Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Test Guideline 474 (1998). GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Remarks:
OECD GLP, US FDA GLP (21 CFR 58); US EPA GLP (40 CFR 160 and 40 CFR 792), UK GLP and Japanese GLP Standard
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Bisphenol Dianhydride (BPA-DA; CAS No. 38103-06-9); Lot No. UI0054; Purity: 98.2%; from GE Plastics

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
The ICR mice were obtained from Harlan Sprague Dawley, Inc. and were approximately 6-8 weeks of age at study initiation, weighing 25.6-33.4 g (males) and 19.8-27.6g (females). Up to five mice of the same sex were group-housed in polycarbonate cages. The controlled environment parameters were 72 ± 3°F, 50 ± 20% relative humidity and a 12-hour light/dark cycle. The mice had free access to certified rodent chow and tap water.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Corn oil was determined to be the vehicle of choice based on a solubility determination of BPA-DA and compatibility of the vehicle with the test system. BPA-DA was a workable suspension in corn oil at 200 mg/mL, the maximum concentration tested in the solubility test.
Details on exposure:
In the pilot toxicity assay, one group of five male and five female mice were exposed to BPA-DA at a dose of 2000 mg/kg body weight and four groups of two male mice each to 1, 10, 100, or 1000 mg/kg. BPA-DA dosing formulations were administered at a volume of 20 mL/kg by a single IP injection. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration and 1 and 3 days after dose administration.
For the toxicity assay and supplement, all mice were weighed immediately prior to dose administration and the dose volume was based on individual body weight. In the toxicity study, animals were exposed to 1200, 1400, 1600 or 1800 mg/kg BPA-DA. Since mortality was observed at all these doses, two lower doses, 700 and 1000 mg/kg, were tested in the supplemental toxicity study. BPA-DA dosing formulations were administered at a volume of 20 mL/kg by a single IP injection. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration and 1 and 3 days after dose administration.

For the definitive micronucleus assay, ICR mice were assigned to seven groups, each containing 5 males and 5 females. Animals in five of these groups were treated either with the controls (negative or positive) or with BPA-DA at a dose of 200, 400 or 800 mg/kg and were euthanized 24 hours after treatment. Animals in the other two groups were treated either with the negative control or BPA-DA at a dose of 800 mg/kg and were euthanized 48 hours after treatment. BPA-DA vehicle mixture, the vehicle alone or the positive control was administered by a single IP injection at a dose volume of 20 mL/kg. IP injection was selected to maximize delivery of BPA-DA to the test system. All mice in the experi¬mental and control groups were weighed immediately before dose administration, and the dose volume was based on individual body weight. Mice were observed after dose administration for clinical signs of toxicity.
Frequency of treatment:
Single treatment
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Preliminary assay: of 1, 10, 100, 1000 or 2000 mg/kg body weight
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Toxicity Assay and Supplement: 700, 1000, 1200, 1400, 1600, or 1800 mg/kg body weight
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Definitive Assay: 200, 400 or 800 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
Preliminary Assay: 5/sex at 2000 mg BPA-DA/kg; 2 males/group at 1, 10, 100, or 1000 mg BPA-DA/kg
Toxicity and Supplement Assay: 5/sex/group
Definitive Assay: 5/sex/group
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes, Cyclophosphamide monohydrate (CP, CAS number 6055-19-2)

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
At the scheduled sacrifice times, five mice per sex per dose were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes, and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May Gruenwald Giemsa and permanently mounted.
Bone marrow cells [polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)], were analyzed for the presence of micronuclei. Polychromatic erythrocytes are young, immature red blood cells that stain bluish while normochromatic erythrocytes or normocytes are mature red blood cells that stain pink. Micronuclei are round, darkly-staining nuclear fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei can occur in both PCEs (MPCEs) and NCEs (MNCEs).
To control for bias, slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification (10 x 40), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (10 x 100), 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
Evaluation criteria:
To quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and dose group.
As a guide to interpretation of the data, BPA-DA was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p is less than or equal to 0.05, Kastenbaum Bowman Tables) at any sampling time. However, values that were statistically significant but did not exceed the range of historical negative or vehicle controls were judged as not biologically significant. BPA-DA was judged negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response were observed at any sampling time.
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and dose group. Statistical significance was determined using the Kastenbaum Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
males greater than 400 mg/kg and females greater than 800 mg/kg
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
In the pilot study, mortality was observed in 4/5 male mice and 4/5 female mice at 2000 mg/kg. Clinical signs following dose administration included: piloerection in males at doses ≥10 mg/kg and in females at 2000 mg/kg. Lethargy was seen in males at 1000 mg/kg and in males and females at 2000 mg/kg and hunched position in males and females at 2000 mg/kg.
In the toxicity study, mortality was observed in 5/5 male and 5/5 female mice at 1800 mg/kg, 5/5 males and 2/5 females at 1600 mg/kg, 4/5 males and 3/5 females at 1400 mg/kg, in 4/5 males and 3/5 females at 1200 mg/kg and in 1/5 males and 1/5 females at 1000 mg/kg. Clinical signs following dose administration included: lethargy and piloerection in males and females at all doses and hunched position in males at 1200 mg/kg and in males and females at all doses ≥1400 mg/kg. Based upon these results, the high dose for the micronucleus test was set at 800 mg/kg, which was estimated to be the maximum tolerated dose.
In the definitive micronucleus study, no mortality was observed in any male or female. Clinical signs following dose administration included: piloerection in males and females at all doses and lethargy in males at 400 mg/kg and in males and females at 800 mg/kg. All other animals treated with control articles appeared normal during the course of the study.
Bone marrow cells (polychromatic erythrocytes, PCEs and normochromatic erythrocytes, NCEs), collected 24 and 48 hours after treatment were examined microscopically for presence of micronuclei (MPCEs or MNCEs). Reductions of 15%, 37% and 56% in the ratio of polychromatic erythrocytes to total erythrocytes were observed at 800 mg/kg in female group 24 hours postdose and in male and female group 48 hours postdose, respectively. These reductions demonstrate that there was bioavailability of BPA-DA to the bone marrow target tissue. The number of micronucleated polychromatic erythrocytes per 10,000 polychromatic erythrocytes in BPA-DA-treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose or bone marrow collection time (p is greater than 0.05, Kastenbaum-Bowman Tables).

In this study, all criteria for a valid test were met as specified in the protocol. CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p is less than 0.05, Kastenbaum-Bowman Tables). The negative and positive controls were consistent with the historical control data, indicating that there was no problem with the test system or the quality of the test.

Any other information on results incl. tables

Summary of Bone Marrow Micronucleus Analysis Following a Single Dose of Bisphenol A Dianhydride (BPA-DA; CAS# 38103-06-9) in ICR Mice

Treatment (20 mL/kg)

Sex

Time (hr)

Number of Mice

PCE/Total             Erythrocytes        (Mean +/- SD)

Change from Control (%)

Micronucleated Polychromatic Erythrocytes

Number per 1000 PCEs (Mean +/- SD)

Number per PCEs Scored

Corn oil*

M

24

5

0.485 ± 0.06

---

0.8 ± 0.27

8 / 10000

F

24

5

0.464 ± 0.09

---

0.4 ± 0.42

4 / 10000

BPA-DA

200 mg/kg

M

24

5

0.464 ± 0.07

-4

0.4 ± 0.42

4 / 10000

F

24

5

0.460 ± 0.05

-1

0.4 ± 0.22

4 / 10000

400 mg/kg

M

24

5

0.457 ± 0.08

-6

0.5 ± 0.35

5 / 10000

F

24

5

0.456 ± 0.07

-2

0.6 ± 0.42

6 / 10000

800 mg/kg

M

24

5

0.471 ± 0.05

-3

0.3 ± 0.45

3 / 10000

F

24

5

0.396 ± 0.08

-15

0.6 ± 0.22

6 / 10000

CP*

50 mg/kg

M

24

5

0.341 ± 0.04

-30

20.1 ± 3.34

*201 / 10000

F

24

5

0.324 ± 0.01

-30

19.4 ± 3.15

*194 / 10000

Corn oil*

M

48

5

0.480 ± 0.03

---

0.7 ± 0.27

7 / 10000

F

48

5

0.519 ± 0.02

---

0.5 ± 0.00

5 / 10000

BPA-DA

800 mg/kg

M

48

5

0.301 ± 0.06

-37

0.7 ± 0.27

7 / 10000

F

48

5

0.228 ± 0.09

-56

0.9 ± 0.55

9 / 10000

*Statistically significant, p less than or equal to 0.05 (Kastenbaum-Bowman Tables)

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 24 Hours Following a Single Dose of Bisphenol A Dianhydride (BPA-DA; CAS# 38103-06-9) in ICR Mice

Treatment (20 mL/kg)

Sex

Animal Number

PCE/Total Erythrocytes

Micronucleated PCE (Number/PCE scored)

Corn oil*

M

1

0.467

1 / 2000

2

0.435

2 / 2000

3

0.420

2 / 2000

4

0.564

1 / 2000

5

0.540

2 / 2000

F

6

0.340

0 / 2000

7

0.571

1 / 2000

8

0.519

2 / 2000

9

0.407

1 / 2000

10

0.485

0 / 2000

BPA-DA

200 mg/kg

M

11

0.464

1 / 2000

12

0.411

2 / 2000

13

0.510

0 / 2000

14

0.377

1 / 2000

15

0.556

0 / 2000

F

16

0.416

1 / 2000

17

0.474

1 / 2000

18

0.528

0 / 2000

19

0.461

1 / 2000

20

0.421

1 / 2000

400 mg/kg

M

21

0.461

1 / 2000

22

0.322

1 / 2000

23

0.467

1 / 2000

24

0.482

0 / 2000

25

0.552

2 / 2000

F

26

0.461

2 / 2000

27

0.414

1 / 2000

28

0.411

1 / 2000

29

0.568

2 / 2000

30

0.425

0 / 2000

800 mg/kg

M

31

0.417

0 / 2000

32

0.519

0 / 2000

33

0.531

2 / 2000

34

0.472

0 / 2000

35

0.417

1 / 2000

F

36

0.409

1 / 2000

37

0.470

1 / 2000

38

0.317

1 / 2000

39

0.310

2 / 2000

40

0.474

1 / 2000

CP*

50 mg/kg

M

71

0.314

35 / 2000

72

0.383

33 / 2000

73

0.319

42 / 2000

74

0.376

41 / 2000

75

0.314

50 / 2000

F

76

0.310

44 / 2000

77

0.338

36 / 2000

78

0.332

43 / 2000

79

0.323

29 / 2000

80

0.316

42 / 2000

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 48 Hours Following a Single Dose of  Bisphenol A Dianhydride (BPA-DA; CAS# 38103-06-9) in ICR Mice

Treatment (20 mL/kg)

Sex

Animal Number

PCE/Total Erythrocytes

Micronucleated PCE (Number/PCE scored)

Corn oil*

M

81

0.478

2 / 2000

82

0.458

2 / 2000

83

0.473

1 / 2000

84

0.524

1 / 2000

85

0.465

1 / 2000

F

86

0.520

1 / 2000

87

0.536

1 / 2000

88

0.535

1 / 2000

89

0.481

1 / 2000

90

0.523

1 / 2000

BPA-DA

800 mg/kg

M

91

0.251

1 / 2000

92

0.312

1 / 2000

93

0.343

2 / 2000

94

0.367

2 / 2000

95

0.231

1 / 2000

F

96

0.309

2 / 2000

97

0.112

3 / 2000

98

0.200

2 / 2000

99

0.194

2 / 2000

100

0.326

0 / 2000

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the assay described in this report, a single intraperitoneal administration of BPA-DA at doses up to 800 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, Bisphenol A Dianhydride (BPA-DA; CAS# 38103-06-9) was concluded to be negative in the micronucleus test using male and female ICR mice.