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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 407 (1995); GLP

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Remarks:

OECD GLP

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:

2-Phenyl, 3-3-Bis (4-Hydroxy Phenyl) Phthalimidine (PPP-BP; CAS RN 6607-41-6); Purity: 99.8%

Test animals

Species:

rat

Strain:

other: Crl:CD®(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animals and environment: Forty seven male and 47 female Crl:CD(SD)IGS BR rats were received in good health from Charles River Laboratories, Inc., Raleigh, North Carolina. The animals were approximately 40 days old at receipt. Each animal was examined by a qualified technician on the day of receipt and weighed two days later. Each animal was uniquely identified by a metal ear tag displaying the permanent identification number. All animals were housed for a 10-day acclimation/pretest period. During this period, each animal was observed twice daily for mortality and changes in general appearance or behavior and food consumption and body weights were recorded. All animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study, except during the period of fasting prior to blood collection when food, but not water, was withheld. All animals were housed throughout the acclimation period and during the study in an environmentally controlled room. Actual mean daily temperature ranged from 70.4°F to 72.4°F (21.4°C to 22.5°C) and mean daily relative humidity ranged from 39.6% to 54.9% during the study. Light timers were set to provide a 12-hour light (6 a.m. to 6 p.m.)/12-hour dark photoperiod. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Test article formulations were prepared as weight/volume mixtures at 20, 60 and 200 mg/ml. The appropriate amount of the test article for each formulation was weighed into a tared, calibrated storage container. A sufficient volume of vehicle (corn oil) was added to each container. The formulations were mixed until uniform using a magnetic stirrer. The formulations were then homogenized using an electric homogenizer until uniform mixtures were obtained. Vehicle was then added to each container to bring the formulations to the calibration mark. The test article formulations were prepared weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored refrigerated. The test article formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the initiation of dose administration, triplicate samples (1 mL each) for homogeneity determination were collected from the top, middle and bottom strata of the 100 and 1000 mg/kg/day dosing formulations. In addition, triplicate samples (1 mL each) for stability determinations were collected from the top, middle and bottom strata of representative aliquots of these same dosing suspensions sufficient for 1 day of test article administration and after 8 days of refrigerated storage. Duplicate samples (1 mL each) for concentration analyses were collected from the first and third weekly dosing formulations (including the control group). A high performance liquid chromatography method using ultraviolet detection at a wavelength of 230 nm was used for the determination of PPP-BP concentration in the dose formulations. Each formulation met the requirements for homogeneity, i.e., the RSD for the overall mean concentration was less than or equal to 10% at a concentration that was within the acceptable limits (within 85% to 115% of target concentration). The test article in the low and high concentration formulation groups was stable during 8 days of refrigerated storage, since the post-storage concentration was greater than or equal to 90% of the corresponding zero-time value. The analyzed formulations used for dose administration met the requirement for concentration acceptability for suspension formulations, i.e., the analyzed concentrations were within 85% to 115% of the target dose concentrations.

Duration of treatment / exposure:

a minimum of 28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg/day (measured)
Basis:

No. of animals per sex per dose:

8/sex/group; Additional animals: 5/sex at 0 and 1000 mg/kg/day for a 14-day recovery period

Control animals:

yes

Details on study design:

Doses were selected based on a 10-day oral gavage study in which PPP-BP was administered at 100, 300 and 1000 mg/kg/day. Based on this study, the NOEL for oral administration of PPP-BP for 10 consecutive days was 1000 mg/kg/day. Therefore, the high dose of 1000 mg/kg/day was selected for the present study.

For the main study, PPP-BP in the vehicle, corn oil, was administered orally by gavage at 0, 100, 300 or 1000 mg/kg/day once daily for a minimum of 28 consecutive days to Crl:CD(SD)IGS BR rats. The vehicle was corn oil and the dose volume was 5 mL/kg for all groups. Groups 1 and 4 each consisted of 13 animals/sex and Groups 2 and 3 each consisted of 8 animals/sex. Individual doses were based on the most recently recorded body weights. Study day 0 and study week 0 were the first day and week of dosing, respectively. Following 28 consecutive days of dose administration, 8 rats/sex/group were euthanized (primary necropsy; study day 28); the remaining 5 rats/sex in the control and high dose groups were euthanized following a 14-day recovery period (recovery necropsy; study day 42).

Examinations

Observations and examinations performed and frequency:

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed twice daily, at the time of dosing and at approximately 1 to 2 hours following dose administration. During the recovery period animals were observed once daily. Detailed physical examinations were performed weekly beginning one week prior to test article administration and concluding on the day of necropsy. Individual body weights and food consumption were recorded weekly. Functional observational battery (FOB) (observations included: home cage; handling; open field; sensory; neuromuscular and physiological) and locomotor activity (MA) data were recorded for all animals prior to the initiation of dose administration and during the fourth week of dose administration (prior to the daily dose). Hematology, serum chemistry and urinalysis parameters (see lists below) were evaluated in all study animals from the primary necropsy (study week 4). The animals were fasted overnight prior to blood collection. Blood was collected at the time of necropsy via the vena cava. Blood was collected into tubes containing EDTA (hematology), sodium citrate (coagulation tests) or no anticoagulant (serum chemistry). Urine samples were collected overnight using metabolism cages prior to the day blood samples were collected.

Sacrifice and pathology:

A complete necropsy was conducted on all animals. Animals were euthanized at the scheduled necropsies by isoflurane anesthesia and exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities, including viscera. Select tissues and organs were collected and/or weighed and placed in 10% neutral-buffered formalin (except as noted) (see lists below). After fixation, the specified tissues were trimmed and processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides and stained with hematoxylin and eosin. Microscopic examination was performed on all tissues listed below from all animals in the control and 1000 mg/kg/day groups at the scheduled primary necropsy. Gross lesions were examined from all animals in the 100 and 300 mg/kg/day at the primary necropsy. Microscopic examination was performed at the laboratory.

Statistics:

All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-treated group to the control group by sex. Body weight, body weight change, food consumption, clinical pathology, organ weight (absolute and relative) and applicable functional observational battery and locomotor activity data were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p is less than 0.05) intergroup variance, Dunnett's test was used to compare the test article-treated groups to the control group. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test. Clinical pathology values for white blood cell types that occur at a low incidence (i.e., monocytes, eosinophils and basophils) were not subjected to statistical analysis.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

All animals survived to the scheduled necropsies. There were no test article-related clinical observations. All clinical findings in the test article-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner and/or were common findings for laboratory rats of this age and strain.

There were no test article-related effects on body weight. Statistically significantly (p is less than 0.05) lower mean body weight gains were observed in the 300 and 1000 mg/kg/day group males during the first week of dosing (study days 0-7) when compared with control group values. These lower body weight gains did not occur in a dose-related manner and therefore, these differences from the control group were not attributed to toxicity of the test article. A statistically significantly (p is less than 0.01) lower body weight gain was also observed in the 1000 mg/kg/day males during the first week of the recovery period (study days 27-34) which resulted in lower mean body weights on study days 34 and 41 in this group. The weight differences during the recovery period were clearly related to the selection process for the 5 males/group. That is, the mean weight for all 13 males from the control group at study day 27 was 403.6 g compared to the randomly selected animals mean weight of 412.0 g, while the corresponding weights for the 13 high dose animals and 5 recovery animals were 378.9 g and 370.1 g, respectively. There were no other statistically significant differences when the control and test article-treated groups were compared.

There were no test article-related effects on food consumption. A statistically significantly (p is less than 0.05) lower food consumption value was observed in the 1000 mg/kg/day group males during the first week of the recovery period (study days 27-34) when compared to control group values. As this lower food consumption value was observed during the recovery period and was higher than previous weekly food consumption values for this group, it was not attributed to the test article. There were no other statistically significant differences when the control and test article-treated groups were compared.

There were no test article-related effects on the following functional observational battery parameters: home cage, handling, open field, neuromuscular and physiological observations. Motor activity patterns (total and ambulatory activity counts) were unaffected by test article administration. There were no statistically significant differences for the test article-treated males and females when compared to the control group at the evaluation at study week 3 for home cage and handling observations or at the evaluation during the fourth week of dosing for all other parameters.

Sensory observations were unaffected by test article administration. A statistically significant (p less than 0.05) difference in tail pinch response (animal may turn slowly, walk away) was observed in 3/8 males in the 300 mg/kg/day group compared to 12/13 males in the control group. This isolated statistical difference was considered a spurious finding unrelated to treatment. There were no statistically significant differences when the test article-treated males and females were compared to the control group at the evaluation during the fourth week of dosing.

Hematology, serum chemistry and urinalysis parameters were unaffected by test article administration. Statistically significantly (p is less than 0.05) lower mean total protein values were observed in the 100 and 1000 mg/kg/day group males when compared to control group values. Statistically significant (p is less than 0.05 or p is less than 0.01) decreases were also observed in mean glucose levels in the 100 and 300 mg/kg/day group males and mean creatinine levels in the 1000 mg/kg/day group females. These differences were of minimal magnitude and not present in a dose-related manner and therefore, were not considered test article-related, but rather the result of normal biological variation. There were no other biologically relevant statistically significant differences when the control and test article-treated groups were compared.

There were no test article-related macroscopic findings at the scheduled necropsies. All macroscopic findings noted were considered to be spontaneous and/or incidental in nature and unrelated to test article administration.

There were no clear test article-related effects on organ weights. A statistically significant (p is less than 0.01) minimal decrease in relative (to final body weight) liver weight was observed in the 1000 mg/kg/day group males at the primary necropsy when compared to control group values. This change was potentially test article-related, but of unknown, if any, toxicological significance as no microscopic correlates were observed in conjunction with the lower liver weights. There were no other statistically significant differences when the control and test article-treated groups were compared.

There were no test article-related microscopic findings. All findings observed were consistent with normal background lesions in clinically normal rats of the age and strain used on this study and were considered spontaneous and/or incidental in nature and unrelated to test article administration.

Effect levels

Dose descriptor:

NOAEL

Effect level:

> 1 000 other: mg/kg/day

Sex:

male/female

Basis for effect level:

other: Maximum dose tested

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Summary of Body Weights for Recovery Males (g)

Group:	Dose (mg/kg/day)
	0	100	300	1000
Day 34
Mean	457.4	NA	NA	402.0*
S.D.	17.28			41.66
N	5			5
Day 41
Mean	478.0	NA	NA	419.4*
S.D.	21.21			44.45
N	5			5
* = Significantly different from the control group at 0.05 using Dunnett’s test S.D. = Standard deviation N = Number of animals NA = Not applicable

Summary of Body Weight Changes (g)

Group:	Dose (mg/kg/day)
	0	100	300	1000
Males
Days 0-7
Mean	63.6	59.7	50.2*	53.6*
S.D.	9.58	10.53	7.71	10.51
N	13	8	8	13
Recovery Males
Days 27-34
Mean	45.3	NA	NA	31.9**
S.D.	7.33			4.31
N	5			5
* = Significantly different from the control group at 0.05 using Dunnett’s test ** = Significantly different from the control group at 0.01 using Dunnett’s test S.D. = Standard deviation N = Number of animals NA = Not applicable

Summary of Food Consumption for Recovery Males (g)

Group:	Dose (mg/kg/day)
	0	100	300	1000
Day 27 - 34
Mean	30.5	NA	NA	27.5*
S.D.	1.57			2.25
N	5			5
* = Significantly different from the control group at 0.05 using Dunnett’s test S.D. = Standard deviation N = Number of animals NA = Not applicable

Summary of Select FOB Observations

Group:	Dose (mg/kg/day)
	0	100	300	1000
Open Field Observations
Females
N	13	8	8	13
Grooming
Mean	1.2	0.4*	0.1**	0.3**
S.D.	1.01	0.52	0.35	0.48
Sensory Observations
Males
N	13	8	8	13
Tail Pinch Response
No Reaction	1	0	3	0
Slowly turns, walks away	12	7	3*	10
More energetic response, with/without vocalization	0	1	2	3
* = Significantly different from the control group at 0.05 using Dunnett’s test ** = Significantly different from the control group at 0.01 using Dunnett’s test S.D. = Standard deviation N = Number of animals

Serum Chemistry Values

Group:	Dose (mg/kg/day)
	0	100	300	1000
Males (Day 28)
Total protein (g/dL)
Mean	6.4	6.1*	6.2	6.0*
S.D.	0.24	0.28	0.21	0.40
N	8	8	8	8
Glucose (mg/dL)
Mean	182.	142.**	142.**	162.
S.D.	23.7	17.8	13.0	31.3
N	8	8	8	8
Females (Day 28)
Creatinine (mg/dL)
Mean	0.2	0.2	0.2	0.2*
S.D.	0.05	0.7	0.04	0.05
N	8	8	8	8
* = Significantly different from the control group at 0.05 using Dunnett’s test ** = Significantly different from the control group at 0.01using Dunnett’s test S.D. = Standard deviation N = Number of animals

Organ Weights Relative to Final Body Weights (g/100 g)

Group:	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
Males
Final Body Weight (Day 28)
Mean	380.	374.	358.	367.
S.D.	28.6	25.0	31.6	33.0
N	8	8	8	8
Liver
Mean	3.255	3.036	3.046	2.926**
S.D.	0.2336	0.1727	0.2455	0.1086
N	8	8	8	8
Recovery Males
Final Body Weight (Day 42)
Mean	450.	NA	NA	391.*
S.D.	21.4			42.0
N	5			5
* = Significantly different from the control group at 0.05 using Dunnett’s test ** = Significantly different from the control group at 0.01 using Dunnett’s test S.D. = Standard deviation N = Number of animals NA = Not applicable

Applicant's summary and conclusion

Conclusions:

No clear evidence of test article-related effects was identified in this study. The decrease in relative liver weight in the 1000 mg/kg/day group males, in the absence of test article-related microscopic changes in the liver, is of unknown significance and is not considered to be an adverse effect.

Based on the results of this study, the no-observed-adverse-effect level (NOAEL) for oral (gavage) administration of PPP-BP to rats for 28 consecutive days was > 1000 mg/kg/day.

Executive summary:

The test substance was formulated and administered daily as a suspension in corn oil to rats by oral gavage at target doses of 0, 100, 300 and 1000 mg/kg/day for a minimum of 28 consecutive days. Control rats received corn oil alone. Test organisms were euthanatized following the 28-day period in the control and high dosage groups, following a further 2 -week post-treatment recovery period with gross necropsy effects examined. No clear evidence of test article-related effects was identified in this study within the range of test substance concentrations. The NOAEL (rat) was determined to be >1000 mg/kg/day.