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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro tests were performed on Ethoxylated Bisphenol A dimethacrylate. The Ames test, the in vitro micronucleus assay and the HPRT test showed negative results in presence and in absence of metabolic activation. Therefore, Ethoxylated Bisphenol A dimethacrylate is considered to be not mutagenic.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 March 2018 - 07 May 2018
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:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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, TA 100 and TA 102
Details on mammalian cell type (if applicable):
n/a
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 poorly soluble in the final treatment medium in the preliminary test and toxic only at dose levels close to the highest recommended one, the selection of the highest dose level to be used in the main experiments was based on the level of emulsion or was 5000 µg/plate, according to the criteria specified in the international guidelines.
The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were six analysable dose levels for each strain and test condition. The study was therefore considered to be valid.

Experiments without S9 mix
Selected dose levels
-6.9, 20.6, 61.7, 185.2, 555.6 and 1666.7 µg/plate for the five strains in both mutagenicity experiments, except for the TA 1537 strain in the 2nd experiment,
-6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1537 strain in the 2nd experiment.
A moderate to strong emulsion and/or precipitate was observed in the Petri plates when scoring the revertants at dose levels = 555.6 µg/plate in both experiments.
No noteworthy toxicity was noted at any dose levels, in any strains, in either experiment.

Experiments with S9 mix
Selected dose levels
-20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the 5 strains in both mutagenicity experiments, except for the TA 1537 strain in the 2nd experiment,
-6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1537 strain in the 2nd experiment.
A moderate to strong emulsion or precipitate was observed in the Petri plates when scoring the revertants at dose levels = 555.6 µg/plate in both experiments.
No noteworthy toxicity was noted at any dose levels, in any strains, in either experiment.

The test item did not induce any noteworthy increase in the number of revertants, in any of the 5 strains, in either experiment. These results met thus the criteria of a negative response.
Vehicle / solvent:
According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control plates was dimethylsulfoxide (DMSO).
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 (without S9 mix) 2-Anthramine, Benzo(a)pyrene (with S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

DURATION
- Exposure duration: 48 to 72 hours.

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

NUMBER OF REPLICATIONS: three plates/dose level
Evaluation criteria:
In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item is considered to have shown mutagenic activity in this study if:
- 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 mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose level,
- and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose levels,
- nor any evidence of a dose-response relationship is noted.
Statistics:
no
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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:
TEST-SPECIFIC CONFOUNDING FACTORS
Emulsion and/or precipitate

RANGE-FINDING STUDY:
A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose levels = 555.6 µg/plate with and without S9 mix.
A moderate toxicity (thinning of the bacterial lawn) was noted at dose levels = 1666.7 µg/plate in the three strains used in the presence of S9 mix. In the absence of S9 mix, no noteworthy toxicity was noted at any dose levels.

RESULTS OF CYTOTOXICITY and GENOTOXICITY:
Since the test item was found poorly soluble in the final treatment medium in the preliminary test and toxic only at dose levels close to the highest recommended one (i.e. 5000 µg/plate), the selection of the highest dose level to be used in the main experiments was based on the level of emulsion or was 5000 µg/plate, according to the criteria specified in the international guidelines.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were six analysable dose levels for each strain and test condition. The study was therefore considered to be valid.

Experiments without S9 mix
The selected dose levels were:
- 6.9, 20.6, 61.7, 185.2, 555.6 and 1666.7 µg/plate for the five strains in both mutagenicity experiments, except for the TA 1537 strain in the second experiment,
- 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1537 strain in the second experiment.

A moderate to strong emulsion and/or precipitate was observed in the Petri plates when scoring the revertants at dose levels ¿ 555.6 µg/plate in both experiments.
No noteworthy toxicity was noted at any dose levels, in any strains, in either experiment.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in either experiment. These results met thus the criteria of a negative response.

Experiments with S9 mix
The selected dose levels were:
- 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the five strains in both mutagenicity experiments, except for the TA 1537 strain in the second experiment,
- 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1537 strain in the second experiment.

A moderate to strong emulsion or precipitate was observed in the Petri plates when scoring the revertants at dose levels = 555.6 µg/plate in both experiments.
No noteworthy toxicity was noted at any dose levels, in any strains, in either experiment.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in either experiment. These results met thus the criteria of a negative response.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see attached document.
Conclusions:
Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, 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 reverse mutations in Salmonella typhimurium.

The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14) 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, diluted in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both 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.

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

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least six 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.

Thetreatment of the TA 1537 strain in the second experiment with and without S9 mix was performed at the test site.

Results

Since the test item was found poorly soluble in the final treatment medium in the preliminary test and toxic only at dose levels close to the highest recommended one (i.e. 5000 µg/plate), the selection of the highest dose level to be used in the main experiments was based on the level of emulsion or was 5000 µg/plate, according to the criteria specified in the international guidelines.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were six analysable dose levels for each strain and test condition. The study was therefore considered to be valid.

 

The selected dose levels were:

. 6.9, 20.6, 61.7, 185.2, 555.6 and 1666.7 µg/plate for the five strains in both mutagenicity experiments without S9 mix, except for the TA 1537 strain in the second experiment,

. 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the five strains in both mutagenicity experiments with S9 mix, except for the TA 1537 strain in the second experiment,

. 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate for the TA 1537 strain in the second experiment with and without S9 mix.

 

A moderate to strong emulsion and/or precipitate was observed in the Petri plates when scoring the revertants at dose levels =555.6 µg/plate in both experiments with and without S9 mix.

No noteworthy toxicity was noted at any dose levels, in any strains or test conditions.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in either experiment with or without S9 mix. These results met thus the criteria of a negative response.


Conclusion

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

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 March 2018 - 17 May 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:
26 September 2014 updtaed 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 poorly soluble in the final treatment medium and cytotoxic in the preliminary test, the highest dose levels selected for the main experiments were based on the presence of emulsion and/or on the level of cytotoxicity, according to the criteria specified in the international regulations.

Experiments without S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the treatments were as follows:
- 0.23, 0.69, 2.1, 6.3, 12.5, 25, 50 and 100 µg/mL in the first experiment (3- and 24-hour treatments),
- 1.46, 4.39, 13.2, 19.8, 29.6, 44.4, 66.7 and 100 µg/mL in the second experiment (24-hour treatment).

Experiments with S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the treatment were 0.55, 1.6, 4.9, 14.8, 44.4, 133.3, 250 and 400 µg/mL.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: based on available solubility data, the test item was prepared as a solution at a concentration of 125 mg/mL (higher concentrations leading to important precipitate and emulsion in the final treatment medium). Therefore, using this stock solution and a maximal treatment volume of 1% (v/v) in culture medium, the dose levels selected for the treatment of the preliminary cytotoxicity test were 5.1, 15.4, 46.3, 138.9, 416.7 and 1250 µg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Clastogen, Cyclophosphamide (+S9 mix) Clastogen, Mitomycin C, Aneugen, Colchicine (-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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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:
Based on available solubility data, the test item was prepared as a solution at a concentration of 125 mg/mL (higher concentrations leading to important precipitate and emulsion in the final treatment medium). Therefore, using this stock solution and a maximal treatment volume of 1% (v/v) in culture medium, the dose levels selected for the treatment of the preliminary cytotoxicity test were 5.1, 15.4, 46.3, 138.9, 416.7 and 1250 µg/mL.

At the highest dose level of 1250 µg/mL, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 452 mOsm/kg H2O (457 mOsm/kg H2O for the vehicle control). Therefore, none of the tested dose levels was considered to produce extreme culture conditions.

An emulsion was observed in the culture medium at dose levels = 138.9 µg/mL at the end of the 3-hour treatment period (observation undertaken in the absence of S9 mix) and at dose levels = 416.7 µg/mL at the end of the 24-hour treatment period.

Following the 3-hour treatment (with and without S9 mix), a slight to severe cytotoxicity was observed from the lowest tested dose level of 5.1 µg/mL, as shown by a 28 to 100% decrease in the Population Doubling (PD).
Following the 24-hour treatment without S9 mix, a noteworthy cytotoxicity was observed at dose levels = 15.4 µg/mL, as shown by a 53 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:
See tables 1 to 9.
Since the test item was found poorly soluble in the final treatment medium and cytotoxic in the preliminary test, the highest dose levels selected for the main experiments were based on the presence of emulsion and/or 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.
 
Experiments without S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose levels selected for the treatments were as follows:
. 0.23, 0.69, 2.1, 6.3, 12.5, 25, 50 and 100 µg/mL in the first experiment (3- and 24-hour treatments),
. 1.46, 4.39, 13.2, 19.8, 29.6, 44.4, 66.7 and 100 µg/mL in the second experiment (24-hour treatment).
An emulsion was observed in the culture medium at dose levels >= 25 µg/mL at the end of the 3-hour treatment period, whereas no emulsion was observed at the end of the 24-hour treatment period, at any dose levels.

Cytotoxicity
Following the 3-hour treatment, a slight to severe cytotoxicity was induced at dose levels >= 50 µg/mL, as shown by a 30 to 70% decrease in the PD.
Following the 24-hour treatment, a slight to severe cytotoxicity was induced at dose levels = 12.5  µg/mL in the first experiment or at dose levels = 44.4 µg/mL in the second experiment, as shown by a 26 to 100% decrease in the PD.
 
Micronucleus analysis
The dose levels selected for the micronucleus analysis were as follows:
.  6.3, 12.5 and 25 µg/mL for the 3-hour treatment, the latter being the lowest dose level showing emulsion in the culture medium at the end of the treatment period,
. 12.5, 25 and 50 µg/mL for the 24-hour treatment in the first experiment, the latter inducing only a 37% decrease in the PD and higher dose level being too cytotoxic,
. 19.8, 44.4 and 100 µg/mL for the 24-hour treatment in the second experiment, the latter inducing a 59% decrease in the PD (i.e. the recommended level of cytotoxicity of 55 ± 5% cytotoxicity).
 
Following the 3- or 24-hour treatment without S9 mix, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the corresponding vehicle control, in either experiment. Moreover, no dose-response relationship was demonstrated by the linear regression (p > 0.05) and none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle historical range. These results met 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 treatment were 0.55, 1.6, 4.9, 14.8, 44.4, 133.3, 250 and 400 µg/mL.
An emulsion was observed in the culture medium at the end of the 3-hour treatment period, at dose levels >= 44.4 µg/mL.
 
Cytotoxicity
A noteworthy cytotoxicity was induced at dose levels = 250 µg/mL, as shown by a 46 to 88% decrease in the PD.
 
Micronucleus analysis
The dose levels selected for the micronucleus analysis were 4.9, 14.8 and 44.4 µg/mL, the latter being the lowest dose level showing emulsion in the culture medium at the end of the treatment period.
 
No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression (p > 0.05) and none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle historical range. These results met 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 cellsin the mouse cell line L5178Y TK+/-.

The study was performed according to the OECD guideline No.487 and in compliance with the principles of Good Laboratory Practice.

 

Methods

After a preliminary cytotoxicity test, the test item diluted in dimethylsulfoxide (DMSO), was tested in two independent experiments, with or 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

 

24 h treatment + 0 h recovery

With S9 mix

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, cells from at least four 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 poorly soluble in the final treatment medium and cytotoxic in the preliminary test, the highest dose levels selected for the main experiments were based on the presence of emulsion and/or 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.

 

Experiments without S9 mix

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

. 0.23, 0.69, 2.1, 6.3, 12.5, 25, 50 and 100 µg/mL in the first experiment (3- and 24-hour treatments),

. 1.46, 4.39, 13.2, 19.8, 29.6, 44.4, 66.7 and 100 µg/mL in the second experiment (24-hour treatment).

An emulsion was observed in the culture medium at dose levels >= 25 µg/mL at the end of the 3-hour treatment period, whereas no emulsion was observed at the end of the 24-hour treatment period, at any dose levels.

Cytotoxicity

Following the 3-hour treatment, a slight to severe cytotoxicity was induced at dose levels >= 50 µg/mL, as shown by a 30 to 70% decrease in the PD.

Following the 24-hour treatment, a slight to severe cytotoxicity was induced at dose levels = 12.5  µg/mL in the first experiment or at dose levels = 44.4 µg/mL in the second experiment, as shown by a 26 to 100% decrease in the PD.

 

Micronucleus analysis

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

.  6.3, 12.5 and 25 µg/mL for the 3-hour treatment, the latter being the lowest dose level showing emulsion in the culture medium at the end of the treatment period,

. 12.5, 25 and 50 µg/mL for the 24-hour treatment in the first experiment, the latter inducing only a 37% decrease in the PD and higher dose level being too cytotoxic,

. 19.8, 44.4 and 100 µg/mL for the 24-hour treatment in the second experiment, the latter inducing a 59% decrease in the PD (i.e. the recommended level of cytotoxicity of 55 ± 5% cytotoxicity).

 

Following the 3- or 24-hour treatment without S9 mix, no statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the corresponding vehicle control, in either experiment. Moreover, no dose-response relationship was demonstrated by the linear regression (p > 0.05) and none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle historical range. These results met 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 treatment were 0.55, 1.6, 4.9, 14.8, 44.4, 133.3, 250 and 400 µg/mL.

An emulsion was observed in the culture medium at the end of the 3-hour treatment period, at dose levels >= 44.4 µg/mL.

 

Cytotoxicity

A noteworthy cytotoxicity was induced at dose levels = 250 µg/mL, as shown by a 46 to 88% decrease in the PD.

 

Micronucleus analysis

The dose levels selected for the micronucleus analysis were 4.9, 14.8 and 44.4 µg/mL, the latter being the lowest dose level showing emulsion in the culture medium at the end of the treatment period.

 

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression (p > 0.05) and none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle historical range. These results met 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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
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:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro gene mutation study in mammalian cells
Target gene:
hprt locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.
- Storage at Covance: as frozen stocks in liquid notrogen.
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 at 37+/-1°C. When the cells were growing well, subcutltures were established in an appropriate number of flasks.

MEDIA USED
- Type and identity of media: RPMI 1640 media containing L-glutamine and HEPES
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)
Test concentrations with justification for top dose:
Range finder (+/-S9): six concentrations ranging from 62.5 to 2000 µg/mL (limited by precipitate).
Mutation experiment (-S9): ten concentrations, ranging from 25 to 300 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9.
Vehicle / solvent:
DMSO

Preliminary solubility data indicated that Poly(oxy-1,2-ethanediyl), a,a'-[(1-methylethylidene)di-4,1-phenylene]bis[¿-[(2-methyl-1-oxo-2propen-1yl)oxy]] was miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 576.4 mg/mL. The solubility limit in culture medium was in the range of 360.3 to 720.5 µg/mL, as indicated by precipitation at the higher concentration which persisted for approximately 24 hours after test article addition. A maximum concentration of 2000 µg/mL was selected for the cytotoxicity Range-Finder Experiment. This is the maximum concentration generally required for in vitro studies of this type according to current regulatory test guidelines (OECD, 2016). As the test article was a UVCB, the maximum concentration may have been increased to 5000 µg/mL but this could not be achieved due to the limited solubility of the formulated test article in culture medium. Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
DURATION
- Preincubation period: 3h
- Exposure duration: 7d
- Expression time (cells in growth medium): 7d

NUMBER OF CELLS EVALUATED: At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Cloning Efficiency (CE) in any given culture is therefore: CE = P/No of cells plated per well, and as an average of 1.6 cells/well were plated on all survival and viability plates, CE = P/1.6.
Percentage Relative Survival (% RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus: % RS = [CE (test)/CE (control)] x 100.
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival values for each concentration of test article were adjusted as follows: Adjusted % RS = [% RS x Post-treatment cell concentration for test article treatment] / Post-treatment cell concentration for vehicle control


- OTHER: metabolic activation system
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of S-9 were stored frozen in aliquots at <-50°C prior to use (Booth et al., 1980). Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities).
The S-9 mix was prepared in the following way: G6P (180 mg/mL), NADP (25 mg/mL), KCl (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, an aliquot of the mix was added to each cell culture to achieve the required final concentration of test article in a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%.
Rationale for test conditions:
Acceptance Criteria: The assay was considered valid if the following criteria were met:
1. The MF in the concurrent negative control was considered acceptable for addition to the laboratory historical negative control database,
2. The MF in the concurrent positive controls induced responses that were compatible with those generated in the historical positive control database and give a clear, unequivocal increase in MF over the concurrent negative control,
3. The test was performed with and without metabolic activation,
4. Adequate numbers of cells and concentrations were analysable.
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 MF at one or more concentrations was significantly greater than that of the vehicle control (p=0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p=0.05)
3. The results were outside the historical vehicle control range.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 62.5 to 2000 µg/mL (a precipitating concentration). Upon addition of the test article to the cultures, precipitate was observed at the highest five concentrations tested in the absence and presence of S-9 (125 to 2000 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest four concentrations tested in the absence of S-9 (250 to 2000 µg/mL) and at the highest three concentrations tested in the presence of S-9 (500
to 2000 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded. The highest concentration analysed in the absence of S-9 and which gave =10% RS in the presence of S-9 was 250 µg/mL, which gave 46% and 32% RS, respectively.
In the Mutation Experiment, ten concentrations, ranging from 25 to 300 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested. Upon addition of the test article to the cultures, precipitate was observed at the highest eight concentrations tested in the absence of S-9 (75 to 300 µg/mL) and at the highest nine concentrations tested in the presence of S-9 (100 to 500 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations tested in the absence of S-9 (200 to 300 µg/mL) and at the highest two
concentrations tested in the presence of S-9 (450 and 500 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded. Seven days after treatment, two intermediate concentrations (125 and 175 µg/mL) in the absence of S-9 were not selected to determine viability and 6TG resistance as there were sufficient concentrations to determine the toxicity profile. In addition, in the presence of S-9 concentrations of 100 µg/mL (non-toxic) and
400 µg/mL (excessively toxic) were not selected to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 200 µg/mL in the absence of S-9 and 450 µg/mL in the presence of S-9, which gave 72% and 10% RS, respectively

Mutation
The acceptance criteria were met and the study was accepted as valid.
No marked changes in osmolality or pH were observed in the Mutation Experiment at the highest concentrations retained at the end of the 3 hour treatment incubation period (200 µg/mL in the absence of S-9 and 450 µg/mL in the presence of S-9), compared to the concurrent vehicle controls (measured data not reported).
When tested up to precipitating and/or toxic concentrations, no statistically significant increases in MF were observed following treatment with the test substance at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends.
Conclusions:
It is concluded that the test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to precipitating and/or toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.
Executive summary:

The test substance 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 a Mutation Experiment, 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 each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 62.5 to 2000 µg/mL (limited by

precipitate). The highest concentration to give =10% relative survival (RS) was 250 µg/mL (limited by the appearance of post-treatment precipitate in the absence of S-9), which gave 46% and 32% RS in the absence and presence of S-9, respectively.

In the Mutation Experiment, ten concentrations, ranging from 25 to 300 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested. The highest concentrations analysed were 200 µg/mL in the absence of S-9 and 450 µg/mL in the presence of S-9 (limited by the appearance of post-treatment precipitate and/or toxicity), which gave 72% and 10% RS, respectively.

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

When tested up to precipitating and/or toxic concentrations, no statistically significant increases in MF were observed following treatment with test substance at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends.

It is concluded that the test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to precipitating and/or

toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

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 assay (Ames test) (Chevallier 2018)

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

A preliminary toxicity test was performed to define the dose levels of the test item, diluted in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both 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. Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102.

Since the test item was found poorly soluble in the final treatment medium in the preliminary test and toxic only at dose levels close to the highest recommended one (i.e.5000 µg/plate), the selection of the highest dose level to be used in the main experiments was based on the level of emulsion or was 5000 µg/plate, according to the criteria specified in the international guidelines.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were six analysable dose levels for each strain and test condition.

A moderate to strong emulsion and/or precipitate was observed in the Petri plates when scoring the revertants at dose levels equal or higher than 555.6 µg/plate in both experiments with and without S9 mix.

No noteworthy toxicity was noted at any dose levels, in any strains or test conditions.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in either experiment with or without S9 mix. These results met thus the criteria of a negative response.

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimuriumstrains, either in the presence or absence of a rat liver metabolizing system.

In vitro mammalian cell gene mutation test (HPRT) (Lloyd 2018)

The test substance 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 a Mutation Experiment, 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 each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 62.5 to 2000 µg/mL (limited by

precipitate). The highest concentration to give =10% relative survival (RS) was 250 µg/mL (limited by the appearance of post-treatment precipitate in the absence of S-9), which gave 46% and 32% RS in the absence and presence of S-9, respectively.

In the Mutation Experiment, ten concentrations, ranging from 25 to 300 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested. The highest concentrations analysed were 200 µg/mL in the absence of S-9 and 450 µg/mL in the presence of S-9 (limited by the appearance of post-treatment precipitate and/or toxicity), which gave 72% and 10% RS, respectively.

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

When tested up to precipitating and/or toxic concentrations, no statistically significant increases in MF were observed following treatment with test substance at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends.

It is concluded that the test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to precipitating and/or

toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

In vitro mammalian cell micronucleus test (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 cellsin the mouse cell line L5178Y TK.

The study was performed according to the OECD guideline No.487 and in compliance with the principles of Good Laboratory Practice.

After a preliminary cytotoxicity test, the test item diluted in dimethylsulfoxide (DMSO), was tested in two independent experiments, with a metabolic activation (3h treatment, one experiment) or without a metabolic activation system (3h and 24h treatment, 2 experiment for 24h treatment), the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Since the test item was found poorly soluble in the final treatment medium and cytotoxic in the preliminary test, the highest dose levels selected for the main experiments were based on the presence of emulsion and/or 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.

Experiments without S9 mix:

An emulsion was observed in the culture medium at dose levels >= 25 µg/mL at the end of the 3-hour treatment period, whereas no emulsion was observed at the end of the 24-hour treatment period, at any dose levels.

Cytotoxicity : Following the 3-hour treatment, a slight to severe cytotoxicity was induced at dose levels >= 50 µg/mL, as shown by a 30 to 70% decrease in the PD.

Following the 24-hour treatment, a slight to severe cytotoxicity was induced at dose levels = 12.5  µg/mL in the first experiment or at dose levels = 44.4 µg/mL in the second experiment, as shown by a 26 to 100% decrease in the PD.

Micronucleus analysis : Following the 3- or 24-hour treatment without S9 mix,no statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the corresponding vehicle control,in either experiment. Moreover, no dose-response relationship was demonstrated by the linear regression (p > 0.05) andnone of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle historical range. These results met the criteria of a negative response.

Experiments with S9 mix:

An emulsion was observed in the culture medium at the end of the 3-hour treatment period, at dose levels >= 44.4 µg/mL.

Cytotoxicity : A noteworthy cytotoxicity was induced at dose levels = 250 µg/mL, as shown by a 46 to 88% decrease in the PD.

Micronucleus analysis: The dose levels selected for the micronucleus analysis were 4.9, 14.8 and 44.4 µg/mL, the latter being the lowest dose level showing emulsion in the culture medium at the end of the treatment period

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression (p > 0.05) andnone of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle historical range. These results met 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 available data, no classification for genetic toxicity is required for Ethoxylated Bisphenol A dimethacrylate according to the Regulation EC N°1272/2008.