Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

DGEBA diacylate showed no gene mutation in the bacterial reverse mutation test (Ames) and in the in vitro mammalian gene mutation assay (HPRT).

Link to relevant study records

Referenceopen allclose all

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
n/a
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:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
24.0 µg - 6,000 µg/plate (SPT); 4.8 µg - 3000 µg/plate (PIT)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which has been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
with S-9 mix for strains TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: N-methyl-N'nitro-N-nitrosoguanidine (MNNG)
Remarks:
without S-9 mix for strains TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine (NOPD)
Remarks:
without S-9 mix for strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S-9 mix for strain TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S-9 mix for strain E.coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48-72 hours

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one
tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments
carried out independently of each other.
Statistics:
no data
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:
cytotoxicity
Remarks:
A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 600 µg - 3000 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 600 µg - 3000 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 600 µg - 3000 µg/plate onward.

The maximum increase in the number of revertant colonies of 1.6, which was observed in a single incidence without a dose response relationship was well below a doubling of the number of revertant colonies and as the criteria for a positive result were not met, the test substance is not mutagenic in the Ames test.
Conclusions:
Under the test conditions, DGEBA diacrylate is not considered as mutagenic in bacterial reverse mutation test.
Executive summary:

In a GLP study performed according to OECD guideline 471, DGEBA diacrylate was tested for mutagenicity using Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvrA with the plate incorporation and preincubation methods in the presence and absence of metabolic activation system.

A concentration range from 24.0 µg to 6000 µg/plate was used with the plate incorporation technique and concentrations from 4.8 µg to 3000 pg/plate were tested with the preincubation method. Precipitation was found from about 3000 µg/plate and higher. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 600 pg to 3000 pg/plate.

The positive controls induced the appropriate responses in the corresponding strains. DGEBA diacrylate showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S-9.

Under the test conditions, DGEBA diacrylate is not considered as mutagenic in this bacterial system.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 14, 2009 to February 24, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
during viability scoring, erroneous counts noted in single culture (with S-9, in experiment 1) due to dilution error
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co. Cells
- Type and identity of media: RPMI 1640 medium
RPMI A: Penicillin (100 units/mL), streptomycin (100 µg/mL), amphotericin B (2.5 µg/mL) and pluronic acid (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10% v/v), penicillin (100 units/mL), streptomycin (100 µg/mL), amphotericin B (2.5 µg/mL) and pluronic acid (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20% v/v), penicillin (100 units/mL), streptomycin (100 µg/mL) and amphotericin B (2.5 µg/mL)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Yes; each batch was purged of TK- mutants
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
2% S9 fraction of Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Range-finder experiment: 0, 46.88, 93.75, 187.5, 375 or 750 µg/mL (with and without S-9)
Main study:
- Experiment 1: Without S-9: 0, 0.0977, 0.1953, 0.3906, 0.7813, 1.563, 3.125, 6.25, 12.5, 25 or 50 µg/mL; with S-9: 0, 3.75, 7.5, 15, 30, 35, 40, 45, 50, 60 or 75 µg/mL
- Experiment 2: Without S-9: 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 µg/mL; with S-9: 0, 15, 30, 40, 50, 60, 70, 80, 90, 100 or 125 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test material was soluble in anhydrous DMSO, with the aid of warming at 80 °C, at concentrations up to at least 545.5 mg/mL; solubility limit in the culture medium: 340.9-681.8 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation Migrated to IUCLID6: 2 or 3 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation Migrated to IUCLID6: 0.10, 0.15, 10 or 15 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours, 37 ± 1 ºC
- Expression time (cells in growth medium): 7 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
- Selection time (if incubation with a selection agent): 12-13 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Duplicates (single cultures only used for positive control treatments)

NUMBER OF CELLS EVALUATED: 20000 cells/well plated for survival, viability and 6TG resistance

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency

OTHER: Cell viability were identified by eye using background illumination and counted; cell densities were determined using a coulter counter
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.

- Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
The experimental data was analysed using UKEMS recommended statistical guidelines
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 applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No data
- Water solubility: No data; solubility limit in the culture medium: 340.9-681.8 µg/mL
- Precipitation: Yes; in the range-finding study, precipitation was observed at concentrations of 93.75-1500 µg/mL tested in the absence and presence of S-9

RANGE-FINDING/SCREENING STUDIES: Six concentrations were tested with and without S-9 ranging from 46.88-1500 µg/mL (limited by solubility in culture medium).
- Precipitation was observed at concentrations of 93.75 to 1500 µg/mL tested with and without S-9. Lowest precipitating concentration was retained and the higher concentration was discarded.
- Without S-9: complete toxicity was observed at all concentrations; with S-9: highest concentration providing > 10% RS was 46.88 µg/mL
- See table 1 for more details


COMPARISON WITH HISTORICAL CONTROL DATA: Yes; historical mean mutant frequencies for the vehicle controls were 4.34 without S-9 and 4.22 with S-9

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the range-finding study, complete toxicity was observed at all concentrations in the absence of S-9. In the presence of S-9, highest concentration providing > 10% RS was 46.88 µg/mL which gave 21% RS.

Table 1: RS values - range-finder experiment

Treatment (µg/mL)

-S-9 %RS

+S-9 %RS

0

100

100

46.88

0

21

93.75 P

0

0

187.5 P

0

0

375 P

0

0

750 P, PP

0

0

% RS: Percentage Relative Survival

P: Precipitation observed at time of treatment

PP: Precipitation observed following treatment incubation period

Table 2: Summary of mutation data

Experiment 1: (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

2.21

 

0

100

1.34

 

0.0977

91

4.25

NS

3.75

103

NE

 

0.1953

84

2.3

NS

7.5

113

NE

 

0.3906

105

NE

 

15

107

NE

 

0.7813

90

NE

 

30

98

3.53

NS

1.563

84

NE

 

35

92

1.72

NS

3.125

79

3.81

NS

40

77

NE

 

6.25

75

2.24

NS

45

81

3.45

NS

12.5

58

6.66

*

50

83

NE

 

25

4

2.97

NS

60

61

9.53

*

50

0

NE

 

75

45

5.23!

NS

Linear trend

 

NS

Linear trend

**

NQO

 

 

 

B[a]P

 

 

 

0.1

61

45.16

 

2

30

97.71

 

0.15

47

49.83

 

3

11

190.6

 

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

2.89

 

0

100

3.56

 

3

101

4.05

NS

15

86

5.71

NS

6

71

4.69

NS

30

63

3.61

NS

9

76

7.85

*

40

29

4.24

NS

12

57

3.38

NS

50

21

6.69

NS

15

59

3.15

NS

60

6

4.67

NS

18

27

3.66

NS

70

4

NE

 

21

18

6.87

NS

80

3

NE

 

24

5

NE

 

90

1

NE

 

27

2

NE

 

100

0

NE

 

30

0

NE

 

125

0

NE

 

Linear trend

 

NS

Linear trend

NS

NQO

 

 

 

B[a]P

 

 

 

 10 

 34 

 29.68 

 

 2 

 70 

 85.43 

 

 15 

 40 

 52.00 

 

 3 

 25 

 61.29 

 

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

% RS: Percent relative survival adjusted by post treatment cell counts

* Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level

NS: Not significant

*, **, *** Test for linear trend: T2 (one-sided), significant at 5%, 1% and 0.1% level respectively

NE: Not evaluated for mutant frequency due to excessive toxicity. Presented for information only

! Based on one replicate only

Conclusions:
Under the test conditions, 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid is not considered as mutagenic in this HPRT test.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD guideline 476, in compliance with GLP, mouse lymphoma L5178Y TK+/- (3.7.2C) cells were exposed to 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid in DMSO at concentrations of 0, 46.88, 93.75, 187.5, 375 or 750 µg/mL in RPMI 1640 medium with and without 2% S-9 metabolic activation for a preliminary cytotoxicity test. In the main test, two experiments were performed at the following concentrations:

- Experiment 1 (3 hour exposure): Without S-9: 0, 0.0977, 0.1953, 0.3906, 0.7813, 1.563, 3.125, 6.25, 12.5, 25 or 50 µg/mL; with S-9: 0, 3.75, 7.5, 15, 30, 35, 40, 45, 50, 60 or 75 µg/mL

- Experiment 2 (3 hour exposure): Without S-9: 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 µg/mL; with S-9: 0, 15, 30, 40, 50, 60, 70, 80, 90, 100 or 125 µg/mL 

Mutant frequencies in negative control cultures fell within normal 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). In Experiment 1, statistically significant increases in mutant frequency were observed at 12.5 (without S-9) and 60 µg/mL (with S-9). A significant linear trend was observed in the presence of S-9 only. In Experiment 2, a statistically significant increase in mutant frequency was observed at 9 µg/mL in the absence of S-9 only. However, no significant increases were observed following treatment with S-9 and there were no significant linear trends in either the absence or presence of S-9. Further, in both experiments, the increases in mutant frequency at the significant concentrations were all within three times the historical mean value and the increases were not considered concentration related or biologically relevant.

 

Under the test conditions, 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid is not considered as mutagenic in L5178Y cells according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008). 

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

Genetic toxicity in vivo

Description of key information

In the in vivo mouse bone marrow micronucleus assay, DGEBA diacrylate showed negative results.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 03 to August 07, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: CD-1® (ICR) BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, USA
- Age at study initiation: 8 weeks
- Weight at study initiation: Preliminary study: 32.9-39.4 g (males) or 25.8-29.4 g (females); Main study: 32.1-40.0 g (males)
- Assigned to test groups randomly: Yes, animals randomized using a computer program
- Housing: Individually housed in sanitary polycarbonate cages
- Diet (e.g. ad libitum): PMI Certified Rodent Diet® #5002, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-79 °F
- Humidity (%): 30-70%
- Air changes (per hour): 10/hour
- Photoperiod (hours dark / hours light): 12 hours dark/12 hours light

IN-LIFE DATES: From: April 12, 2007 To: April 19, 2007
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Concentration of test material in vehicle: 25 or 50 mg/mL
- Amount of vehicle (if gavage): 20 mL/kg
- Lot/batch no. (if required): 12-455
- Source: Welch, Holme, & Clarke
- Storage: Refrigerated at 0-10 °C
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material was emulsified with corn oil to give stock solution which was then diluted further to give the desired dose levels; formulations were held in a waterbath at ~60 to 70 °C prior to dosing and stirred during the dosing procedure to prevent emulsion breakdown
Duration of treatment / exposure:
24 or 48 hours
Frequency of treatment:
Once for dose levels of 500 and 1000 mg/kg bw, and twice, approximately one hour apart, for the dose level of 2000 mg/kg bw (1000 mg/kg/dose, BID)
Post exposure period:
Not applicable
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:
- Range-finding study: 3 mice/sex/dose
- Main study: Five male mice/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Lot no.: 076K1050
- Storage: Refrigerated at 0-10 °C
- Route of administration: Oral (gavage)
- Doses / concentrations: 80 mg/kg bw
Tissues and cell types examined:
- Hind limb bones (tibias) were removed for marrow extraction and the prepared slides were examined for polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs) and total erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose selection was based on a preliminary range-finding test conducted on 3 mice/sex/dose at 500, 1000 and 2000 mg/kg bw. No mortality and/or toxic signs were recorded after 2 days of exposure.

TREATMENT AND SAMPLING TIMES: Hind limb bones (tibias) were removed for marrow extraction from five surviving animals in each treatment and control group at 24 or 48 hours after exposure.

DETAILS OF SLIDE PREPARATION: Bone marrow cells extracted, preparations spread on slides and air-dried. The slides were fixed in methanol, stained with May-Grunwald solution and Giemsa and coded.

METHOD OF ANALYSIS: Slides were scanned to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) per animal. In addition, PCE:NCE ratio was determined in a population of 500 erythrocytes/animal.
Evaluation criteria:
- Criteria for a positive response: Detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response.
- Test article that does not induce both of these responses is considered negative.
- Biological relevance of the results should be considered first.
Statistics:
- Statistical analysis was performed using ANOVA/Program Trend computer system.
- ANOVA followed by Dunnett’s t-test performed on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-2000 mg/kg bw
- Solubility: Solubility limit in corn oil: 50 mg/mL
- Clinical signs of toxicity in test animals: No
Based on the results of the dose range-finding study, the high dose chosen was 2000 mg/kg, the formulation limit dose and maximum dose required by regulatory guidelines. In the micronucleus assay, the test article was formulated in corn oil and administered once to the 500 and 1000 mg/kg dose groups, or twice, approximately 1-hour apart, to the 2000 mg/kg dose group (1000 mg/kg/dose),

RESULTS OF DEFINITIVE STUDY (table 1)
The test article did not induce signs of clinical toxicity in the animals treated at dose levels up to 2000 mg/kg (the limit dose based on regulatory guidelines).
BADGEDA did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (500, 1000, and 2000 mg/kg).
A statistically significant decrease in the PCE:NCE ratios at a dose level of 1000 mg/kg of the test article was observed. Since the high dose at 2000 mg/kg/dose did not show a similar trend, this statistical difference is not considered biological significance




Table 1: Micronucleus Assay - Summary Table

Treatment 

 Dose

 Harvest Time (hours) 

% Micronucleated PCEs

Mean of 2000 per

Animal ± S.E.(Males)

 Ratio PCE:NCE

Mean ± S.E.(Males)

 Controls 

 

 

 

 

 Vehicle 

 

 Corn Oil 20 mL/kg 

 

 24 

0.04 ± 0.02

0.51 ± 0.06

 48 

0.03 ± 0.02

0.35 ± 0.01

 Positive 

 CP 80 mg/kg 

 24

2.77 ± 0.28* 

0.55 ± 0.1

 Test Article 

 

 

 

 500 mg/kg 

 24

0.04 ± 0.03

0.43 ± 0.05

 1000 mg/kg 

 24

0.06 ± 0.02

0.32 ± 0.07** 

 2000 mg/kga 

 

 24

0.02 ± 0.01

0.59 ± 0.06

 48

0.05 ± 0.02

0.4 ± 0.08

* Significantly greater than the corresponding vehicle control, p = 0.01.

** Significantly less than the corresponding vehicle control, p = 0.05.

CP = Cyclophosphamide; PCE = Polychromatic erythrocyte; NCE = Normochromatic erythrocyte

a Due to solubility limit, the high dose was dosed at 1000 mg/kg BID (~1 hr apart) to achieve a dose of 2000 mg/kg.

Conclusions:
Under the test conditions, BADGEDA is not considered as mutagenic in the mouse bone marrow micronucleus test.
Executive summary:

In a bone marrow micronucleus test, performed according to OECD guideline 474, in compliance with GLP, CD-1® (ICR) BR male mice (5/dose) were given a single oral (gavage) dose of Bisphenol A diglycidyl ether diacrylate (BADGE Diacrylate) in corn oil at concentrations of 500 and1000 mg/kg bw and twice, approximately one hour apart, at concentration of 2000 mg/kg bw (1000 mg/kg/dose, BID). Bone marrow was extracted after 24 or 48 hours of exposure and the prepared slides were scanned to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) per animal. In addition, the number of PCEs and normochromatic erythrocytes (NCEs) in a population of 500 erythrocytes was determined as a measure of cytotoxicity. A preliminary range-finding test was also conducted on 3 mice/sex/dose at 500, 1000 and 2000 mg/kg bw and animals were observed for 2 days. In this previous test, no mortality and/or toxic signs were recorded.

 

No statistically significant increases in the frequency of micronucleated PCEs were observed at any dose levels. A statistically significant decrease in the PCE:NCE ratios at a dose level of 1000 mg/kg bw was observed but was not considered biologically significant as the highest dose of 2000 mg/kg bw did not show a similar trend.

 

Under the test conditions, BADGEDA is not considered as mutagenic in the mouse bone marrow micronucleus test according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008). 

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

Additional information

In vitro Ames test (2002)

In a GLP study performed according to OECD guideline 471, test item (DGEBADA) was tested for mutagenicity using Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvrA with the plate incorporation and preincubation methods in the presence and absence of metabolic activation system.

A concentration range from 24.0 µg to 6000 µg/plate was used with the plate incorporation technique and concentrations from 4.8 µg to 3000 pg/plate were tested with the preincubation method. Precipitation was found from about 3000 µg/plate and higher. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 600 pg to 3000 pg/plate.

The positive controls induced the appropriate responses in the corresponding strains. The test item showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S-9.

Under the test conditions, the test item (DGEBADA) is not considered as mutagenic in this bacterial system.

In vitro gene mutations test on mammalian cells (HPRT, 2010)

In an in vitro mammalian cell gene mutation testperformed according to OECD guideline 476, in compliance with GLP, mouse lymphoma L5178Y TK+/- (3.7.2C) cells were exposed to 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid (DGEBADA) in DMSO. Mutant frequencies in negative control cultures fell within normal 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). In Experiment 1, statistically significant increases in mutant frequency were observed at 12.5 (without S-9) and 60 µg/mL (with S-9). A significant linear trend was observed in the presence of S-9 only. In Experiment 2, a statistically significant increase in mutant frequency was observed at 9 µg/mL in the absence of S-9 only. However, no significant increases were observed following treatment with S-9 and there were no significant linear trends in either the absence or presence of S-9. Further, in both experiments, the increases in mutant frequency at the significant concentrations were all within three times the historical mean value and the increases were not considered concentration related or biologically relevant.

Under the test conditions, DGEBADA is not considered as mutagenic in L5178Y cells.

In vivo micronucleus assay (2007)

In a bone marrow micronucleus test, performed according to OECD guideline 474, in compliance with GLP, CD-1® (ICR) BR male mice (5/dose) were given a single oral (gavage) dose of Bisphenol A diglycidyl ether diacrylate (BADGE diacrylate) in corn oil at concentrations of 500 and 1000 mg/kg bw and twice, approximately one hour apart, at concentration of 2000 mg/kg bw (1000 mg/kg/dose, BID). Bone marrow was extracted after 24 or 48 hours of exposure and the prepared slides were scanned to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) per animal. In addition, the number of PCEs and normochromatic erythrocytes (NCEs) in a population of 500 erythrocytes was determined as a measure of cytotoxicity. A preliminary range-finding test was also conducted on 3 mice/sex/dose at 500, 1000 and 2000 mg/kg bw and animals were observed for 2 days. In this previous test, no mortality and/or toxic signs were recorded. 

No statistically significant increases in the frequency of micronucleated PCEs were observed at any dose levels. A statistically significant decrease in the PCE:NCE ratios at a dose level of 1000 mg/kg bw was observed but was not considered biologically significant as the highest dose of 2000 mg/kg bw did not show a similar trend. Under the test conditions, DGEBADA is not considered as mutagenic in the mouse bone marrow micronucleus test .

Justification for classification or non-classification

As DGEBA diacrylate showed no genotoxic activity in all the performed studies covering mutagenicity and clastogenicity endpoints, no classification for mutagenicity is required according to the CLP Regulation (EC) N° (1272-2008).