Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
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




Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).