Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

Carcinogenicity

Description of key information

Supporting studies

There are several available carcinogenicity studies on the main components rutoside and quercetin.

Two in vivo carcinogenicity experiments performed with quercetin and rutin did not show any carcinogenic activity in ACI rats at doses up to 10% in diet of each substance for both sexes (5000 mg/Kg) after 850 days (Hirono I, 1981).

Another in vivo carcinogenicity test performed also with quercetin and rutin did not show any carcinogenic activity in golden hamster forestomach or adrenal gland at doses up to 10% of each test item in diet for both sexes after 733 days (15 g/kg bw) (Morono K, 1982).

An in vivo carcinogenicity study performed in quercetin did not show significant increases in tumor incidence in rats at the single dose tested of 0.1% in diet for both sexes (50 mg/kg bw) (Takanashi H, 1983). Another in vivo carcinogenicity study performed in quercetin did not show significant increases in tumor incidence in rats in doses up to 5% in both sexes (Ito N, 1989).

However, in a 2-year in vivo carcinogenicity study performed with quercetin in Fischer 344/N rats at doses of 1000, 10000 and 40000 ppm in diet, the test item showed carcinogenic activity in the kidney of the male rat, causing primarily benign tumors of the renal tubular epithelium. The NOAEL of the test item was set at 10000 ppm for males and 40000 ppm for females (Dunnick JK, 1992, NTP 1992).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

Principles of method if other than guideline:

- Principle of test: Test item is widely distributed in natural foods, so it was administered to rats by dosed feed because human exposure is by dietary consumption.
- Short description of test conditions: Groups of 70 rats of each sex were administered 0, 1000, 10000 or 40000 ppm of the test item. Since 1000 ppm was the dose level used in the one study reporting carcinogenic results in rats, this concentration was selected as the low dose for these studies. Ten male and ten female rats per dose group were randomly selected and necropsied for interim evaluation after 6 months and 15 months of chemical administration.
- Parameters analysed / observed: carcinogenicity (NOAEL descriptor)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: from Freeman Industries (Tuckahoe, NY) in two lots.
- Purity, including information on contaminants, isomers, etc.: Both lots were greater than 95% pure. The largest impurity was identified as ellagic acid (2.6%)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stable as a bulk chemical for at least 2 weeks at temperatures to 60" C when protected from light in a nitrogen atmosphere. Based on the results of a stability study, the bulk chemical was stored at 0±5ºC throughout the study period. The bulk chemical was monitored periodically by the study laboratory using high-performance liquid chromatography and infrared spectroscopy. No change in the study material was detected.

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, MI)
- Age at study initiation: 7 weeks old
- Housing: Rats were housed five per cage. Solid-bottom polycarbonate cages (Lab Products, Inc., Rochelle Park, NJ). Aspen bed, heat-treated hardwood chips (American Excelsior Co., Baltimore, MD), changed twice weekly. Non-woven fiber filters (Snow Filtration, Cincinnati, OH)
- Diet: NIH47 Rat and Mouse Ration, Open formula, pellets (Zeigler Bros., Inc., Gardners, PA), available ad Iibitum
- Water: Tap water (City of Worcester Water Supply) via outside-the cage automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5 ± 1.5 ºC
- Humidity (%): 47.6% ± 5.8%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hours/day fluorescent light

IN-LIFE DATES:
6-month interim evaluation: Males: 28 -30December 1982; Females: 12 -14 January 1983.
15-month interim evaluation: Males: 28 -30 September 1983; Females: 12 -14 October 1983.
2-year studies: Males: 15 -21 June 1984; Females: 28 June -5 July 1984

Route of administration:

oral: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared by mixing appropriate amounts of test item and feed.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Chemical-feed premix prepared by grinding test item and feed with mortar and pestle; premix and remaining feed layered in a blender with intensifier bar and mixed for 15 minutes.
- Storage temperature of food: 4ºC

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Studies were conducted by the analytical chemistry laboratory to determine the homogeneity and stability of 10000 ppm test item in feed. Homogeneity was confirmed by UV-Vis. Stability of dose formulations stored at temperatures up to 25ºC for at least 14 days was confirmed by HPLC. During the studies, the dose formulations were stored in opaque plastic bags at approximately 4ºC for no longer than 2 weeks.
The study laboratory conducted periodic analyses of the test item dose formulations using UV-Vis. During the 2-year studies, the dose formulations were analyzed at approximately 8-week intervals and all formulations were within 10% of the target concentrations. Results of periodic referee analyses of the dose formulations performed by the analytical chemistry laboratory were in agreement with the results from the study laboratory.

Duration of treatment / exposure:

Main Study: 104 weeks. Interim studies: There were two groups that were tested for 6 and 15 months for interim evaluation

Frequency of treatment:

daily

Dose / conc.:

0 ppm

Remarks:

Control group

Dose / conc.:

1 000 ppm

Remarks:

Main and interim studies

Dose / conc.:

10 000 ppm

Remarks:

Main and interim studies

Dose / conc.:

40 000 ppm

Remarks:

Main and interim studies

No. of animals per sex per dose:

70 rats of each sex

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: These doses were selected based on the literature reports which showed that the test item administered in the diet at levels up to approximately 40000 ppm caused a minor body weight decrement, and that this effect was more severe at doses higher than this concentration. Since 1000 ppm was the dose level used in the one study reporting carcinogenic results in rats, this concentration was selected as the low dose for these studies.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: abnormalities, mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings were recorded weekly for 13 weeks and monthly thereafter.

DERMAL IRRITATION (if dermal study): No

BODY WEIGHT: Yes
- Time schedule for examinations: Rats were weighed at study initiation, once per week for 14 weeks, and once every 4 weeks thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined: Yes, weekly

FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the experiment (6 and 15 months for interim evaluations)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all of them
- Parameters checked: erythrocytes, total leukocyte count, leukocyte differential counts, and nucleated erythrocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the experiment (6 and 15 months for interim evaluations)
- Animals fasted: No data
- How many animals: all of them
- Parameters checked: blood urea nitrogen, creatinine, sodium, potassium, chloride, alanine aminotrans- ferase, aspartate aminotransferase, and sorbitol dehydrogenase

URINALYSIS: Yes
- Time schedule for collection of urine: One week prior to the 6-or 15-month interim evaluations, urine was collected over a 24-hour period
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: chloride, potassium, and sodium

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Animals found moribund, selected for the 6-or 15-month interim evaluations, or surviving to the end of the 2-year studies were killed. Necropsy was performed on all animals. At necropsy, all organs and tissues were examined for gross lesions.

HISTOPATHOLOGY: Yes
All major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic examination. Histopathology examinations of the tissues were performed according to an "inverse pyramid" design (McCoMell, 1983a,b). Complete histopathologic examinations were performed on all grossly visible lesions in all dose groups, on all control animals, and on animals receiving 40000 ppm. Selected histopathology examinations were performed on 1000 and 10000 ppm dose group animals dying before the end of the study period.

Statistics:

-Survival analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier. Statistical analyses for a possible dose-related effect on survival used the method of Cox for testing two groups for equality and Tarone’s life table test to identify dose- related trends. All reported P values for the survival analyses are two sided.
-Calculation of incidence: The incidence of neoplasms or nonneoplastic lesions is given as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which that site was examined. However, when macroscopic examination was required to detect lesions prior to histologic sampling, or when lesions had multiple potential sites of occurrence, the denominators consist of the number of animals on which a necropsy was performed.
-Analysis of neoplasm incidence: The primary statistical method used was a logistic regression analysish. In this approach, neoplasm prevalence was modeled as a logistic function of chemical exposure and time. Both linear and quadratic terms in time were incorporated initially, and the quadratic term was eliminated if it did not significantly enhance the fit of the model. The dosed and control groups were compared on the basis of the likelihood score test for the regression coefficient of dose. This method of adjusting for intercurrent mortality is the prevalence analysis of Dinse and Lagakos. When neoplasms are incidental, this comparison of the time-specific neoplasm prevalence also provides a comparison of the time- specific neoplasm incidences. In addition to logistic regression, alternative methods of statistical analysis were used, including the life table test (Cox; Tarone), and the Fisher exact test and the Cochran-Armitage trend test (Armitage; Gart), procedures based on the overall proportion of tumor-bearing animals. Continuity-corrected tests were usedin the analysis of tumor incidence, and reported P values are one sided

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A yellowish discoloration of the hair coat, especially in the perineal area, was present in all mid- and high-dose animals, presumably due to the urinary and/or fecal excretion of test item and/or its metabolites.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Exposure to test item had no significant effect on survival.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weights in females observed at high doses in in interim evaluations. Male and female rats given 40000 ppm test item in the main study had lower body weight gains than those of the controls.

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

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

presence of calcium oxalate crystals in 7 of 10 high-dose males at 15 months.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The relative kidney and liver weights of rats in both sexes that received 40000 ppm were
significantly greater than those of the controls at both 6 and 15 months.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the main study, nephropathy was significantly more severe in male rats receiving 40000 ppm than in the controls, whereas there was no significant increase in dosed female rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No non-neoplastic lesions related to test item administration in male or female rats at 6 or 15 months. In the main study, there was a dose-related increase in the incidence of renal pelvic transitional epithelial hyperplasia in males. This change is associated with severe nephropathy. Other non-neoplastic lesions occurred with statistically significant increased incidence, but the biological significance of their occurence is uncertain.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No neoplasms related to test item administration in male or female rats at 6 or 15 months. However, in the main study a few lesions were observed, particularly in male kidneys. Renal tubule neoplasms were seen in four high-dose male rats and slight dose-related increase in the incidence of renal tubule hyperplasia in males. There was no evidence of a chemical-related increased incidence in kidney neoplasms in females.
Fibroadenomas of the mammary gland occurred with a highly significant, dose-related, negative trend in female rats. Although the incidence of fibroadenoma in the high-dose group is about one-half of the mean rate of historical controls. The lower number of female rats with fibroadenomas in the high-dose group is considered chemically related, and may be associated with the lower body weights in this group. Uterine stromal polyps occurred more frequently in mid-dose female rats than in controls, but these neoplasms were not considered related to the test item.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Yellow-brown pigmentation occurred in several tissues and was most prevalent in the glandular stomach and the distal segments of the small intestine. The incidence and severity of pigmentation increased with dose concentration and duration. There was a significant dose-related accumulation of a fine granular yellow to light brown pigment in the epithelial cells lining the glandular stomach, jejunum, ileum, and, to a lesser extent, the duodenum and colon

Details on results:

High-dose female rats had a marginally increased incidence of chronic inflammation involving the liver and high-dose males had a reduced incidence of bile duct hyperplasia, an increased incidence of lymphatic ectasia within mesenteric lymph nodes, and a marginal dose-related increased incidence in testicular interstitial cell hyperplasia

Relevance of carcinogenic effects / potential:

The increased incidences of renal tubule hyperplasia and renal tubule neoplasms in high-dose males is supportive of some evidence of carcinogenicity.

Key result

Dose descriptor:

NOAEL

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: equivalent to 500 mg/kg bw

Key result

Dose descriptor:

NOAEL

Effect level:

40 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no significant effects observed

Remarks on result:

other: equivalent to 2000 mg/kg bw

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

40 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1. Survival of Rats in the 2-YearFeed Studies of test item.

	0 ppm	1000 ppm	10000 ppm	40000 ppm
Male
Animals initially in study	70	70	70	70
6-Month interim evaluation^a	10	10	10	10
15-Month interim evaluation^a	10	10	10	10
Natural deaths	3	7	3	6
Moribund kills	21	15	22	21
Animals surviving until end of the study	26	28	25	23
Percent survival at end of study^b	52	56	50	47
Mean survival (days)^c	576	581	577	576
survival analyses^d	P=0.603	P=0.838N	P=0.940	P=0.824
Female
Animals initially in study	70	70	70	70
6-Month interim evaluation^a	10	10	10	10
15-Month interim evaluation^a	10	10	10	10
Natural deaths	1	4	2	3
Moribund kills	19	18	13	19
Animals surviving until end of the study	30	28	35	28
Percent survival at end of study^b	60	56	71	56
Mean survival (days)^c	590	574	586	576
survival analyses^d	P=0.709	P=0.612	P=0.445N	P=0.656

^aCensored from survival analyses

^bKaplan-Meier determinations. Survival rates adjusted for interim evaluations.

^cMean of all deaths (uncensored, censored, terminal sacrifice).

^dThe result of the life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparisons (Cox, 1972) with the controls are in the dosed columns. A lower mortality in a dose group is indicated by N.

 

Table 2. Selected Kidney Lesions in Male Rats in the 2-Year Feed Study of test item.

	0 ppm	1000 ppm	10000 ppm	40000 ppm
Initial Evaluation (Single Sections)
Renal Tubule: Hyperplasia
Overall rates^a	1/50 (2%)	2/50 (4%)	3/50 (6%)	4/50 (8%)
Logistic regression^b	P=0.079	P=0.752N	P=0.492	P=0.182
Renal Tubule: Adenoma
Overall rates	0/50 (0%)	0/50 (0%)	0/50 (0%)	3/50 (6%)
Adjusted rates^c	0.0%	0.0%	0.0%	11.1%
Terminal rates^d	0/26 (0%)	0/28 (0%)	0/25 (0%)	2/23 (9%)
First incidence (days)	-^e	-	-	676
Logistic regression	P=0.042	-	-	P=0.122
Renal Tubule: Adenocarcinoma
Overall rates	0/50 (0%)	0/50 (0%)	0/50 (0%)	1/50 (0%)
Renal Tubule: Adenoma or Adenocarcinoma^f
Overall rates	0/50 (0%)	0/50 (0%)	0/50 (0%)	4/50 (8%)
Adjusted rates^c	0.0%	0.0%	0.0%	15.3%
Terminal rates^d	0/26 (0%)	0/28 (0%)	0/25 (0%)	3/23 (13%)
First incidence (days)	-	-	-	676
Logistic regression	P=0.002	-	-	P=0.064
Evaluation of Step Sections
Renal Tubule: Hyperplasia
Overall rates	2/50 (4%)	2/50 (4%)	6/50 (12%)	8/50 (16%)
Renal Tubule: Adenoma
Overall rates	1/50 (2%)	2/50 (4%)	7/50 (14%)	6/50 (12%)
Single and Step Sections Combined
Renal Tubule: Hyperplasia
Overall rates	3/50 (6%)	3/50 (6%)	8/50 (16%)	11/50 (22%)
Logistic regression	P=0.006	P=0.655	P=0.099	P=0.022
Renal Tubule: Adenoma
Overall rates	1/50 (2%)	2/50 (4%)	7/50 (14%)	8/50 (16%)
Logistic regression	P=0.012	P=0.526	P=0.032	P=0.018
Renal Tubule: Adenoma or Adenocarcinoma
Overall rates	1/50 (2%)	2/50 (4%)	7/50 (14%)	9/50 (18%)
Logistic regression	P=0.005	P=0.526	P=0.032	P=0.010

^aNumber of lesion-bearing animals/number of animals examined at site

^bBeneath the control incidence are the P values associated with the trend test. Beneath the dosed group incidence are the P values correspondingto pairwise comparisons between the controls and that dosed group. The logistic regression tests regard these lesions as nonfatal. A lower incidence in a dose group is indicated by N.

^cKaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality

^dObserved incidence at terminal kill
^eNot applicable; no neoplasms in animal group

^fHistorical incidence for 2-year NTP feed studies with untreated control group (mean ± standard deviation): 4/499 (0.8% ± 1.1%, range 0%-4%)

 

Table 3. Incidences and Severity of Nephropathy in Male and Female Rats in the 2-Year Feed Studies of test item.

	0 ppm	1000 ppm	10000 ppm	40000 ppm
Male
Minimal (grade 1)	1/48	2/50	1/50	2/49
Mild (grade 2)	18/48	19/50	13/50	7/49
Moderate (grade 3)	19/48	20/50	23/50	16/49
Marked (grade 4)	10/48	9/50	13/50	24/49
Average severity grade	2.7±0.14	2.7±0.11	3.0±0.11	3.2±0.14^**
Female
Minimal (grade 1)	12/48	7/48	9/50	8/48
Mild (grade 2)	20/48	25/48	30/50	21/48
Moderate (grade 3)	13/48	12/48	10/50	14/48
Marked (grade 4)	3/48	4/48	1/50	5/48
Average severity grade	2.1±0.13	2.2±0.12	2.1±0.10	2.2±0.14

^**Statistically significant (P≤0.01)from the control group by the Mann-Whitney U test
^aNumber of animals with severity grade/number of animals with nephropathy. Severity grade was based on the percentage of parenchyma involved: Minimal -usually less than 25% of cortex; mild -25%to 50% of cortex; moderate -50% to 75% of cortex; marked -greater than 75% of cortex. Average seventy grade given as the mean ± standard error.

 

Table 4. Mammary Gland Neoplasms in Female Rats in the 2-Year Feed Studies of test item.

	0 ppm	1000 ppm	10000 ppm	40000 ppm
Fibroadenoma^a
Overall rates^b	29/50 (58%)	27/50 (54%)	16/50 (32%)	9/50 (18%)
Adjusted rates^c	66.4%	72.3%	38.4%	30.1%
Terminal rates^d	16/30 (53%)	18/28 (64%)	10/35 (29%)	8/28 (29%)
First incidence (days)	597	597	605	549
Logistic regression tests^c	P<0.001N	P=0.553N	P=0.008N	P<0.001N

^aHistorical incidence for 2-year NTP feed studies with untreated control group (mean ± standard deviation): 178/500 (35.6% ± 15.0%, range 8%-56%)

^bNumber of neoplasm-bearing animals/number of animals necropsied

^cKaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality

^dObserved incidence at terminal kill
^eBeneath the control incidence are the P values associated with the trend test. Beneath the dosed group incidence are the P values correspondingto pairwise comparisons between the controls and that dosed group. The logistic regression tests regard these lesions as nonfatal. A lower incidence in a dose group is indicated by N.

Conclusions:

The no-observed-adverse-effect level (NOAEL) of the test item in rat was 10000 ppm (500 mg/kg bw) in diet for males and 40000 ppm (2000 mg/kg bw) in diet for females.

Executive summary:

An in vivo carcinogenicity study was conducted with the test item in Fischer 344/N rats. The administration of the test item was achieved orally, by mixing it with the diet in three different concentrations : 1000, 10000 and 40000 ppm. Also, a control group was monitored. 70 males and 70 females were distributed per group and were administered the test item on a daily basis for 104 weeks. Two interim evaluations were also carried out for 6 and 15 months, with 10 males and 10 females for each one.

Survival, clinical sign, haematological, clinical-chemistry and urinalysis examinations were performed with no relevant adverse effects observed. High-dose group in both sexes had reduced body weight gain in comparison to controls. Necropsy was performed for all surving animals and the organ relative weights calculated. The relative kidney and liver weights of rats in both sexes that received 40000 ppm were significantly greater than those of the controls at both 6 and 15 months. After two years of treatment, hispathological examinations revealed an increase of renal tubule hyperplasia and the severity of nephropathy in exposed male rats. Therefore, the no-observed-adverse-effect level (NOAEL) of the test item in rat was 10000 ppm (500 mg/kg bw) in diet for males and 40000 ppm (2000 mg/kg bw) in diet for females. (conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives). The increased incidence of renal tubule cell adenomas in high-dose males is supportive of some evidence of carcinogenicity.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The administration of the test item was achieved orally, by mixing it with the diet in three different concentrations : 1000, 10000 and 40000 ppm. Also, a control group was monitored. 70 males and 70 females were distributed per group and were administered the test item on a daily basis for 104 weeks.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Freeman Industries (Tuckahoe, NY). Lots 969-3790-05 and 969-0483-18BL. The first lot was used during the first year of the study and the second lot during the last half of the study.
- Purity, including information on contaminants, isomers, etc.: >95%. The largest impurity was ellagic acid (1.1-2.6%)

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, MI)
- Age at study initiation: 7 week old
- Weight at study initiation: Males: 155.5±2.2 - 161.8±2.5 g / Females: 138.2±1.4 - 140.8±1.2 g
- Housing: Animals were assigned to groups using a table of random numbers, were housed by sex, five per cage, in polycarbonate cages covered with fiber filters, and were provided with heat-treated hardwood chips as bedding (American Excelsior, Co., Baltimore, MD).
- Diet: NIH 07 feed (Zeigler Bras., Gardners, PA) ad libitum
- Water: Tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C):21-24ºC
- Air changes (per hr):12
- Photoperiod (hrs dark / hrs light): 12h-fluorescent light cycle.

Route of administration:

oral: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item was mixed in the feed to obtain concentrations of 1000, 10,000 or 40,000 ppm.

DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 weeks
- Mixing appropriate amounts with (Type of food): feed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the 2-year studies, the dose formulations were analyzed at approximately 8-week intervals and all formulations were within ±10% of the target concentrations

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

daily

Dose / conc.:

1 000 ppm

Dose / conc.:

10 000 ppm

Dose / conc.:

40 000 ppm

No. of animals per sex per dose:

70

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: These doses were selected based on information in the literature which showed that the test item administered in the diet at levels of more than 4% caused a significant decrease in body weight (Hirono et al., 1981). A low dose of 1000 ppm was included in this study, because this was the dose level used by Pamucku et al, 1980. Ten male and ten female rats per dose group were randomly selected and necropsied for interim evaluation after 6 and 15 months of chemical administration.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a week during the first 13 weeks of study and thereafter every 4 weeks.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the 6th and 15th month from the rectroorbital sinus.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all of them
- Parameters checked: Erythrocytes, Leukocytes, Segmented neutrophils, Lymphocytes, Monocytes, Eosinophils, Nucleated erythrocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Not specified
- How many animals: all of them
- Parameters checked: BUN, Creatinine, Sodium, Potassium, Chloride, SDH.

PLASMA/SERUM HORMONES/LIPIDS: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy was performed on all animals. The initial evaluation of the kidney consisted of a single section from each of the right and left kidneys. Because of the low number of tumors in the high-dose male rats additional kidney sections were taken from the remaining half of the formalin-fixed kidneys from the control and treated rats. In this evaluation additional sections were taken at approximately 1mm intervals from both (right and left) remaining kidney halves. This provided six to eight additional kidney sections per animal for microscopic examination.

HISTOPATHOLOGY: Yes
Tissues were preserved in 10% neutral-buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. At the 6- and 15-months interim termination periods complete histopathology was performed on control and high-dose animals. At 2 years complete histopathology was performed on all control and high-dose animals, on all animals dying during the course of the study, and on selected target organs in the lower dosed groups. Complete histopathology included the examination of adrenal, brain, cecum, colon, duodenum, epididymis, heart, ileum, jejunum, kidney, liver, lungs, lymph nodes, nasal cavity and turbinates. ovaries, pancreas, parathyroid, pituitary, prostate, rectum, seminal vesicle, spleen, stomach, testes, thyroid, trachea, urinary bladder, uterus, and gross lesions and tissue masses. The selected tissues examined in the low-dose groups included kidney, liver, pancreas, parathyroid, pituitary gland, small intestine, tongue, urinary bladder, and uterus.

Statistics:

Differences in survival were analyzed by life-table methods. For the analysis of tumor incidence data, survival adjusted procedures were used to assess dose-response trends and to make pairwise comparisons between dosed groups and controls. Fisher exact tests and Cochran-Armitage tests were also utilized to assess tumor incidence data. Reported p values for tumor incidence are one-sided. For all hematologic and clinical chemistry variables, significant differences between animals in treated and control groups were analyzed by nonparametric comparisons methods of Shirley ( 1977) and Dunn (1964).

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There are no treatment-related effects on survival in these rodent studies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High-dose group had reduced body weight gain in comparison to controls during the last half of the study and the final mean bw of both sexes in the high-dose were 87% of the corresponding controls

Food consumption and compound intake (if feeding study):

not examined

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Pigmentation of the superficial epithelium of the glandular stomach and the distal segments of the small intestine.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

-Kidney: there was a statistically significant increase in hyperplasia and neoplasia of the renal tubule epithelium as well as mild exacerbation of the chronic nephropathy in males of the treated groups when compared to the control group.
-Kidney: increase in the severity of nephropathy in males relative to controls in dosed animals.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- Males also had a dose-related increase in renal pelvic epithelial hyperplasia and parathyroid gland hyperplasia.
- Fibroadenomas of the mammary gland occurred with a significant dose-related negative trend in female rats, but the incidence rates in all groups were within the historical range for these tumors.

Key result

Dose descriptor:

NOAEL

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 410 mg/kg bw

Key result

Dose descriptor:

NOAEL

Effect level:

40 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no significant effects observed

Remarks on result:

other: equivalent to 1900 mg/kg bw

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The no-observed-adverse-effect level (NOAEL) of the test item in rat was 10000 ppm (410 mg/kg bw) in diet for males and 40000 ppm (1900 mg/kg bw).

Executive summary:

An in vivo carcinogenicity study was conducted with the test item in Fischer 344/N rats. The administration of the test item was achieved orally, by mixing it with the diet in three different concentrations : 1000, 10000 and 40000 ppm. Also, a control group was monitored. 70 males and 70 females were distributed per group and were administered the test item on a daily basis for 104 weeks.

Survival, clinical sign, haematological and clinical-chemistry examinations were performed with no relevant adverse effects observed. High-dose groups had reduced body weight gain in comparison to controls during the last half of the study. Necropsy was performed for all surving animals and the organ relative weights calculated. After two years, hispathological examinations revealed neoplastic lesions in the kidneys of male rats, including increased severity of chronic nephropathy, hyperplasia, and neoplasia of the renal tubular epithelium. Under these conditions, the test item showed carcinogenic activity in the kidney of the male rat, causing primarily benign tumors of the renal tubular epithelium. Therefore, the no-observed-adverse-effect level (NOAEL) of the test item in rat was 10000 ppm (410 mg/kg bw) in diet for males and 40000 ppm (1900 mg/kg bw) in diet for females.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Experiment 1: A group of 20 males and 20 females of hamsters were administered the test item orally with the diet in a daily manner for 733 days. This group received a dose of 10% test item in diet. A control one (basal diet) was also tested.
Experiement 2: The hamsters were divided into five groups: G1, 4% test item diet for 709 days. G2, 1% test item diet for 351 days and then basal diet, CE-2, for 350 days. G3, 1% test item diet for 351 days and then 1% croton oil diet for 350 days. G4, basal diet for 351 days and then 1% croton oil diet for 350 days. G5, basal diet for 701 days.
Body weight changes and the presence of tumors in forestomach and adrenal gland were investigated in both experiments

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan.
- Purity, including information on contaminants, isomers, etc.: 95% pure;

Species:

hamster

Strain:

other: Golden

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Cooperative Society of Experimental Animals, Shizuoka, Japan
- Age at study initiation: 6 weeks
- Weight at study initiation: 75 - 80 g
- Assigned to test groups randomly: yes
- Housing: They were housed 5 to a cage
- Diet: ad libitum
- Water: ad libitum

Route of administration:

oral: feed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of test item in diets:

The pellet diets used in this study were ground in a mortar. Two grams of the diet was suspended in 20 ml of methanol and shaken for 5 min at room temperature. Then the suspensions were centrifuged at 3000 r.p.m. for 10 min, and the precipitates were extracted twice with 20 ml of methanol. The pooled methanol extracts were evaporated to dryness and the dried materials were dissolved in methanol for measurement of quercetin and test item by HPLC ( Conditions: instrument, Toyo Soda HLC-803A; column, LS-410 ODS SIL, 4 mm ID x 300 mm; mobile phase, 60% methanol-0. 2% acetic acid for test item; temperature, ambient; flow rate, 1.0 ml/min; detector, UV366 ran for the test item. The concentrations of the test item was 10.6% ± 0.73 (Mean ± S.D.) in 10% test item diet. The test item content of basal diet, CE-2, was <0.0002%.

Duration of treatment / exposure:

733 days for experiment 1 and 351 or 709 days for experiment 2

Frequency of treatment:

daily

Dose / conc.:

0 other: % in diet

Remarks:

Experiment 1 (733 days) - Group 3- control.
Experiment 2 (701 days) - Group 4- control (351d) + 1% croton oil and Group 5- control (350d)

Dose / conc.:

10 other: % in diet

Remarks:

Experiment 1 (733 days) - Group 1

Dose / conc.:

4 other: % in diet

Remarks:

Experiment 2 (709 days) -Group 1

Dose / conc.:

1 other: % in diet

Remarks:

Experiment 2 (701 days)- Group 2 -test item (351d) + basal diet (350d) and Group 3 -test item (351d)+ 1% croton oil (350d)

No. of animals per sex per dose:

Experiment 1: 20 males and 20 females per each group;
Experiment 2: G1, 15 males and 15 females; G2, 7 males and 7 females; G3, 8 males and 8 females; G4, 7 males and 7 females; G5, 8 males and 8 females

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: mortality

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: once a month

DERMAL IRRITATION (if dermal study): No
FOOD CONSUMPTION AND COMPOUND INTAKE: No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
OTHER: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Forestomach, adrenal gland

HISTOPATHOLOGY: Yes
All animals were carefully autopsied when they died or were killed because they became moribund or at the end of the experiment. Intestine was opened longitudinally and Swiss roll technique was used for the histological examination. The urinary bladder was filled with 0.5-1.0 ml of 15% buffered formalin for fixation, then it was cut vertically. The tissues were fixed in 15% buffered-formalin, embedded in paraffin, processed, and stained routinely with hematoxylin and eosin. Other organs were also examined histologically in all animals.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Exp. 1: The survival rate on day 480 was 55% in females of control Group 3 but >70% in males in Group 3 and in both sexes in all groups.
Exp. 2: The survival curves of Groups 1-5 were not significantly different throughout the experiment

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Exp. 1: There was no significant difference in the body weight increases in groups given 10% test item diet, and basal diet.
Exp 2: There was no significant difference in the body weight in males, but in females of groups 1-3 had slightly larger body weights than Groups 4 and 5 during the initial 351 days, when
the animals in Groups 1 — 3 were receiving test item diets

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Exp 1: all the tumors in the forestomach were papillomas, and tumors in adrenal were cortical adenomas. However, there were no significant differences in the incidences in groups fed on 10% test item diet, and basal diet.
Exp 2: All the tumors found were in the forestomach except for one leiomyoma of the uterus in a female in G1 on day 709 and a cortical adenoma of the adrenal in a male of G5 on day 671 and a female of G4 on day 701. Papillomas of the forestomach were found in 7% in G1, 14% in G2, and 7% in G5. G3 and G4 had a higher tumor incidence, as they were both treated with croton oil, but they were not significantly different between them.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

10 other: % n diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: equivalent to 15000 mg/kg bw

Key result

Critical effects observed:

no

Table 1. Mean cumulative intakes of test item and incidences of tumors of hamsters

Experimental group	Dietary treatment	Sex	Estimated cumulative intake (g) /hamster	No. of hamsters used	Effective No.	Papilloma in the forestomach	Cortical adenoma in the adrenal	Others
			Test item
1	10% Test item	M	680	20	16^a	3(19)^b	2(13)^c	0
		F	671	20	16	2(13)	0(0)	1 (adenocarcinoma of the ileum)
		Total		40	32	5(16)	2(6)	1
3	Basal diet	M	<0.013	20	15	1(7)	2(13)	0
		F	<0.013	20	11	2(18)	1(9)	3 (mesothdioma, cortical adenocarcinoma of the adrenal, granulosa-theca cell tumor)
		Total		40	26	3(12)	3(12)	3

^aNumbers of survivors for >480 experimental days.
^b,cNumbers in parentheses show the percentages of tumor bearing animals.

 

Table 2. Mean cumulative intakes of test item and incidences of tumors of hamsters in experiment II.

Experimental group	Dietary treatment	Sex	Estimated cumulative intake (g) /hamster		No. of hamsters used	Effective No.	Papilloma in the forestomach	Others
			Test item	Croton oil
1	4% Test item (709 days)	M	246	0	15	15^a	0(0)^b	0
		F	244	0	15	12	2(7)	1 (leiomyoma of the uterus)
		Total			30	27	2(7)	1
2	1% Test item (351 days), then Basal diet (350 days)	M	35	0	7	7	2(29)	0
		F	37	0	7	7	0(0)	0
		Total			14	14	2(14)	0
3	1% Test item (351 days), then 1% croton oil (350 days)	M	35	31	8	7	6(86)	0
		F	34	25	8	7	1(14)	0
		Total			16	14	7(50)	0
4	Basal diet (351 days), then 1% croton oil (350 days)	M	<0.007	29	7	7	3(43)	0
		F	<0.007	27	7	7	1(14)	1 (cortical adenoma of the adrenal)
		Total			14	14	4(29)	1
5	Basal diet (701 days)	M	<0.013	0	8	8	1(13)	1 (cortical adenoma of the adrenal)
		F	<0.013	0	8	7	0(0)	0
		Total			16	15	1(7)	1

^aNumbers of survivors for >480 experimental days.
^bNumbers in parentheses show the percentages of tumor bearing animals.

Conclusions:

The in vivo carcinogenicity test for the test item did not show any carcinogenic activity in golden hamster forestomach or adrenal gland at doses up to 10% of the test item in diet for both sexes after 733 days (15 g/kg bw) in experiment I and also at doses up to 4% in diet in experiment II.

Executive summary:

Two in vivo carcinogenicity experiments in Golden hamsters were performed for the test item. In the first experiment, a group of 20 males and 20 females were administered the test item orally with the diet in a daily manner for 733 days. This group received a dose of 10 w/w% test item in diet. A control group was also tested (basal diet). In the second experiment, five groups were tested: G1, 4% test item diet for 709 days. G2, 1% test item diet for 351 days and then basal diet, CE-2, for 350 days. G3, 1% test item diet for 351 days and then 1% croton oil diet for 350 days. G4, basal diet for 351 days and then 1% croton oil diet for 350 days. G5, basal diet for 701 days. Body weight changes and the presence of tumors in forestomach and adrenal gland were investigated for all groups in both experiments.

In the first experiment, there was no significant difference in the body weight increases between the groups and even though tumors were found in both groups, it was concluded that there was no increase in the incidence of tumors by the test item and the no-observed-adverse-effect level (NOAEL) of the test item in Golden Hamsters is 10 % in diet (15 g/Kg bw) for both sexes. (conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives taking into account the mouse data).

In the second experiment, almost all tumors were found in the forestomach. Papillomas of the forestomach were found in 2 of 27 animals (7%) in Group 1, 2 of 14 animals (14%) in Group 2, and 1 of 15 animals (7%) in Group 5. There were no significant differences in the incidences of papillomas in test item treated groups and the control group. Groups 3 and 4 had a higher tumor incidence, but since they were not significantly different, treatment with test item had no effect on the incidence of papillomas. 

Reference 4

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two experiments were made on the carcinogenicity of flavonoids. In Experiment I, 110 ACI rats of 1-1.5 months of age were divided into 4 groups. In Experiment II, 146 rats of the same strain and age were divided into 3 groups. Rats in each group were given the prescribed diets until the end of the experiment, for 540 days in Experiment I and for 850 days in Experiment II.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan.
- Purity, including information on contaminants, isomers, etc.: 99.0% pure;

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of flavonoids in the 10% diet was examined after preservation for 2 months, i.e., the test item was extracted with 90% ethanol, purified by thin-layer chromatography (TLC), and determined by UV spectrum. Test item showed the same RF values as their authentic samples on TLC, and the recovery was 108 ± 10% for test item pellets. Thus, no sign of the degradation of the test item was observed.

Species:

rat

Strain:

other: ACI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 1-1.5 months
- Assigned to test groups randomly: yes
- Diet: CE-2 (CLEA Japan Inc., Tokyo, Japan)
- Water: ad libitum

Route of administration:

oral: feed

Details on exposure:

The rats were given pellets which were prepared by mixing quercetin and test item with basal diet CE-2 using a ‘California pellet mill’. The ground material was dampened by steaming at 100--110°C for 30 s, and the pellets were then compressed and dried.

Duration of treatment / exposure:

540 days in Experiment I and for 850 days in Experiment II.

Frequency of treatment:

daily

Dose / conc.:

1 other: % in diet

Remarks:

Experiment I

Dose / conc.:

5 other: % in diet

Remarks:

Experiment I

Dose / conc.:

10 other: % in diet

Remarks:

Experiment II

No. of animals per sex per dose:

Experiment I: 11 males and 10 females treated, 30 males and 22 females for control.
Experiment II: 20 males and 20 females treated, 33 males and 33 females for control.

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: mortality

DETAILED CLINICAL OBSERVATIONS: No
DERMAL IRRITATION (if dermal study): No

BODY WEIGHT: Yes
- Time schedule for examinations: not specified

FOOD CONSUMPTION AND COMPOUND INTAKE: No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
cecum, colon, adrenal glands, adrenal medulla, pituitary gland, urinary bladder, testis, breasts, uterus

HISTOPATHOLOGY: Yes
Tissues were fixed in 10% formalin, sectioned, and stained with hematoxylin and eosin.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

For the 1% group, 19 of 20 rats survived for more than 335 days and 17 survived until the end of experiment I, whereas in group 2 all rats survived for more than 388 days and 14 survived until the end of the experiment . In experiment II, 37 of 40 rats survived for more than 349 days and 27 for more than 600 days.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant change of body weights were shown between the experimental and the control group in both experiments.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In both experiments, adrenal cortical adenomas, pituitary adenomas, urinary bladder papillomas and interstitial cell tumors of the testis were found in both the experimental and control groups but there was no significant difference in the incidence of these tumors in the experimental
and control groups (P > 0.05).

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

10 other: % in diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no significant effects observed

Remarks on result:

other: equivalent to 5000 mg/kg bw

Key result

Critical effects observed:

no

Table 1. Tumors in the 5 groups of rats in experiment 1

Group	Diet	Sex	Initial no.of rats	Effective^a no. of  rats	No. of rats with tumors
					Cecal adenoma	Adrenal cortical adenoma	Pheochromocytoma	Pituitary adenoma	Urinary bladder papilloma	testis interstitial cell tumor
1	1% Test item	M	10	10						1
		F	10	9
2	5% Test item	M	8	8	1	1	1			3
		F	9	9
3	5% rutin	M	11	11		2				3
		F	10	9				1	1
4	Control	M	30	29		2		1	1	3
		F	22	21			1

^a No, of rats surviving for more than 300 days. M, male; F, female.

 

Table 2. Tumors in the 3 groups of rats in experiment 2

Group	Diet	Sex	Initial no. of rats	Effective no. of rats	No. of rats with tumors
					Ileal adenoma	Cecal		Colonic adenoma	Adrenal cortical adenoma	Pheochromocytoma	Pituitary adenoma	Urinary bladder		Breast fibroadenoma	Testis interstitial cell tumor	Uterus adenocarcinoma	Miscellaneous
						Adenoma	Adenocarcinoma					Papilloma	Carcinoma
1	10% Test item	M	20	19		1	2		3						6		1^a
		F	20	18					1		2					1	1^b
2	10% Rutin	M	20	20	1			1	1						6
		F	20	19						1				1		1	1^c
3	Control	M	33	30					3				2		10
		F	33	31					1		3	1	1	2		4	1^d

^a Pancreatic exocrine adenoma.
^b Sarcoma of the thigh.
^c Heart sarcoma.
^d Renal pelvic papilloma.

Conclusions:

Two in vivo carcinogenicity experiments for the test item did not show any carcinogenic activity in ACI rats at doses up to 10% in diet for both sexes (5000 mg/Kg) after 850 days.

Executive summary:

An in vivo carcinogenicity test in ACI rats was performed for the test item. Two different experiments were carried out, where the animals were orally administered: I) With 5% content of test item and rutin in meal for 540 days (daily), and II) With 10% content of test item and rutin in meal for 850 days (daily). Control groups were used in both experiments. Cecum, colon, adrenal glands, adrenal medulla, pituitary gland, urinary bladder, testis, breasts and uterus were investigated for tumors at the end of the experiments. In all experimental groups, no significant change of body weights or tumoral activity was observed with the control group (P > 0.05). Therefore, it was concluded that there was no increase in the incidence of tumors by the test item and the no-observed-adverse-effect level (NOAEL) of the test item in ACI rats is 10% in diet for both sexes (5000 mg/Kg bw, conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives).

 

Reference 5

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

-Principle of test: Clarify the equivocal carcinogenicity, particularly for the ileum, urinary bladder and liver of the test item quercetin and evaluate its toxic potential.
- Short description of test conditions: The administration of the test item was achieved orally, by mixing it with the diet in two different concentrations : 1.25 and 5.0 %. Also, a control group was monitored. 50 males and 50 females were distributed per group and were administered the test item on a daily basis for 104 weeks and then normal diet for a further 8 weeks.
- Parameters analysed / observed: Incidences of tumors.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) of test material: supplied by the National Institute of Hygienic Sciences, Tokyo and munfactured by Tokyo Kasei Co Ltd, Tokio.
- Purity: 99.4%

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 6 weeks old
- Housing: Five animals to a plastic cage with hardwood chips for bedding.
- Diet: CRF-1 Diet (Charles River Japan, Inc., Kanagawa)

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22±2ºC
- Humidity (%):60±10%
- Air changes (per hr):15 per hour
- Photoperiod (hrs dark / hrs light):12h light/dark cycle

Route of administration:

oral: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item was incorporated into powdered CRF-1 diet at concentrations of 0, 1.25 and 5.0%. Analyses of the samples showed that the actual levels of the test item in food, nominally containing 1.25% and 5.0%, were 1.24±0.04 and 5.02±0.15%, respectively.

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): CRF-1 Diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the samples showed that the actual levels of test item in food, nominally containing 1.25 and 5.0%, were 1.24±0.04 and 5.02±0.15% (analyzed by Japan Food Research Laboratories)

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

daily

Post exposure period:

8 weeks with normal diet

Dose / conc.:

0 other: % in diet

Dose / conc.:

1.25 other: % in diet

Dose / conc.:

5 other: % in diet

No. of animals per sex per dose:

50 males and 50 females per dose

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: abnormalities, mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 14 weeks and then every other week.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: measured over 2-day period before each weighing.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: measured over 2-day period before each weighing.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 112 weeks
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: erythrocyte counts, leukocyte counts, measurement of hemoglobin concentrations and hematocrit values, platelet counts and differential leukocyte counts.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 112 weeks
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: glutamic-pyruvic transaminase, alkaline phosphatase, total cholesterol, total protein, albumin:globulin ratio, urea nitrogen, glucose and albumin.

URINALYSIS: Yes
- Time schedule for collection of urine: During week 112 urine samples were obtained from 10 rats in each group.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked: protein, glucose, bilirubin, ketones, occult blood and urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
They were performed at autopsy, and detailed examinations of the luminar surfaces of intestines and urinary bladder were also carried out using a dissecting microscope after fixation. the following organs of each rat were weighed and organ-to-body weight ratios were determined: brain, heart, liver, spleen, kidneys, adrenals and testes or ovaries.

HISTOPATHOLOGY: Yes
Samples of the above mentioned organs and of the salivary glands, trachea, lungs, thymus, lymph nodes, stomach, small intestines, large intestines, pancreas urinary bladder, pituitary, thyroid, adrenals, prostate, seminal vesicle, epididymis, skin, mammary gland, skeletal mucle, eyes, Harderian glands, spinal cord, sciatic nerve and any other tissues of abnormal appearance were fixed in 10% buffered formalin. Preserved tissues to be examined microscopically were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. Histopathological examinations were also performed on rats that died spontaneously and those were killed upon becoming moribund.

Statistics:

Data on tumor incidence were analyzed by the two-sided Fisher's exact probability test. Other data were analyzed using Student's t test.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no significant differences in mortality between controls and treated animals

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights were significantly reuced in both sexes receiving the 5.0% dose from week 1 to the termination, and in 1.25% males and females sporadically.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Although statistically significant increases of relative organ weights of brain in both sexes at 5%, and of the kidneys at 5% in the male group were observed, all values were within the normal range of this rat strain and seemed to be related to the reduced body weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

a higher incidence of raised areas of the cecum, which occurred as single lesions, was recorded in the 5.0% males when compared to the controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The hyperplastic polyps of the cecum which were found in both sexes receiving the 5.0% dose, and at statistically significant incidences in males, appeared to correlate with the raised areas observed on detailed gross examination with a dissecting microscope. The several instances ofreduced non-neoplastic lesion incidences which achieved statistical significance might be related to the test item, but the biological significance remains unclear.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Adenocarcinomas of the small intestine were found in a control and the 1.25% groups in males. An adenoma and two adenocarcinomas of the cecum in the 5% males as well as two colon adenomas in the 5% females were observed. However the differences from the zero control values were not statistically significant.

Other effects:

no effects observed

Relevance of carcinogenic effects / potential:

The present investigation of the carcinogenicity of the test item revealed a treatment-related growth retardation suggesting toxicity, althogh there were no biologically significant findings with regard to other clinical signs, survival rate, urinalyses, hematology, and relative organ weights.

Key result

Dose descriptor:

NOAEL

Effect level:

1.25 other: % in diet

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

Remarks on result:

other: equivalent to 427 mg/kg bw

Key result

Dose descriptor:

NOAEL

Effect level:

5 other: % in diet

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no significant effects observed

Remarks on result:

other: equivalent to 2372 mg/kg bw

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 other: %diet

System:

gastrointestinal tract

Organ:

other: Cecum

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1. Noneoplastic-proliferative and neoplastic lesions developing in the intestines, liver and urinary bladder of F344/DuCrj rats fed test item-containing diet.

Site and type of tumor	Males			Females
	0%	1.25%	5.0%	0%	1.25%	5.0%
Effective no.of rats	50	50	50	50	50	50
Small intestine
Adenocarcinoma	1(2)	1(2)	0	0	0	0
Leiomyoma	1(2)	0	0	0	0	0
Cecum
Hyperplastic polyp	0	0	11(22)^a	0	0	2(4)
Inflammatory polyp	0	0	0	0	0	1(2)
Adenoma	0	0	1(2)	0	0	0
Adenocarcinoma	0	0	2(4)	0	0	0
Sarcoma, NOS	0	0	1(2)	0	0	0
Colon
Adenoma	0	0	0	0	0	2(4)
Liver
Focus (area) of cellular alteration	19(38)	21(42)	14(28)	25(50)	22(44)	10(20)^b
Hyperplastic (neoplastic) nodule	2(4)	2(4)	3(6)	2(4)	0	0
Bile duct hyperplasia	47(94)	44(88)	0^a	1(2)	0	0
Urinary bladder
Transitional cell hyperplasia	0	1(2)	1(2)	0	0	3(6)
Papillomatosis	0	0	1(2)	0	0	1(2)
Transitional cell papilloma	1(2)	0	0	0	1(2)	0
Leiomyoma	1(2)	0	0	0	0	0

^a,b Significantly different from control values at P<0.001 or 0.01, respectively.

 

Table 2. Noneoplastic-proliferative and neoplastic lesions developing in the intestines, liver and urinary bladder of F344/DuCrj rats fed test item-containing diet.

Site and type of tumor	Males			Females
	0%	1.25%	5.0%	0%	1.25%	5.0%
Effective no. of rats	50	50	50	50	50	50
Heart
Sarcoma, NOS	0	0	0	0	1(2)	0
Spleen
Lymphoma	0	0	0	1(2)	0	0
Fibroma	0	1(2)	0	0	0	0
Hemangiosarcoma	0	0	1(2)	0	0	0
Leiomyosarcoma	0	0	1(2)	0	0	0
Pituitary
Adenoma, pars distalis	14(28)	12(24)	9(18)	20(40)	19(38)	17(34)
Adenoma, pars intermedia	1(2)	1(2)	1(2)	0	1(2)	0
Craniopharyngioma	0	1(2)	0	0	0	0
Carcinoma, pars distalis	0	2(4)	1(2)	2(4)	4(8)	0
Thyroid
Follicular cell adenoma	1(2)	0	0	0	0	0
C-cell adenoma	5(10)	8(16)	3(6)	3(6)	0	2(4)
Follicular cell carcinoma	0	0	1(2)	0	0	0
C-cell carcinoma	0	3(6)	1(2)	1(2)	1(2)	1(2)
Parathyroid
Adenoma	1(2)	0	0	0	1(2)	0
Adrenals
Pheochromocytoma	4(8)	8(16)	6(12)	2(4)	1(2)	0
Ganglioneuroma	2(4)	0	0	0	0	0
Cortical carcinoma	0	0	0	2(4)	0	0
Malignant pheochromocytoma	2(4)	1(2)	2(4)	1(2)	0	0
Malig. ganglioneuroma-pheochromocytoma	0	0	0	2(4)	0	0
Lungs
Adenoma	1(2)	2(4)	0	1(2)	0	0
Adenocarcinoma	1(2)	0	1(2)	0	0	0
Esophagus
Fibroma	0	0	0	0	1(2)	0
Stomach
Squamous cell papilloma	0	0	0	0	1(2)	0
Squamous cell carcinoma	0	0	0	1(2)	0	0
Pancreas
Acinar cell adenoma	0	0	1(2)	0	0	0
Islet-cell adenoma	2(4)	1(2)	2(4)	0	0	0
Kidneys
Adenoma	1(2)	0	0	0	0	0
Adenocarcinoma	0	1(2)	0	0	0	0
Transtional cell carcinoma	0	1(2)	0	0	0	0
Nephroblastoma	1(2)	0	0	0	0	0
Testes
Interstitial cell tumor	48(96)	47(94)	47(94)	-	-	-
Mlignant interstitial cell  tumor	0	1(2)	1(2)	-	-	-
Prostate
Adenoma	9(18)	12(24)	6(12)	-	-	-
Adenocarcinoma	3(6)	1(2)	1(2)	-	-	-
Preputial/clitoral gland
Adenoma	2(4)	4(8)	3(6)	1(2)	2(4)	4(8)
Adenocarcinoma	1(2)	1(2)	2(4)	0	0	0
Mammary gland
Adenoma	1(2)	0	0	1(2)	0	0
Fibroadenoma	6(12)	11(22)	9(18)	15(30)	8(16)	4(8)^a
Adenocarcinoma	0	0	1(2)	0	2(4)	0
Ovaries
Granulosa/Theca cell tumor	-	-	-	1(2)	0	0
Uterus
Adenoma	-	-	-	1(2)	0	0
Endometrial stromal polyp	-	-	-	8(16)	10(20)	12(24)
Adenomatous polyp	-	-	-	0	1(2)	0
Adenocarcinoma	-	-	-	1(2)	1(2)	3(6)
Endometrial stromal sarcoma	-	-	-	2(4)	1(2)	1(2)
Leiomyosarcoma	-	-	-	2(4)	0	0
Fibrosarcoma	-	-	-	1(2)	0	0
Sarcoma, NOS	-	-	-	0	1(2)	0
Skin/subcutis
Squamous cell papilloma	3(6)	2(4)	3(6)	0	0	0
Fibroma	11(22)	9(18)	10(20)	3(6)	2(4)	4(8)
Lipoma	3(6)	0	0	0	0	0
Lymphangioma	0	1(2)	0	0	0	0
Squamous cell carcinoma	1(2)	0	0	0	0	0
Basal cell carcinoma	0	1(2)	1(2)	0	0	1(2)
Sebaceous carcinoma	1(2)	0	0	0	0	0
Fibrosarcoma	1(2)	0	1(2)	0	0	0
Malignant fibrous histiocytoma	2(4)	1(2)	0	1(2)	1(2)	1(2)
Liposarcoma	0	1(2)	0	0	0	0
Sarcoma, NOS	0	0	1(2)	0	0	0
Malignant schwannoma	0	0	0	1(2)	0	0
Brain
Granular cell tumor	0	0	1(2)	0	0	0
Astrocytoma	1(2)	0	0	0	0	0
Mixed glioma	0	0	0	0	0	1(2)
Spinal cord
Astrocytoma	0	1(2)	0	0	0	0
Peripheral nerve
Malignant schwannoma	0	1(2)	0	0	0	0
Thoracic cavity
Mesothelioma	1(2)	0	0	0	0	0
Abdominal cavity
Lipoma	0	0	2(4)	0	0	0
Liposarcoma	0	0	0	0	1(2)	0
Mesothelioma	5(10)	3(6)	0	0	0	0
Malignant fibrous histiocytoma	0	0	0	0	0	1(2)
All sites
Malignant lymphoma/leukemia	6(12)	18(36)^a	14(28)	8(16)	14(28)	17(34)
Site unknown
Malignant fibrous histiocytoma	0	1(2)	0	0	0	0
Others
Odontoma	0	1(2)	0	0	0	0
Lipoma	0	0	1(2)	0	0	0
Osteosarcoma	0	0	0	1(2)	0	0

^a Significantly different from control values at P<0.001 or 0.01, respectively.

Conclusions:

In this in vivo carcinogenicity study, the test item given in the diet did not show significant increases in tumor incidence in rats. The NOAEL of the test item was 1.25% (427 mg/kg bw) in diet for males and 5% (2372 mg/kg bw) in diet for females.

Executive summary:

An in vivo carcinogenicity study was conducted with the test item in Fischer 344/DuCrj rats. The administration of the test item was achieved orally, by mixing it with the diet in two different concentrations : 1.25 and 5.0 %. Also, a control group was monitored. 50 males and 50 females were distributed per group and were administered the test item on a daily basis for 104 weeks and then normal diet for a further 8 weeks. Clinical sign, urinalysis, haematological and clinical-chemistry examinations were performed as well as monitorisation of body weight and food consumption changes, with no relevant adverse effects observed. Necropsy was performed for all surving animals and the organ relative weights calculated. Gross pathological examinations revealed a higher incidence of raised areas of the cecum, which occurred as single lesions, was recorded in the 5.0% males when compared to the controls. Histopatological findings showed a wide range of non-neoplastic and neoplastic lesions, but they were observed in both control and treated groups. Therefore, the no-observed-adverse-effect level (NOAEL) of the test item in rat was 1.25% (427 mg/kg bw) in diet for males and 5% (2372 mg/kg bw) in diet for females. (conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives).

Reference 6

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

An in vivo carcinogenicity test in Fisher 344 rats was performed for the test item. 15 males and 15 females were orally administered 0.1% of the test item with the feed continuously for 540 days (daily). A control group was also tested.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) of test material: Tokyo Kasei Co.
- Purity: 99.0%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Japan Inc., Atsugi
- Age at study initiation: 1.5 month old
- Assigned to test groups randomly: no
- Fasting period before study:
- Diet: basal diet CE-2 (CLEA Japan Inc., Tokyo) ad libitum
- Water: ad libitum

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): daily
- Mixing appropriate amounts with (Type of food): In this experiment, the test item was mixed at 0.1% concentration with basal diet CE-2

Duration of treatment / exposure:

540 days

Frequency of treatment:

daily

Dose / conc.:

0.1 other: % in diet

No. of animals per sex per dose:

15 males and 15 females per treated group, 16 males and 16 females per control group

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: mortality

DETAILED CLINICAL OBSERVATIONS:No
DERMAL IRRITATION (if dermal study): No
BODY WEIGHT: No
FOOD CONSUMPTION AND COMPOUND INTAKE: No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were carefully autopsied when they died or sacrificed because of moribundity or at the termination of the experiment. Testes, adrenal gland, pituitary gland, lung, jejunum, breast were inspected.

HISTOPATHOLOGY: Yes
Tissues were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Most tumors induced in the experimental group were also found in the control group. There was no significant difference in the incidences of any tumors in the experimental and control groups
(P>0.05).

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 other: % in diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no significant effects observed

Remarks on result:

other: equivalent to 50 mg/kg bw

Key result

Critical effects observed:

no

Table 1. Tumors in Fischer rats fed 0.1% test item diet for 540 days

Group	Sex	No. of Rats used	Effective No. of rats^1	No. of rats with tumors
				Testicular Interstitial Cell Tumor	Adrenal Cortical Adenoma	Pituitary adenoma	Lung adenoma	Jejunal Adenocarcinoma	Breast Fibroadenoma
0.1% test item diet	M	15	15	15	2	2	1	1
	F	15	15		1
Control	M	16	15	15
	F	16	15		3	3			1

¹ No. of rats surviving until the termination of experiment. M, male; F, female

Conclusions:

In this in vivo carcinogenicity study, the test item given in the diet did not show significant increases in tumor incidence in rats. The NOAEL of the test item is 0.1% in diet for both sexes (50 mg/kg bw).

Executive summary:

An in vivo carcinogenicity test in Fisher 344 rats was performed for the test item. 15 males and 15 females were orally administered 0.1% of the test item with the feed continuously for 540 days (daily). A control group was also tested. Testes, adrenal gland, pituitary gland, lung, jejunum and breast were investigated for tumors at the end of the experiments. In all experimental groups, no significant tumoral activity was observed with the control group (P > 0.05). Therefore, it was concluded that there was no increase in the incidence of tumors by the test item. The no-observed-adverse-effect level (NOAEL) of the test item in rats is 0.1% in diet for both sexes. (50 mg/kg bw, conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives).

Reference 7

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two experiments were made on the carcinogenicity of flavonoids. In Experiment I, 110 ACI rats of 1-1.5 months of age were divided into 4 groups. In Experiment II, 146 rats of the same strain and age were divided into 3 groups. Rats in each group were given the prescribed diets until the end of the experiment, for 540 days in Experiment I and for 850 days in Experiment II.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan.
- Purity, including information on contaminants, isomers, etc.: 99.5% pure;

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of the test item in the 10% diet was examined after preservation for 2 months, i.e., the test item was extracted with 90% ethanol, purified by thin-layer chromatography (TLC), and determined by UV spectrum. The test item showed the same RF values as their authentic samples on TLC, and the recovery was 100 ± 12% for test item pellets. Thus, no sign of the degradation of the test item was observed.

Species:

rat

Strain:

other: ACI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 1-1.5 months
- Assigned to test groups randomly: yes
- Diet: CE-2 (CLEA Japan Inc., Tokyo, Japan)
- Water: ad libitum

Route of administration:

oral: feed

Details on exposure:

The rats were given pellets which were prepared by mixing quercetin and test item with basal diet CE-2 using a ‘California pellet mill’. The ground material was dampened by steaming at 100--110°C for 30 s, and the pellets were then compressed and dried.

Duration of treatment / exposure:

540 days in Experiment I and for 850 days in Experiment II.

Frequency of treatment:

daily

Dose / conc.:

5 other: % in diet

Remarks:

Experiment I

Dose / conc.:

10 other: % in diet

Remarks:

Experiment II

No. of animals per sex per dose:

Experiment I: 11 males and 10 females treated, 30 males and 22 females for control.
Experiment II: 20 males and 20 females treated, 33 males and 33 females for control.

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: mortality

DETAILED CLINICAL OBSERVATIONS: No
DERMAL IRRITATION (if dermal study): No

BODY WEIGHT: Yes
- Time schedule for examinations: not specified

FOOD CONSUMPTION AND COMPOUND INTAKE: No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
cecum, colon, adrenal glands, adrenal medulla, pituitary gland, urinary bladder, testis, breasts, uterus

HISTOPATHOLOGY: Yes
Tissues were fixed in 10% formalin, sectioned, and stained with hematoxylin and eosin.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

20 of 21 rats survived for more than 404 days and 15 survived until the end of experiment I. 39 of 40 rats survived for more than 410 days and 26 survived for more than 600 days in experiment II.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant change of body weights were shown between the experiemental and the control group in both experiments.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In both experiments, adrenal cortical adenomas, pituitary adenomas, urinary bladder papillomas and interstitial cell tumors of the testis were found in both the experimental and control groups but there was no significant difference in the incidence of these tumors in the experimental
and control groups (P > 0.05).

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

10 other: % in diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no significant effects observed

Remarks on result:

other: equivalent to 5000 mg/kg bw

Key result

Critical effects observed:

no

Table 1. Tumors in the 5 groups of rats in experiment 1

Group	Diet	Sex	Initial no.of rats	Effective^a no. of  rats	No. of rats with tumors
					Cecal adenoma	Adrenal cortical adenoma	Pheochromocytoma	Pituitary adenoma	Urinary bladder papilloma	testis interstitial cell tumor
1	1% Quercetin	M	10	10						1
		F	10	9
2	5% Quercetin	M	8	8	1	1	1			3
		F	9	9
3	5% Test item	M	11	11		2				3
		F	10	9				1	1
4	Control	M	30	29		2		1	1	3
		F	22	21			1

^a No, of rats surviving for more than 300 days. M, male; F, female.

 

Table 2. Tumors in the 3 groups of rats in experiment 2

Group	Diet	Sex	Initial no. of rats	Effective no. of rats	No. of rats with tumors
					Ileal adenoma	Cecal		Colonic adenoma	Adrenal cortical adenoma	Pheochromocytoma	Pituitary adenoma	Urinary bladder		Breast fibroadenoma	Testis interstitial cell tumor	Uterus adenocarcinoma	Miscellaneous
						Adenoma	Adenocarcinoma					Papilloma	Carcinoma
1	10% Quercetin	M	20	19		1	2		3						6		1^a
		F	20	18					1		2					1	1^b
2	10% Test item	M	20	20	1			1	1						6
		F	20	19						1				1		1	1^c
3	Control	M	33	30					3				2		10
		F	33	31					1		3	1	1	2		4	1^d

^a Pancreatic exocrine adenoma.
^b Sarcoma of the thigh.
^c Heart sarcoma.
^d Renal pelvic papilloma.

Conclusions:

Two in vivo carcinogenicity experiments for the test item did not show any carcinogenic activity in ACI rats at doses up to 10% in diet for both sexes (5000 mg/Kg) after 850 days.

Executive summary:

An in vivo carcinogenicity test in ACI rats was performed for the test item. Two different experiments were carried out, where the animals were orally administered: I) With 5% content of test item and quercetin in meal for 540 days (daily), and II) With 10% content of test item and quercetin in meal for 850 days (daily). Control groups were used in both experiments. Cecum, colon, adrenal glands, adrenal medulla, pituitary gland, urinary bladder, testis, breasts and uterus were investigated for tumors at the end of the experiments. In all experimental groups, no significant change of body weights or tumoral activity was observed with the control group (P > 0.05). Therefore, it was concluded that there was no increase in the incidence of tumors by the test item and the no-observed-adverse-effect level (NOAEL) of the test item in ACI rats is 10% in diet for both sexes (5000 mg/Kg bw, conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives).

 

Reference 8

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

A group of 20 males and 20 females were administered the test item orally with the diet in a daily manner for 733 days. This group received a dose of 10 w/w% test item in diet. A control one (basal diet) was also tested. Body weight changes and the presence of tumors in forestomach and adrenal gland were investigated.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan.
- Purity, including information on contaminants, isomers, etc.: 95% pure;

Species:

hamster

Strain:

other: Golden

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Cooperative Society of Experimental Animals, Shizuoka, Japan
- Age at study initiation: 6 weeks
- Weight at study initiation: 75 - 80 g
- Assigned to test groups randomly: yes
- Housing: They were housed 5 to a cage
- Diet: ad libitum
- Water: ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of test item in diets:

The pellet diets used in this study were ground in a mortar. Two grams of the diet was suspended in 20 ml of methanol and shaken for 5 min at room temperature. Then the suspensions were centrifuged at 3000 r.p.m. for 10 min, and the precipitates were extracted twice with 20 ml of methanol. The pooled methanol extracts were evaporated to dryness and the dried materials were dissolved in methanol for measurement of the test item by HPLC ( Conditions: instrument, Toyo Soda HLC-803A; column, LS-410 ODS SIL, 4 mm ID x 300 mm; mobile phase, 45% methanol-0.05 M phosphate buffer pH 2.2 for test item; temperature, ambient; flow rate, 1.0 ml/min; detector, UV254 ran for the test item. The concentration of the test item was 9.8% ± 0.64 in 10% test item diet and that in basal diet was <0.0002%.

Duration of treatment / exposure:

733 days

Frequency of treatment:

daily

Dose / conc.:

0 other: % in diet

Remarks:

Group 3 -Control

Dose / conc.:

10 other: % in diet

Remarks:

Group 2

No. of animals per sex per dose:

20 males and 20 females per each group

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: mortality

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: once a month

DERMAL IRRITATION (if dermal study): No
FOOD CONSUMPTION AND COMPOUND INTAKE: No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
OTHER: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Forestomach, adrenal gland

HISTOPATHOLOGY: Yes

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The survival rate on day 480 was 55% in females of control Group 3 but >70% in males in Group 3 and in both sexes in all groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no significant difference in the body weight increases in groups given 10% test item diet, and basal diet.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

all the tumors in the forestomach were papillomas, and tumors in adrenal were cortical adenomas. However, there were no significant differences in the incidences in groups fed on 10% test item diet, and basal diet.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

10 other: % in diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Remarks on result:

other: equivalent to 15000 mg/kg bw

Key result

Critical effects observed:

no

Table 1. Mean cumulative intakes of test item and incidences of tumors of hamsters

Experimental group	Dietary treatment	Sex	Estimated cumulative intake (g) /hamster	No. of hamsters used	Effective No.	Papilloma in the forestomach	Cortical adenoma in the adrenal	Others
2	10% Test item	M	581	20	17	1(6)	2(12)	0
		F	592	20	14	0(0)	2(14)	1 (leiomyoma of the uterus)
		Total		40	31	1(3)	4(13)	1
3	Basal diet	M	<0.013	20	15	1(7)	2(13)	0
		F	<0.013	20	11	2(18)	1(9)	3 (mesothdioma, cortical adenocarcinoma of the adrenal, granulosa-theca cell tumor)
		Total		40	26	3(12)	3(12)	3

 

Conclusions:

The in vivo carcinogenicity test for the test item did not show any carcinogenic activity in golden hamster forestomach or adrenal gland at doses up to 10% of the test item in diet for both sexes after 733 days (15 g/kg bw).

Executive summary:

An in vivo carcinogenicity test in hamsters was performed for the test item. A group of 20 males and 20 females were administered the test item orally with the diet in a daily manner for 733 days. This group received a dose of 10 w/w% test item in diet. A control group was also tested (basal diet). Body weight changes and the presence of tumors in forestomach and adrenal gland were investigated. There was no significant difference in the body weight increases between the groups and even though tumors were found in both groups, it was concluded that there was no increase in the incidence of tumors by the test item and the no-observed-adverse-effect level (NOAEL) of the test item in Golden Hamsters is 10 % in diet for both sexes. (15 g/Kg bw, conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives taking into account the mouse data).