4,4'-[(isopropylidene)bis(p-phenyleneoxy)]diphthalic dianhydride

Genetic toxicity: in vivo

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

4,4'-[(isopropylidene)bis(p-phenyleneoxy)]diphthalic dianhydride

EC Number:

253-781-7

EC Name:

4,4'-[(isopropylidene)bis(p-phenyleneoxy)]diphthalic dianhydride

Cas Number:

38103-06-9

Molecular formula:

C31H20O8

IUPAC Name:

5,5'-[propane-2,2-diylbis(4,1-phenyleneoxy)]bis(2-benzofuran-1,3-dione)

Details on test material:

Bisphenol Dianhydride (BPA-DA; CAS No. 38103-06-9)

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system 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 experimental 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 / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

800 mg/kg bw/day (nominal)

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 analysed 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 ≤ 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

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

males greater than 400 mg/kg and females greater than 800 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

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 > 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 < 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 ≤ 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:

Under the conditions of the assay described in this report, a single intraperitoneal administration of the test material at doses up to 800 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the test material was concluded to be negative in the micronucleus test using male and female ICR mice.

Executive summary:

The study was conducted in ICR mice in accordance with OECD 474 under GLP conditions (BioReliance, 2003).

Male and female ICR mice were dosed via the intraperitoneal route with test material concentrations of 200, 400 or 800 mg/kg body weight in corn oil.

No mortality was observed. 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 post-dose and in male and female group 48 hours post-dose, respectively. These reductions demonstrate that there was bioavailability of the test material to the bone marrow target tissue.

Under the conditions of the assay, the test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, it was concluded to be negative in the micronucleus test.