 (one experiment), 3h treatment with S9 (two experiments).

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. 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 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.

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:

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: 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:

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, thelatter inducinga 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%versus55 ± 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.

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. 

Justification for classification or non-classification

Based on the negative results in all in vitro mutagenicity tests required by REACH regulation, no classification for Propoxylated neopentylglycol diacrylate is required for genotoxicity according to the Regulation EC n°1272/2008.