Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The three in vitro recommended tests were performed on Ethoxylated Bisphenol A diacrylate. The Ames test and the HPRT test showed negative results in presence and in absence of metabolic activation. However, the in vitro micronucleus test showed a positive response without metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 August 2012 to 15 February 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)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance)
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:
Range finding test: six concentrations were tested in the presence and absence of S-9 ranging from 31.25 to 1000 µg/mL
Experiment 1: eleven concentrations ranging from 6 to 60 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9
Experiment 2: twelve concentrations ranging from 5 to 50 µg/mL in the absence of S-9 and ten concentrations ranging from 50 to 275 µg/mL in the presence of S-9

Positive controls:
4-nitroquinoline 1 oxide (NQO), stock solution: 0.015 and 0.020 mg/mL and final concentration: 0.15 and 0.20 µg/mL, no S-9 present
Benzo[a]pyrene (B[a]P), stock solution: 0.200 and 0.300 mg/mL and final concentration: 2.00 and 3.00 µg/mL, S-9 present
Vehicle / solvent:
DMSO diluted 100-fold in the treatment medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO diluted 100-fold in the treatment area
True negative controls:
no
Positive controls:
yes
Remarks:
4-nitroquinoline-N-oxide without metabolic activation and benzo(a)pyrene with metabolic activation) Positive control substance 4-nitroquinoline-N-oxide without metabolic activation and benzo(a)pyrene with metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: NA
- Exposure duration: 3 Hours
- Selection time (if incubation with a selection agent): NA
- Fixation time (start of exposure up to fixation or harvest of cells): 7 Days

NUMBER OF REPLICATIONS: 9

DETERMINATION OF CYTOTOXICITY
- Method: Osmolality
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:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. 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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes
- Effects of osmolality: No marked changes
- Evaporation from medium: Not reported
- Water solubility: Preliminary solubility data indicated that Ethoxylated bisphenol A diacrylate (CAS Number 64401-02-1) was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 500.0 mg/mL. The solubility limit in culture medium was approximately 156.3 to 312.5 µg/mL, as indicated by precipitation at the higher concentration which persisted for approximately 3 hours after test article addition
- Precipitation: no precipitate was observed in the absence and presence of S-9 following the 3 hour treatment incubation period
- Other confounding effects: Not Applicable

Details of results :
In Experiment 1 eleven concentrations, ranging from 6 to 60 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 time of treatment at the highest nine concentrations (200 to 500 µg/mL) in the presence of S-9 but no precipitate was observed in the absence and presence of S-9 following the 3-hour treatment incubation period. Seven days after treatment, the highest two concentrations in the absence of S-9 (50 and 60 µg/mL) and the highest seven concentrations in the presence of S-9 (300 to 500 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentration analysed in the absence of S-9 was 40 µg/mL, which gave 21% RS and was considered acceptably close to the target toxicity range of 10-20% RTG. In the presence of S-9, steep concentration-related toxicity was observed between 200 and 250 µg/mL, which gave 44% and 2% RS, respectively. Marked heterogeneity was observed at 250 µg/mL but the concentration was included in the analysis for comparative purposes.
In Experiment 2 twelve concentrations, ranging from 5 to 50 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 50 to 275 µg/mL, were tested in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the time of treatment at the highest eight concentrations in the presence of S-9 (150 to 275 µg/mL) but no precipitate was observed in the absence and presence of S-9 following the 3-hour treatment incubation period. Seven days after treatment the highest concentration in the absence of S-9 (50 µg/mL) and the highest five concentrations in the presence of S-9 (220 to 275 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, concentrations of 5 and 15 µg/mL in the absence of S-9 were not selected as there were sufficient non-toxic concentrations to define the toxicity profile. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 45µg/mL in the absence of S-9 and 210 µg/mL in the presence of S-9, which gave 15% and 14% RS, respectively.

Table1: RS Values - Range-Finder Experiment

Treatment

(µg/mL)

-S-9

% RS

+S-9

% RS

0

100

100

31.25

13

190

62.5

0

154

125

0

127

250

0 P

72 P

500

0 P, PP

0 P

1000

NP P, PP

0 P, PP

% RS                      Percent relative survival adjusted by post treatment cell counts

P                             Precipitation observed at time of treatment

PP                          Precipitation observed following treatment incubation period

NP                          Not plated for viability due to precipitation

 

Table 2: Summary of mutation data

Experiment 1 (3 hour treatment in the absence and presence of S-9)

Treatment

(mg/mL)

-S-9

Treatment

(mg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

2.55

 

0

 

100

5.11

 

6

 

93

3.24

NS

50

 

114

6.12

NS

12

 

85

4.19

NS

100

 

106

4.21

NS

16

 

85

4.33

NS

200

P

44

4.95

NS

20

 

74

4.08

NS

250

P,$

2

3.95

NS

24

 

62

5.78

NS

 

 

 

 

 

28

 

50

6.69

NS

 

 

 

 

 

32

 

12

5.28

NS

 

 

 

 

 

36

 

25

6.60

NS

 

 

 

 

 

40

 

21

1.68

NS

 

 

 

 

 

Linear trend

 

NS

Linear trend

 

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.15

 

61

47.14

 

2

 

70

43.33

 

0.2

 

44

52.31

 

3

 

52

71.30

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Experiment 2 (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

9.15

 

0

 

100

10.71

 

10

 

107

8.86

NS

50

 

112

1.90

NS

20

 

76

1.80

NS

100

 

105

3.98

NS

25

 

68

5.56

NS

150

P

71

2.33

NS

30

 

50

10.67

NS

200

P

21

7.64

NS

33

 

41

7.43

NS

210

P

14

6.57

NS

36

 

35

6.10

NS

 

 

 

 

 

39

 

33

3.56

NS

 

 

 

 

 

42

 

20

3.97

NS

 

 

 

 

 

45

 

15

1.92

NS

 

 

 

 

 

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.15

 

58

61.92

 

2

 

50

27.37

 

0.2

 

57

101.97

 

3

 

28

47.20

 

 

 

 

 

 

 

 

 

 

 

§                             6-TG resistant mutants/106viable cells 7 days after treatment

% RS                      Percent relative survival adjusted by post treatment cell counts

P                             Precipitation observed at the time of treatment

$                             Marked heterogeneity observed for mutation, but included in analysis for comparative purposes

NS                          Not significant

 

Conclusions:
It is concluded that Ethoxylated bisphenol A 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:

Ethoxylated bisphenol A 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 31.25 to 1000 µg/mL (limited by solubility in culture medium). The highest concentrations to survive treatment were 31.25 µg/mL in the absence of S-9 and 250 µg/mL in the presence of S-9, which gave 13% and 72% relative survival (RS), respectively. Accordingly, for Experiment 1 eleven concentrations, ranging from 6 to 60 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentration analysed in the absence of S-9 was 40 µg/mL, which gave 21% RS and was considered acceptably close to the target toxicity range of 10-20% RTG. In the presence of S-9, steep concentration related toxicity was observed between 200 and 250 µg/mL, which gave 44% and 2% RS, respectively. Marked heterogeneity was observed at 250 µg/mL but the concentration was included in the analysis for comparative purposes. In Experiment 2 twelve concentrations, ranging from 5 to 50 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 50 to 275 µg/mL, were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed were 45 µg/mL in the absence of S-9 and 210 µg/mL in the presence of S-9, which gave 15% and 14% 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 Ethoxylated bisphenol A diacrylate (CAS Number 64401 -02-1) at any concentration tested in the absence and presence of S-9 and there were no significant linear trends. It is concluded that Ethoxylated bisphenol A diacrylate (CAS Number 64401-02-1) 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 two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

Endpoint:
in vitro gene mutation study in bacteria
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 471 (Bacterial Reverse Mutation Assay)
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.
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
Remarks:
DMSO
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, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated 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
Positive controls validity:
valid
Additional information on results:
The number of revertants for the vehicle and positive controls met the acceptance criteria. The study was therefore considered to be valid.
 
A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels >= 1250 µg/plate in the experiments performed using the direct plate incorporation method and at dose-levels = 2500 µg/plate in the experiment performed using the pre-incubation method.
No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any tested dose-level towards the five strains used, in any experiments, either with or without S9 mix.
The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, either with or without S9 mix.
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 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 Ethoxylated bisphenol A diacrylate to be used for the mutagenicity study. The test item 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, 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.

 

The test item Ethoxylated bisphenol A diacrylate was dissolved in dimethylsulfoxide (DMSO).

 

Results

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 freely soluble and non-cytotoxic in the preliminary test, the highest selected dose-level 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 the five strains used in 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 the experiments performed using the direct plate incorporation method and at dose-levels = 2500 µg/plate in the experiment performed using the pre-incubation method.

 

No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any tested dose-level towards the five strains used, in any experiments, either with or without S9 mix.

 

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


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 liver metabolizing system.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 August 2012 - 08 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 487 (In vitro micronucleus)
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:
other: mouse lymphoma L5178Y TK+/- 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
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatment were as follows:
- 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL in the first experiment,
- 3.13, 6.25, 12.5, 25, 37.5, 50 and 100 µg/mL in the second experiment,
- 10, 15, 17.5, 20, 25, 30, 35 and 40 µg/mL in the third experiment.

Experiments with S9 mix
With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatment were as follows:
- 6.25, 12.5, 25, 50, 100 and 200 µg/mL in the first experiment,
- 1.56, 3.13, 6.25, 12.5, 25 and 50 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 DMSO and the maximal treatment volume of 1% in culture medium, the highest recommended dose-level of 5000 µg/mL was achievable.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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
without S9 mix:
3 h treatment + 24 h recovery (first experiment)
- 24 h treatment + 20 h recovery (second and third experiments).

with S9 mix:
3 h treatment + 24 h recovery (first and second experiments).

NUMBER OF CELLS EVALUATED: 2000/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling
Evaluation criteria:
The biological relevance of the results should be considered first. Statistical methods are used as an aid in evaluating the test results but should not be the only determinant of a positive response. A result is considered as positive if at least a 2.5-fold increase in the number of micronucleated cells in comparison to the concurrent control is observed, with a statistically significant difference, at one or more concentrations. Concentration-related increases in the frequency of micronucleated cells and comparison to the vehicle control historical data will also be taken into account.
Statistics:
The statistical comparison was performed using the ¿2 test, in which p = 0.05 was used as the lowest level of significance.
Key result
Species / strain:
other: mouse lymphoma L5178Y TK+/- cells
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
(only without S9 mix)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The population doublings and the mean frequencies of micronucleated cells for the vehicle control cultures were as specified in the acceptance criteria. Positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.
Since the test item was found severely cytotoxic and poorly soluble in the preliminary test, the selection of the highest dose-level to be tested in the main experiments was based on the level of precipitation for the first experiment with S9 mix and on the level of cytoxicity for all the other experiments, according to the criteria specified in the international guidelines.

EXPERIMENTS WITHOUT S9 MIX
A precipitate was observed in the culture medium at dose-levels = 25 µg/mL at the end of the 3-hour treatment period in the first experiment. No precipitate was observed at the end of the 24-hour treatment period in the second and the third experiment up to the highest tested dose-levels.

Cytotoxicity : Following the 3-hour treatment in the first experiment (Table 2), a severe toxicity was noted at dose-levels = 25 µg/mL, as shown by a 83 to 100% decrease in the Population Doubling (PD).
Following the 24-hour treatment in the second experiment (Table 4), a severe toxicity was noted at dose-levels = 37.5 µg/mL, as shown by a 100% decrease in the PD.
Following the 24-hour treatment in the third experiment (Table 6), a slight to severe toxicity was
noted at dose-levels = 30 µg/mL, as shown by a 28 to 100% decrease in the PD.

Micronucleus analysis :
The dose-levels selected for micronucleus analysis were as follows:
. 3.13, 6.25 and 12.5 µg/mL for the 3-hour treatment, the latter dose-level inducing a 18% decrease in the PD and higher dose-levels being too cytotoxic,
. 6.25, 12.5 and 25 µg/mL for the 24-hour treatment in the second experiment, the latter dose-level inducing a 18% decrease in the PD and higher dose-levels being too cytotoxic,
. 20, 25 and 30 µg/mL for the 24-hour treatment in the third experiment, the latter dose-level inducing a 28% decrease in the PD and higher dose-levels being too cytotoxic.
Following the 3-hour treatment (Table 3), no noteworthy increase in the frequency of micronucleated cells was noted in comparison to the vehicle control cultures.
Following the 24-hour treatment in the second experiment (Table 5), an increase in the frequency of micronucleated cells (reaching or exceeding the threshold of 2.5-fold the vehicle control value) was observed at dose-levels = 12.5 µg/mL. This increase was dose-related and reached statistical
significance at the dose-level of 25 µg/mL (p < 0.05). Also, the corresponding frequencies of micronucleated cells exceeded the historical data range of the vehicle control (3 and 5 micronucleated cells in 1000 cells at 12.5 and 25 µg/mL, respectively, versus 0.5 to 2 for the historical data range). Moreover, this increase was found to be reproducible since significant increases in the frequency of micronucleated cells exceeding the threshold of 2.5-fold the vehicle control value were observed at 25 and 30 µg/mL in a third experiment performed under the same experimental conditions with a narrower range of dose-level (Table 7). Statistical significance was reached at 25 µg/mL. Therefore, these increases being reproducible in independent experiments, they were considered to be biologically significant.


EXPERIMENTS WITh S9 MIX
A precipitate was observed in the culture medium at the end of the 3-hour treatment period at dose-levels = 25 µg/mL and at the dose-level of 50 µg/mL in the first and second experiments, respectively.

Cytotoxicity
In the first experiment (Table 8), a marked toxicity was observed at 200 µg/mL as shown by a 77% decrease in the PD.
In the second experiment (Table 10), no noteworthy toxicity was observed at any tested dose-levels as shown by the absence of notably decrease in the PD.

Micronucleus analysis
The dose-levels selected for micronucleus analysis were as follows:
. 6.25, 12.5 and 25 µg/mL for the first experiment, the latter corresponding to the lowest dose-level showing precipitate in the culture medium,
. 12.5, 25 and 50 µg/mL for the second experiment, the latter corresponding to lowest dose-level showing precipitate in the culture medium.
In the first experiment (Table 9), non dose-related increases in the frequency of micronucleated cells, exceeding the threshold of 2.5-fold the vehicle control value were observed at all analyzed dose-levels. At 6.25 µg/mL, the increase was statistically significant (p < 0.05). At all analyzed dose-levels, the frequencies of micronucleated cells remained within the historical data range of the vehicle control (up to 4 micronucleated cells in 1000 cells versus 0.5 to 5 for the historical data range). Moreover, these increases were neither dose-related nor reproducible since no increase in the frequency of micronucleated cells was noted in the second experiment performed under the same experimental conditions with a narrower range of dose-level (Table 11). Consequently, these increases, being neither reproducible nor dose-related, were considered as non-biologically relevant.
Conclusions:
The test item induced chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells in the absence of rat metabolizing system. In the presence of metabolic activation, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in L5178Y TK+/- cultured mouse lymphoma cells.
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 L5178Y TK+/-mouse lymphoma cells.

The study design was based on the OECD Guideline No. 487 (adopted 22 July 2010) and was in compliance with the GLP compliance.

 

Methods

After a preliminary toxicity test, the test item 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 + 20 h recovery

With S9 mix

3 h treatment + 24 h recovery

3 h treatment + 24 h recovery

 

In order to check the reliability of the significant increase in the frequency of micronucleated cells noted in the second experiment without S9 mix (24 h treatment + 20 h recovery) and in order to exhibit about 55% toxicity, a third experiment was performed using eight dose-levels of the test item (two cultures/dose-level) without metabolic activation and using a 24 h treatment + 20 h recovery period.

 

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 and quality of the cells on the slides has also been taken into account.

 

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

The population doublings and the mean frequencies of micronucleated cells for the vehicle control cultures were as specified in the acceptance criteria. Positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Since the test item was found severely cytotoxic and poorly soluble in the preliminary test, the selection of the highest dose-level to be tested in the main experiments was based on the level of precipitation for the first experiment with S9 mix and on the level of cytoxicity for all the other experiments, according to the criteria specified in the international guidelines.

 

Experiments without S9 mix

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

.           1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL in the first experiment,

.           3.13, 6.25, 12.5, 25, 37.5, 50 and 100 µg/mL in the second experiment,

.           10, 15, 17.5, 20, 25, 30, 35 and 40 µg/mL in the third experiment.


A precipitate was observed in the culture medium at dose-levels = 25 µg/mL at the end of the 3-hour treatment period in the first experiment. No precipitate was observed at the end of the 24-hour treatment period in the second and the third experiment up to the highest tested dose-levels.

 

Cytotoxicity

Following the 3-hour treatment in the first experiment, a severe toxicity was noted at dose-levels = 25 µg/mL, as shown by a 83 to 100% decrease in the PD.

Following the 24-hour treatment in the second experiment, a severe toxicity was noted at dose-levels = 37.5 µg/mL, as shown by a 100% decrease in the PD.

Following the 24-hour treatment in the third experiment, a slight to severe toxicity was noted at dose-levels = 30 µg/mL, as shown by a 28 to 100% decrease in the PD.

 

Micronucleus analysis

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

.           3.13, 6.25 and 12.5 µg/mL for the 3-hour treatment, the latter dose-level inducing a 18% decrease in the PD and higher dose-levels being too cytotoxic,

.           6.25, 12.5 and 25 µg/mL for the 24-hour treatment in the second experiment, the latter dose-level inducing a 18% decrease in the PD and higher dose-levels being too cytotoxic,

.           20, 25 and 30 µg/mL for the 24-hour treatment in the third experiment, the latter dose-level inducing a 28% decrease in the PD and higher dose-levels being too cytotoxic.

 

Following the 3-hour treatment, no noteworthy increase in the frequency of micronucleated cells was noted in comparison to the vehicle control cultures.

 

Following the 24-hour treatment in the second experiment, an increase in the frequency of micronucleated cells (reaching or exceeding the threshold of 2.5-fold the vehicle control value) was observed at dose-levels >= 12.5 µg/mL. This increase was dose-related and reached statistical significance at the dose-level of 25 µg/mL (p < 0.05). Also, the corresponding frequencies of micronucleated cells exceeded the historical data range of the vehicle control (3 and 5 micronucleated cells in 1000 cells at 12.5 and 25 µg/mL, respectively, versus 0.5 to 2 for the historical data range). Moreover, this increase was found to be reproducible since significant increases in the frequency of micronucleated cells exceeding the threshold of 2.5-fold the vehicle control value were observed at 25 and 30 µg/mL in a third experiment performed under the same experimental conditions with a narrower range of dose-level. Statistical significance was reached at 25 µg/mL. Therefore, these increases being reproducible in independent experiments, they were considered as biologically significant.

 

Experiments with S9 mix

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

.           6.25, 12.5, 25, 50, 100 and 200 µg/mL in the first experiment,

.           1.56, 3.13, 6.25, 12.5, 25 and 50 µg/mL in the second experiment.

 

A precipitate was observed in the culture medium at the end of the 3-hour treatment period at dose-levels = 25 µg/mL and at the dose-level of 50 µg/mL in the first and second experiments, respectively.

 

Cytotoxicity

In the first experiment, a marked toxicity was observed at 200 µg/mL as shown by a 77% decrease in the PD.

In the second experiment, no noteworthy toxicity was observed at any tested dose-levels as shown by the absence of notably decrease in the PD.


Micronucleus analysis

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

.           6.25, 12.5 and 25 µg/mL for the first experiment, the latter corresponding to the lowest dose-level showing precipitate in the culture medium,

.           12.5, 25 and 50 µg/mL for the second experiment, the latter corresponding to lowest dose-level showing precipitate in the culture medium.

In the first experiment, non dose-related increases in the frequency of micronucleated cells, exceeding the threshold of 2.5-fold the vehicle control value were observed at all analyzed dose-levels. At 6.25 µg/mL, the increase was statistically significant (p < 0.05). At all analyzed dose-levels, the frequencies of micronucleated cells remained within the historical data range of the vehicle control (up to 4 micronucleated cells in 1000 cells versus 0.5 to 5 for the historical data range). Moreover, these increases were neither dose-related nor reproducible since no increase in the frequency of micronucleated cells was noted in the second experiment performed under the same experimental conditions with a narrower range of dose-level. Consequently, these increases, being neither reproducible nor dose-related, were considered as non-biologically relevant.

 

Conclusion

The test item induced chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells in the absence of rat metabolizing system. In the presence of metabolic activation, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in L5178Y TK+/-cultured mouse lymphoma cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo Comet assay on rats was performed on Ethoxylated (3) Bisphenol A Diacrylate and was negative. The test substance did not cause a significant increase in DNA damage in liver, glandular stomach, and duodenum cells relative to the concurrent vehicle control.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 June 2017 to 29 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
This species has been routinely used as an animal model of choice for the mammalian alkaline Comet assay. This strain is an outbred strain that maximizes genetic heterogeneity, and therefore, tends to eliminate strain-specific responses to the test article.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS, Inc., Frederick, MD
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation:
Dose Range Finder: Males - 186.2 to 210.4 grams; Females - 146.9 to 160.1 grams.
Initial Definitive: Males - 198.8 to 227.3 grams;
Repeat Definitive: Males - 154.3 to 169.7 grams
- Assigned to test groups randomly: Yes, animals were assigned to groups using a randomization procedure. At the time of randomization, the weight variation per sex of all animals assigned to the study did not exceed +/-20% of the mean weight. A randomization function in Excel was used to achieve random placement of animals throughout all groups.
- Fasting period before study: None - Housing: Animals were housed in a controlled environment at 72+/-3 degrees F and 50+/-20% relative humidity with a 12-hour light/dark cycle. The light cycle may have been interrupted for study-related activities. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air per hour. Animals of the same sex were housed up to 5 per Micro-Barrier cage.
- Diet (e.g. ad libitum): A certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet) was provided ad libitum. - Water (e.g. ad libitum): Animals had free access to tap water, which met US EPA drinking water standards.
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72+/-3 degrees F
- Humidity (%): 50+/-20% relative humidity
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

IN-LIFE DATES:
Dose Range Finder From: 05 July 2017 To: 12 July 2017
Repeat Definitive From: 25 Oct 2017 To: 31 Oct 2017
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil for test substance; 0.9% Saline for injection, USP for positive control
- Justification for choice of solvent/vehicle: The vehicle was chosen based on its solubility with the test substance and compatibility with the target cells.
- Amount of vehicle : 10 mL/kg/day - Lot/batch no. of test substance vehicle: MKCC0462
Details on exposure:
Preparation of Control Article
The neat EMS was prepared in 0.9% saline for injection. The dosing formulation was prepared at a concentration of 20 mg/mL, just prior to use. Preparation of Test Substance Dose FormulationsThe test substance dose formulations were prepared fresh on each day of use. Each concentration was prepared by calibrating a suitable size amber vial with a PTFE stir bar to target batch size, which holds the pre-weighed amount of the test substance. An appropriate amount of the test substance was combined with approximately 70% of the total volume of vehicle and stirred with the PTFE stir bar. The vehicle was added to the QS line, sealed and mixed magnetically until homogenous in appearance. Formulations were stirred continuously after their preparation and throughout the dosing procedure. The final dose formulation was stored at room temperature.

All dose formulations were administered at a volume of 10 mL/kg/day by oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles). The route has been routinely used and is widely-accepted for use in the comet assay.
Duration of treatment / exposure:
All animals in Groups 1 through 4 were dosed on 2 consecutive days with the vehicle and test substance. The second dose occurred approximately 21 hours after the first dose and 3-4 hours before euthanasia. Two animals in group 2 (males) were euthanized 4-6 minutes earlier than the 3-hour euthanasia time due to a time scheduling error. This deviates from the protocol, which states the animals were to be euthanized 3-4 hours after the 21-hour dose on Day 3.Animals in Group 5 were dosed with the positive control once approximately 3-4 hours prior to euthanasia on Day 2.
Frequency of treatment:
see above
Post exposure period:
All animals were euthanized 3-4 hours after the last dose (Study Day 2) for tissue collection, except as indicated above.
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
DRF: 3 animals/sex
Definitive: 6 male rats per dose (Groups 1 to 4), two additional animals were dosed in Group 4 to cover for any possible mortality, but were euthanized and discarded without evaluation because no mortality was observed; 3 males (Group 5)
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg/day / 10 mL/kg/day
Tissues and cell types examined:
Liver, stomach, and duodenum
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In the DRF assay, 3 animals/sex were exposed to 2000, 1000, and 500 mg/kg/day of test substance. A vehicle control was also included. Animals were dosed on two consecutive days. The second dose occurred approximately 21 hours after the first dose. Clinical observations were seen in the high dose group in both males and females. Following the last observation, animals were euthanized by exposure to CO2 and discarded without further examination after tissues were collected for histopatholgy. No mortality or differences in clinical observations were seen between the sexes, therefore only males were used in the definitive assay. In the Definitive assay, dose levels were based on the results from the DRF assay and histopathology.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Animals were dosed at a dose volume of 10 mL/kg/day by oral gavage for two consecutive days. All animals were euthanized 3-4 hours after the last dose for tissue collection, except as previously indicated.

DETAILS OF SLIDE PREPARATION: A portion of each dissected liver was placed in 3 mL of cold mincing buffer, then the liver was finely cut (minced) with a pair of fine scissors to release the cells. A portion of each dissected interior glandular stomach and duodenum were placed in 1 mL of cold mincing buffer then glandular stomach and duodenum were scraped using a plastic spatula to release the cells. Each cell suspension was strained through a Cell Strainer into a pre-labeled 50 mL polypropylene conical tube and the resulting liver, stomach and duodenum cell suspensions were placed on wet-ice. An aliquot of the suspensions were used to prepare the comet slides. From each liver, stomach and duodenum suspensions, an aliquot of 2.5 µL (liver) and 7.5 µL (stomach and duodenum) were mixed with 75 µL (0.5%) of low melting agarose. The cell/agarose suspension was applied to microscope slides commercially available pre-treated multi-well slides. Commercially purchased multi-well slides were used and these slides have 3 individual circular areas, referred to as wells in the text below. The slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify. Slides were identified with a random code that reflects the study number, group, animal number, and organ/tissue. At least two Trevigen, Inc 20-well slides were prepared per animal per tissue. Three wells were used in scoring and the other wells were designated as a backup. Following solidification of agarose, the slides were placed in jars containing lysis solution.Following solidification of agarose, the slides were submerged in commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution at least overnight at 2-8oC.After cell lysis, slides/wells were washed with neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, pH ~7.5) and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with alkaline buffer composed of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water. The pH was > 13. All slides remained in the buffer for 20 minutes at 2-10oC and protected from light, allowing DNA to unwind.Using the same buffer, electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-10oC and protected from light. The electrophoresis time was constant for all slides.After completion of electrophoresis, the slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides (gels) were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for at least 4 hours and stored at room temperature with desiccant. Slides were stained with a DNA stain (i.e., Sybr-gold) prior to scoring. The stain solution was prepared by diluting 1 uL of Sybr-gold stain in 15 mL of 1xTBE (tris-boric acid EDTA buffer solution).

METHOD OF ANALYSIS: Slides were evaluated for DNA damage using a fully validated automated scoring system (Comet Assay IV) from Perceptive Instruments Ltd. (UK).
Evaluation criteria:
A test article was considered to have induced a positive response if:a) at least one of the group means for the % Tail DNA of the test article doses exhibited a statistically significant increase when compared with the concurrent negative control (p
Statistics:
The median value of 150 counts of % Tail DNA, Tail moment and Tail migration were determined and presented for each animal in each treatment group for each organ. The mean and standard deviation of the median values only for % Tail DNA were presented for each treatment group. Statistical analysis was performed only for % Tail DNA. In order to quantify the test substance effects on DNA damage, the following statistical analysis was performed: •The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for % tail DNA generated for the vehicle and test substance groups were compared using Levene’s test (significant level of p < 0.05). If the differences and variations between groups were found not to be significant, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p < 0.05). •A linear regression analysis was conducted to assess dose responsiveness in the test substance treated groups (p = 0.01). •A pair-wise comparison (Student’s T-test, p = 0.05) was used to compare the positive control group to the concurrent vehicle control group.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs of piloerection were observed at 1000 and 2000 mg/kg/day in the Definitive Assay.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: in liver, stomach and duodenum cells
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
No mortality occurred at any dose level during the course of the dose range finding assay. No considerable reductions in mean group body weights were seen in the test substance treated groups during the course of the study.The following clinical signs were observed at 2000 mg/kg : Lethargy, Crusty Nose in males, and Hunched Position in females.

RESULTS OF DEFINITIVE STUDY
In the Initial Comet Assay, the vehicle control values were not within the expected range, thus not all criteria for a valid test were met. Therefore, in consultation with the Sponsor, the assay was repeated. The results reported are for the Repeat Comet Assay.
No mortality occurred at any dose level during the course of the definitive assay.
No appreciable reductions in mean group body weights were seen in the test substance treated groups during the course of the study.
The following clinical signs were observed:Piloerection at 1000 and 2000 mg/kg

In liver cells :
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the test substance groups was = 0.3%, which was comparable with the % of clouds in the vehicle control group (0.3%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.
• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).
• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).
• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the vehicle control groups (Student’s t test, p = 0.05).
• In the vehicle control group, % Tail DNA was within the historical vehicle control range for the liver (Appendix I).
These results indicate that all criteria for a valid test, as specified in the protocol, were met.

In stomach cells :
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the test substance groups was = 49.5%, which was lower than the % of clouds in the vehicle control group (59.0%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.
• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).
• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).
• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in glandular stomach cells as compared to the vehicle control groups (Student’s t test, p = 0.05).
• In the vehicle control group, % Tail DNA mean was within the historical vehicle control range for the stomach (Appendix I).
These results indicate that all criteria for a valid test, as specified in the protocol, were met.

In duodenum cells:
The scoring results and a statistical analysis of data indicated the following:
• The presence of ‘clouds’ in the test substance groups was = 10.8%, which was lower than the % of clouds in the vehicle control group (18%).
• Group variances for mean of medians of the % Tail DNA in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.
• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent vehicle control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).
• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).
• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in duodenal cells as compared to the vehicle control groups (Student’s t test, p = 0.05).
• In the vehicle control group, % Tail DNA was within the historical vehicle control range for the duodenum (Appendix I).
These results indicate that all criteria for a valid test, as specified in the protocol, were met.

Conclusions:
Under the conditions of the assay described in this report, Ethoxylated (3) Bisphenol A Diacrylate was concluded to be negative for the induction of DNA damage in liver, glandular stomach and duodenum.
Executive summary:

The test substance,Ethoxylated (3) Bisphenol A Diacrylate, was evaluated for its genotoxic potential in the Comet assay to induce DNA damage in liver, glandular stomach, and duodenum cells of male rats. Corn oil was selected as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg/day by oral gavage.

In the dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg/day. The additional dose levels tested were 1000 and 500 mg/kg/day in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/day, which is the highest guideline recommended dose for this assay, which was estimated to be the maximum tolerated dose (MTD). 

The definitive assay dose levels tested were 500, 1000, and 2000 mg/kg/day.

The definitive assay was repeated due to vehicle values in the initial assay not being within the expected range. Thus, the initial assay did not meet the criteria for a valid assay and was repeated. The results and discussion will only include data from the repeated definitive assay. The test substance gave a negative (non-DNA damaging) response in this assay in liver, glandular stomach, and duodenum cells for males in % Tail DNA. None of the test substance treated animal slides had significant increases in the % Tail DNA compared to the respective vehicle controls. The vehicle control % Tail DNA was within the Testing Facility’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver, glandular stomach, and duodenum cells.

All critiera for a valid study were met as described in the protocol.

Under the conditions of this study, the administration of Ethoxylated (3) Bisphenol A Diacrylate at doses up to and including a dose of 2000 mg/kg/day did not cause a significant increase in DNA damage in liver, glandular stomach, and duodenum cells relative to the concurrent vehicle control. Therefore, Ethoxylated (3) Bisphenol A Diacrylate was concluded to be negative in the in vivo Comet Assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay (Ames test) 2012 :

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

After a preliminary assay, the test item was 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. The test item Ethoxylated bisphenol A diacrylate was dissolved in dimethylsulfoxide (DMSO).

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, 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.

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 freely soluble and non-cytotoxic in the preliminary test, the highest selected dose-level 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 the five strains used in 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 the experiments performed using the direct plate incorporation method and at dose-levels = 2500 µg/plate in the experiment performed using the pre-incubation method. No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any tested dose-level towards the five strains used, in any experiments, either with or without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, either with or without S9 mix. 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 liver metabolizing system.

In vitro mammalian cell gene mutation test (HPRT) 2013:

Ethoxylated bisphenol A 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. For Experiment 1 eleven concentrations, ranging from 6 to 60 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentration analysed in the absence of S-9 was 40 µg/mL, which gave 21% RS and was considered acceptably close to the target toxicity range of 10-20% RTG. In the presence of S-9, steep concentration related toxicity was observed between 200 and 250 µg/mL, which gave 44% and 2% RS, respectively. Marked heterogeneity was observed at 250 µg/mL but the concentration was included in the analysis for comparative purposes. In Experiment 2 twelve concentrations, ranging from 5 to 50 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 50 to 275 µg/mL, were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed were 45 µg/mL in the absence of S-9 and 210 µg/mL in the presence of S-9, which gave 15% and 14% RS, respectively. In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with Ethoxylated bisphenol A diacrylate at any concentration tested in the absence and presence of S-9 and there were no significant linear trends. It is concluded that Ethoxylated bisphenol A 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).

In vitro mammalian cell micronucleus test

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 L5178Y TK+/- mouse lymphoma cells (OECD 487).

After a preliminary toxicity test, the test item 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: first experiment of 3 h treatment/24 h recovery, with and without S9 mix; second experiment of 24 h treatment/20 h recovery without S9 mix, and 3 h treatment/24 h recovery with S9 mix.

 In order to check the reliability of the significant increase in the frequency of micronucleated cells noted in the second experiment without S9 mix (24 h treatment + 20 h recovery) and in order to exhibit about 55% toxicity, a third experiment was performed using eight dose-levels of the test item (two cultures/dose-level) without metabolic activation and using a 24 h treatment + 20 h recovery period.

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 and quality of the cells on the slides has also been taken into account. The test item was dissolved in dimethylsulfoxide (DMSO).

Since the test item was found severely cytotoxic and poorly soluble in the preliminary test, the selection of the highest dose-level to be tested in the main experiments was based on the level of precipitation for the first experiment with S9 mix and on the level of cytoxicity for all the other experiments, according to the criteria specified in the international guidelines.

Following the 3-hour treatment without S9, no noteworthy increase in the frequency of micronucleated cells was noted in comparison to the vehicle control cultures. Following the 24-hour treatment (without S9) in the second experiment, an increase in the frequency of micronucleated cells (reaching or exceeding the threshold of 2.5-fold the vehicle control value) was observed at dose-levels >= 12.5 µg/mL. This increase was dose-related and reached statistical significance at the dose-level of 25 µg/mL (p < 0.05). Also, the corresponding frequencies of micronucleated cells exceeded the historical data range of the vehicle control (3 and 5 micronucleated cells in 1000 cells at 12.5 and 25 µg/mL, respectively,versus 0.5 to 2 for the historical data range). Moreover, this increase was found to be reproducible since significant increases in the frequency of micronucleated cells exceeding the threshold of 2.5-fold the vehicle control value were observed at 25 and 30 µg/mL in a third experiment performed under the same experimental conditions with a narrower range of dose-level. Statistical significance was reached at 25 µg/mL. Therefore, these increases being reproducible in independent experiments, they were considered as biologically significant.

With S9 mix, In the first experiment, a marked toxicity was observed at 200 µg/mL as shown by a 77% decrease in the PD. In the second experiment with S9 mix, no noteworthy toxicity was observed at any tested dose-levels as shown by the absence of notably decrease in the PD.

In the first experiment, non dose-related increases in the frequency of micronucleated cells, exceeding the threshold of 2.5-fold the vehicle control value were observed at all analyzed dose-levels. At 6.25 µg/mL, the increase was statistically significant (p < 0.05). At all analyzed dose-levels, the frequencies of micronucleated cells remained within the historical data range of the vehicle control (up to 4 micronucleated cells in 1000 cellsversus0.5 to 5 for the historical data range). Moreover, these increases were neither dose-related nor reproducible since no increase in the frequency of micronucleated cells was noted in the second experiment performed under the same experimental conditions with a narrower range of dose-level. Consequently, these increases, being neither reproducible nor dose-related, were considered as non-biologically relevant.

To conclude, the test item induced chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells in the absence of rat metabolizing system. In the presence of metabolic activation, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in L5178Y TK+/- cultured mouse lymphoma cells.

In vivo Comet assay in rats (OECD 489) 2018 :

The test substance,Ethoxylated (3) Bisphenol A Diacrylate, was evaluated for its genotoxic potential in the Comet assay to induce DNA damage in liver, glandular stomach, and duodenum cells of male rats. Corn oil was selected as the vehicle. Test and/or control substance formulations were administered at a dose volume of 10 mL/kg/day by oral gavage.

In the dose range finding assay (DRF), the maximum dose tested was 2000 mg/kg/day. The additional dose levels tested were 1000 and 500 mg/kg/day in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/day, which is the highest guideline recommended dose for this assay, which was estimated to be the maximum tolerated dose (MTD). The definitive assay dose levels tested were 500, 1000, and 2000 mg/kg/day.

The definitive assay was repeated due to vehicle values in the initial assay not being within the expected range. Thus, the initial assay did not meet the criteria for a valid assay and was repeated. The results and discussion will only include data from the repeated definitive assay. The test substance gave a negative (non-DNA damaging) response in this assay in liver, glandular stomach, and duodenum cells for males in % Tail DNA. None of the test substance treated animal slides had significant increases in the % Tail DNA compared to the respective vehicle controls. The vehicle control % Tail DNA was within the Testing Facility’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver, glandular stomach, and duodenum cells.

All critiera for a valid study were met as described in the protocol. Under the conditions of this study, the administration ofEthoxylated (3) Bisphenol A Diacrylate at doses up to and including a dose of 2000 mg/kg/day did not cause a significant increase in DNA damage in liver, glandular stomach, and duodenum cells relative to the concurrent vehicle control. Therefore,Ethoxylated (3) Bisphenol A Diacrylate was concluded to be negative in the in vivo Comet Assay.


Justification for classification or non-classification

The Ames test and the HPRT test showed negative results in presence and in absence of metabolic activation. However, the in vitro micronucleus test showed a positive response without metabolic activation. An in vivo Comet assay was performed on Ethoxylated (3) Bisphenol A Diacrylate and was negative. The test substance did not cause a significant increase in DNA damage inliver, glandular stomach, and duodenum cells relative to the concurrent vehicle control.

Based on the available data, Ethoxylated (3) Bisphenol A Diacrylate is considered to be not genotoxic, no classification for genotoxicity is required according to the Regulation EC N°1272/2008.