1H-Isoindol-1-one, 2,3-dihydro-3,3-bis(4-hydroxyphenyl)-2-phenyl-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 474 (1998):GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Remarks:

OECD GLP, US FDA GLP (21 CFR 58); US EPA GLP (40 CFR 160 and 792), UK GLP and Japanese GLP Standard

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

455-890-7

EC Name:

-

Cas Number:

6607-41-6

Molecular formula:

C26H19NO3

IUPAC Name:

455-890-7

Details on test material:

PPP-BP (CAS# 6607-41-6); Purity 99.85%

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., Frederick, MD and were approximately 6-8 weeks of age at study initiation, weighing 27.4 31.9 g (males) and 21.1-23.9 g (females). Up to five mice of the same sex were group housed in polycarbonate cages with heat-treated hardwood chips for bedding. The controlled environmental 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

Details on exposure:

In the pilot toxicity assay, five male and five female mice were exposed to PPP-BP at a dose of 2000 mg/kg and two male mice each to 1, 10, 100, or 1000 mg/kg. PPP-BP 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, 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 PPP-BP at a dose of 500, 1000 or 2000 mg/kg and were euthanized 24 hours after treatment. Animals in the other two groups were treated either with the negative control or PPP-BP at a dose of 2000 mg/kg and were euthanized 48 hours after treatment. Additional replacement animals (5 animals/sex) were included in the high dose group, 10 mg/kg, to ensure that the availability of 5 animals/sex for micronucleus analysis. PPP-BP vehicle mixture, the vehicle alone or the positive control was administered by a single IP injection at a dose volume of 20 mL/kg. 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

Remarks:

Doses / Concentrations:
1, 10, 100, 1000, 2000 mg/kg body weight (Pilot toxicity assay)
Basis:
other: Actual injected

Doses / concentrations 2

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg body weight (Definitive assay)
Basis:
other: Actual injected

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes, Cyclophosphamide monohydrate (CP, CAS No. 6055-19-2); 50 mg/kg

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 (PCEs/ECs ratio).

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, PPP-BP 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 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. PPP-BP 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

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the pilot study, PPP-BP was administered to two male each mice at 1, 10, 100, and 1000 mg/kg and to five male and five and female mice at 2000 mg/kg. No mortality was observed. Clinical signs following dose administration included: piloerection and lethargy in males at 100 and 1000 mg/kg and in males and females at 2000 mg/kg. In addition, piloerection was seen in males at 10 mg/kg, while animals at 1 mg/kg appeared normal. In the absence of mortality, the high dose for the micronucleus test was set at 2000 mg/kg.

In the definitive micronucleus study, PPP-BP was administered to male and female mice (5/sex/group) at 500, 1000 or 2000 mg/kg. No mortality occurred at any dose during the course of the micronucleus study. Clinical signs following dose administration included: lethargy and piloerection in all males and females at 500,1000, and 2000 mg/kg. All animals treated with the vehicle or positive control article 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 (up to 38%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in the 24-hour test article-treated groups relative to the respective vehicle controls, demonstrating bioavailability of the test article to the bone marrow target. No reduction in PCEs/ECs ratio was observed in the 48-hour high dose test article-treated group. The number of micronucleated polychromatic erythrocytes per 10,000 polychromatic erythrocytes in PPP-BP-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 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 less than or equal to 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 ofPPP-BP(CAS No.6607-41-6) 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	0.506 ± 0.06	---	0.7 ± 0.27	7 / 10000
	F	24	5	0.527 ± 0.01	---	0.6 ± 0.42	6 / 10000
PPP-BP
500 mg/kg	M	24	5	0.471 ± 0.06	-7	0.4 ± 0.42	4 / 10000
	F	24	5	0.424 ± 0.02	-20	0.5 ± 0.35	5 / 10000
1000 mg/kg	M	24	5	0.355 ± 0.04	-30	0.4 ± 0.22	4 / 10000
	F	24	5	0.338 ± 0.02	-36	0.6 ± 0.22	6 / 10000
2000 mg/kg	M	24	5	0.363 ± 0.04	-28	1.1 ± 0.89	11 / 10000
	F	24	5	0.326 ± 0.02	-38	0.7 ± 0.45	7 / 10000
CP
50 mg/kg	M	24	5	0.348 ± 0.01	-31	23.5 ± 4.09	*235 / 10000
	F	24	5	0.288 ± 0.04	-45	21.3 ± 3.80	*213 / 10000
Corn oil	M	48	5	0.448 ± 0.04	---	0.6 ± 0.42	6 / 10000
	F	48	5	0.470 ± 0.04	---	0.5 ± 0.35	5 / 10000
PPP-BP
2000 mg/kg	M	48	5	0.491 ± 0.04	10	0.7 ± 0.27	7 / 10000
	F	48	5	0.484 ± 0.05	3	0.3 ± 0.27	3 / 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 PPP-BP (CAS No. 6607-41-6)in ICR Mice

Treatment (20 mL/kg)	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Corn oil	M	101	0.494	1 / 2000
		102	0.424	2 / 2000
		103	0.590	1 / 2000
		104	0.513	2 / 2000
		105	0.508	1 / 2000
	F	106	0.524	1 / 2000
		107	0.528	2 / 2000
		108	0.540	2 / 2000
		109	0.525	0 / 2000
		110	0.520	1 / 2000
PPP-BP
500 mg/kg	M	111	0.434	0 / 2000
		112	0.408	1 / 2000
		113	0.477	0 / 2000
		114	0.537	2 / 2000
		115	0.498	1 / 2000
	F	116	0.430	1 / 2000
		117	0.417	1 / 2000
		118	0.448	2 / 2000
		119	0.404	0 / 2000
		120	0.420	1 / 2000
1000 mg/kg	M	121	0.338	1 / 2000
		122	0.372	1 / 2000
		123	0.345	1 / 2000
		124	0.411	1 / 2000
		125	0.307	0 / 2000
	F	126	0.320	1 / 2000
		127	0.341	1 / 2000
		128	0.364	2 / 2000
		129	0.310	1 / 2000
		130	0.357	1 / 2000
2000 mg/kg	M	131	0.308	2 / 2000
		132	0.363	2 / 2000
		133	0.421	0 / 2000
		134	0.380	2 / 2000
		135	0.343	5 / 2000
	F	136	0.317	1 / 2000
		137	0.353	2 / 2000
		138	0.334	2 / 2000
		139	0.310	2 / 2000
		140	0.315	0 / 2000
CP 50 mg/kg	M	141	0.340	48 / 2000
		142	0.333	46 / 2000
		143	0.349	36 / 2000
		144	0.351	59 / 2000
		145	0.367	46 / 2000
	F	146	0.228	40 / 2000
		147	0.261	44 / 2000
		148	0.335	33 / 2000
		149	0.294	54 / 2000
		150	0.324	42 / 2000

Induction of Micronucleated Polychromatic Erythrocytes inBone Marrow Cells Collected 48 Hours Following a Single Dose of PPP-BP (CAS No. 6607-41-6) in ICR Mice

Treatment (20 mL/kg)	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Corn oil	M	151	0.416	0 / 2000
		152	0.506	1 / 2000
		153	0.437	2 / 2000
		154	0.428	1 / 2000
		155	0.452	2 / 2000
	F	156	0.482	1 / 2000
		157	0.456	2 / 2000
		158	0.423	1 / 2000
		159	0.536	1 / 2000
		160	0.453	0 / 2000
PPP-BP
2000 mg/kg	M	161	0.437	2 / 2000
		162	0.496	1 / 2000
		163	0.515	1 / 2000
		164	0.473	1 / 2000
		165	0.536	2 / 2000
	F	166	0.565	0 / 2000
		167	0.466	1 / 2000
		168	0.431	1 / 2000
		169	0.494	1 / 2000
		170	0.465	0 / 2000

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of this study, a single intraperitoneal administration of PPP-BP at doses up to 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, PPP-BP (CAS No. 6607-41-6) was concluded to be negative in the micronucleus test using male and female ICR mice.