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Administrative data

Description of key information

Repeated dose toxicity: via oral route. Weight of evidence approach.


Several repeated dose toxicity studies by oral route are available on the main components of the substance. Furthermore, some studies on analogue substances have been included in the dossier in order to strengthen the weight of evidence approach.


Rutoside, isoquercetin and kaempferol-3-O-rutinoside, three of the main components of the substance (91% of total content) are flavonol glycosides. Quercetin, the fourth main component, is a flavonol and it is the aglycone form of the two main components, rutoside and isoquercetin (90% of total content).


Glycosides, when ingested, are hydrolysed by the intestinal microflora, yielding the corresponding aglycones. Thus, absorption of these substances in significant amount will only occur via their hydrolysed derivatives (i.e., quercetin for the two main components).


Likewise, analogues naringin or methyl hesperidin are flavanone glycosides which are also hydrolysed to their corresponding aglycone (naringenin or methyl hesperetin) and further absorbed in this form.


These aglycones are absorbed mainly in the bacterially colonized segments of the gastrointestinal tract and are partly conjugated with glucuronic acid and/or sulphate and partly further metabolized by bacterial ring cleavage (of the flavonoids C-ring). Then, the glucuronates, sulphates and bacterial degradation products (phenols and phenolic compounds) are all excreted via the bile and urine (EFSA Journal 2010; 8(9):1065)


As the metabolic pathway is very similar, all these substances are expected to have similar toxicological properties.


Of all available studies, the 104-weeks chronic study with quercetin (Dunnick JK, 1992) in rats was selected as the key study because it represents the study with the longest duration, it has acceptable reliability and also some effects were seen which resulted in the lowest NOAEL. Furthermore, quercetin, as stated above, is the first step hydrolysis product in the metabolic pathway of the main components of the substance.


In this chronic study with quercetin, histopathological examinations revealed neoplastic lesions in the kidneys of male rats, including increased severity of chronic nephropathy, hyperplasia and neoplasia of the renal tubular epithelium in the high dose (40000 ppm in diet, ca. 1900 mg/kg bw/day). Therefore, NOAEL of the test item in rat were determined to be the mid dose of 10000 ppm (ca. 410 mg/kg bw/day) for males and 40000 ppm (ca.1900 mg/kg bw/day) for females.


Based on this result, the NOAEL for the substance is set at 410 mg/kg bw/day as worst case approach and without further conversion assuming that quercetin represents the aglycone form to which most part of the substance will hydrolysed before absorption in the body.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The analogue substance naringin which shares the same functional groups with the main components of the target substance Extract of fava d'anta also has comparable values for the relevant molecular properties. See attached the reporting format.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See attached the reporting format.

3. ANALOGUE APPROACH JUSTIFICATION
The source substance and main components of the target substance are all naturally occurring flavonoids found in different plants and foods.
Naringin is a flavanone glycoside contained in citrus fruits, especially in grapefruit, where naringin is responsible for the fruit's bitter taste.
Rutoside, isoquercetin and kaempferol-3-O-rutinoside are flavonol glycosides. Quercetin is a flavonol and it is the aglycone form of rutoside and isoquercetin.
Rutoside is found in many plants including buckwheat, the leaves and petioles of Rheum species, and asparagus. Isoquercetin is a compound naturally found in various plants and foods such as tea, wine and strawberries. Quercetin is widely distributed in many edible plants. Kaempferol-3-O-rutinoside is a bitter-tasting flavonol that can be isolated from the rhizomes of the fern Selliguea feei.

Ingestion of these substances results in the formation of the same range of metabolites. Since the β-glycosidic bond is generally resistant to the action of the mammalian hydrolysing enzymes, glycosides are hydrolysed by the intestinal microflora, yielding the corresponding aglycones. Thus, absorption of these substances in significant amount will only occur via their hydrolysed derivatives (naringenin or quercetin). These aglycones are absorbed mainly in the bacterially colonized segments of the gastrointestinal tract and are partly conjugated with glucuronic acid and/or sulphate and partly further metabolized by bacterial ring cleavage (of the flavonoids C-ring). Then, the glucuronates, sulphates and bacterial degradation products (phenols and phenolic compounds) are all excreted via the bile and urine. As the metabolic pathway is very similar, they are expected to have similar toxicological properties.

4. DATA MATRIX
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Limit test:
no
Key result
Dose descriptor:
NOAEL
Effect level:
1 225.22 mg/kg bw/day (nominal)
Based on:
other: Read-across from an analogue
Sex:
male/female
Basis for effect level:
other: No significant effects observed
Remarks on result:
other: read-across from an analogue for which NOAEL=1250 mg/kg bw/day
Key result
Critical effects observed:
no
Conclusions:
Based on read across from the analogue naringin, the substance extract of fava d'anta has a NOAEL > 1225.22 mg/kg bw/d in rats, after 90-day oral administration.

Executive summary:

The 90d repeated oral dose toxicity was studied on Sprague-Dawley rats according to the ‘‘Technical Guideline for Acute Toxicity Test of chemical drugs’’(SFDA, 2004, China), similar to OECD 408 (GLP study, certificate not available). The test item was orally administered to 22 male and 22 female Sprague-Dawley rats, at doses of 0 (control), 50, 250 or 1250 mg/kg bw/d. All animals were thoroughly observed immediately after administration for the onset of any toxic signs and once daily thereafter. Survival, feed intake, and body weight were monitored. During the subchronic oral toxicity study, no mortality and toxicologically significant changes in clinical signs, food consumption, opthalmoscopic examination, hematology, clinical biochemistry, serum sex hormone, macroscopic findings, organ weights and histopathological examination except for slight body weight decrease were noted and attributed to naringin administration. Under test conditions, the test item was found to have a NOAEL of 1250 mg/kg in rats.


Based on these results, the read-across approach was applied and the substance extract of fava d'anta was determined to have a NOAEL of 1225.22 mg/kg bw/d in rats, after 90-day oral administration.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The analogue substance naringin which shares the same functional groups with the main components of the target substance Extract of fava d'anta also has comparable values for the relevant molecular properties. See attached the reporting format.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See attached the reporting format.

3. ANALOGUE APPROACH JUSTIFICATION
The source substance and main components of the target substance are all naturally occurring flavonoids found in different plants and foods.
Naringin is a flavanone glycoside contained in citrus fruits, especially in grapefruit, where naringin is responsible for the fruit's bitter taste.
Rutoside, isoquercetin and kaempferol-3-O-rutinoside are flavonol glycosides. Quercetin is a flavonol and it is the aglycone form of rutoside and isoquercetin.
Rutoside is found in many plants including buckwheat, the leaves and petioles of Rheum species, and asparagus. Isoquercetin is a compound naturally found in various plants and foods such as tea, wine and strawberries. Quercetin is widely distributed in many edible plants. Kaempferol-3-O-rutinoside is a bitter-tasting flavonol that can be isolated from the rhizomes of the fern Selliguea feei.

Ingestion of these substances results in the formation of the same range of metabolites. Since the β-glycosidic bond is generally resistant to the action of the mammalian hydrolysing enzymes, glycosides are hydrolysed by the intestinal microflora, yielding the corresponding aglycones. Thus, absorption of these substances in significant amount will only occur via their hydrolysed derivatives (naringenin or quercetin). These aglycones are absorbed mainly in the bacterially colonized segments of the gastrointestinal tract and are partly conjugated with glucuronic acid and/or sulphate and partly further metabolized by bacterial ring cleavage (of the flavonoids C-ring). Then, the glucuronates, sulphates and bacterial degradation products (phenols and phenolic compounds) are all excreted via the bile and urine. As the metabolic pathway is very similar, they are expected to have similar toxicological properties.

4. DATA MATRIX
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
1 225.22 mg/kg bw/day (nominal)
Based on:
other: Read-across from an analogue
Sex:
male/female
Basis for effect level:
other: No significant effects observed.
Remarks on result:
other: read-across from an analogue for which NOAEL=1250 mg/kg bw/day
Key result
Critical effects observed:
no
Conclusions:
Based on read across from the analogue naringin, the substance extract of fava d'anta has a NOAEL > 1225.22 mg/kg bw/d in rats, after 6 months oral administration.
Executive summary:

The subchronic toxicity of naringin was studied on rats for 6 months, by a method according to "Technical Guideline for Long Term Toxicity Test of chemical drugs" (SFDA, 2005), similar to OECD TG 408, with GLP (no certificate available). 8 male and 8 female Sprague Dawley rats were administered the test substance 6 days a week by gavage at doses of 0 (control), 50, 250, or 1250 mg/kg for the 6 -months study, plus 4 males and 4 females that were kept for a 1 -month recovery period (same treatment). During the 6-month treatment period and one month recovery period, no mortality and toxicologically significant changes in clinical signs, opthalmoscopic examination, hematology, clinical biochemistry, serum sex hormone, macroscopic findings, organ weights and histopathological examination were noted and attributed to naringin administration. Although consecutive and/or isolated periods of significant body weights and food consumption decreases were relevant to naringin administration, they were not considered toxicologically significant. In addition, slight, non-pathological and reversible hair loss was noted during the 6-month treatment period and considered as a kind of change possibly relevant to naringin administration; however, it was not considered adverse change and to be of toxicological significance. Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) of naringin in rats is greater than 1250 mg/kg/day when administered orally for 6 consecutive months.


Based on these results, the read-across approach was applied and the substance extract of fava d'anta was determined to have a NOAEL of 1225.22 mg/kg bw/d in rats, after 6 months oral administration.


 

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The analogue substance neohesperidin dihydrochalcone which shares the same functional groups with the main components of the target substance Extract of fava d'anta also has comparable values for the relevant molecular properties. See attached the reporting format.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See attached the reporting format.

3. ANALOGUE APPROACH JUSTIFICATION
The source substance and main components of the target substance are synthetic or naturally occurring flavonoids found in different plants and foods.
Neohesperidin dihydrochalcone is a dihydrochalcone glycoside mainly used as a sweetener and flavouring agent. It can be prepared by the hydrogenation of neohesperidin, a bitter flavanone found in citrus fruits.
Rutoside, isoquercetin and kaempferol-3-O-rutinoside are naturally occurring flavonol glycosides. Quercetin is a flavonol and it is the aglycone form of rutoside and isoquercetin.
Rutoside is found in many plants including buckwheat, the leaves and petioles of Rheum species, and asparagus. Isoquercetin is a compound naturally found in various plants and foods such as tea, wine and strawberries. Quercetin is widely distributed in many edible plants. Kaempferol-3-O-rutinoside is a bitter-tasting flavonol that can be isolated from the rhizomes of the fern Selliguea feei.

Ingestion of these substances results in the formation of the same range of metabolites. Since the β-glycosidic bond is generally resistant to the action of the mammalian hydrolysing enzymes, glycosides are hydrolysed by the intestinal microflora, yielding the corresponding aglycones. Thus, absorption of these substances in significant amount will only occur via their hydrolysed derivatives (hesperetin dihydrochalcone or quercetin). These aglycones are absorbed mainly in the bacterially colonized segments of the gastrointestinal tract and are partly conjugated with glucuronic acid and/or sulphate and partly further metabolized by bacterial ring cleavage (of the flavonoids C-ring). Then, the glucuronates, sulphates and bacterial degradation products (phenols and phenolic compounds) are all excreted via the bile and urine. As the metabolic pathway is very similar, they are expected to have similar toxicological properties.

4. DATA MATRIX
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOEL
Effect level:
0.93 other: % in diet
Based on:
other: Read-across from an analogue
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Remarks on result:
other: read-across from an analogue for which NOEL = 1% in diet
Key result
Dose descriptor:
NOAEL
Effect level:
4.64 other: % in diet
Based on:
other: Read-across from an analogue
Sex:
male/female
Basis for effect level:
other: no significant effects were observed.
Remarks on result:
other: read-across from an analogue for which NOAEL=5% in diet
Key result
Critical effects observed:
no
Conclusions:
Based on read across from the analogue neohesperidin dihydrochalcone, the substance extract of fava d'anta has a NOAEL of 4.64% in diet (equivalent to 3715.86 mg/kg bw) in rats, and a NOEL of 0.93% in diet (equivalent to 696.72 mg/kg bw), based on clinical findings and biochemistry.
Executive summary:

A study on the sub-chronic toxicity of the test item was performed by a method similar to OECD 408, without GLP. The test item was administered to groups of 20 male and 20 female Wistar rats at dietary levels of 0 (control), 0.2, 1.0 and 5.0% for 91 days. No treatment-related ophthalmoscopical, haematological or histopathological effects were observed. In the high-dose group, a marked caecal enlargement occurred in both sexes, accompanied by soft stools in the early stages of the study, somewhat lower plasma urea concentrations and increased plasma alkaline phosphatase activity and a decreased urinary pH. This group also showed slight growth depression accompanied by transient reduction in food intake; in males the body weights remained relatively low throughout the experimental period. Furthermore, bilirubin level was increased in females and total protein level was decreased in males of the high-dose group. The above changes were considered adaptive responses or chance effects rather than manifestations of clear toxicity. Therefore the NOAEL could be set at 5% in diet (4000 mg/kg bw). The low- and intermediate-dose groups did not show any compound-related untoward effect, so it was concluded that the 1% group (750 mg/kg bw), was the NOEL. (conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives).


Based on these results, the read-across approach was applied and the substance extract of fava d'anta was determined to have a NOAEL of 4.64% in diet (equivalent to 3715.86 mg/kg bw) in rats, and a NOEL of 0.93% in diet (equivalent to 696.72 mg/kg bw), based on clinical findings and biochemistry.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The analogue substance methyl hesperidin which shares the same functional groups with the main components of the target substance Extract of fava d'anta also has comparable values for the relevant molecular properties. See attached the reporting format.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See attached the reporting format.

3. ANALOGUE APPROACH JUSTIFICATION
The source substance and main components of the target substance are all naturally occurring flavonoids found in different plants and foods.
Methyl hesperidin is a flavanone glycoside contained in citrus fruits such as lemons and oranges.
Rutoside, isoquercetin and kaempferol-3-O-rutinoside are flavonol glycosides. Quercetin is a flavonol and it is the aglycone form of rutoside and isoquercetin.
Rutoside is found in many plants including buckwheat, the leaves and petioles of Rheum species, and asparagus. Isoquercetin is a compound naturally found in various plants and foods such as tea, wine and strawberries. Quercetin is widely distributed in many edible plants. Kaempferol-3-O-rutinoside is a bitter-tasting flavonol that can be isolated from the rhizomes of the fern Selliguea feei.

Ingestion of these substances results in the formation of the same range of metabolites. Since the β-glycosidic bond is generally resistant to the action of the mammalian hydrolysing enzymes, glycosides are hydrolysed by the intestinal microflora, yielding the corresponding aglycones. Thus, absorption of these substances in significant amount will only occur via their hydrolysed derivatives (methyl hesperetin or quercetin). These aglycones are absorbed mainly in the bacterially colonized segments of the gastrointestinal tract and are partly conjugated with glucuronic acid and/or sulphate and partly further metabolized by bacterial ring cleavage (of the flavonoids C-ring). Then, the glucuronates, sulphates and bacterial degradation products (phenols and phenolic compounds) are all excreted via the bile and urine. As the metabolic pathway is very similar, they are expected to have similar toxicological properties.

4. DATA MATRIX
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Limit test:
no
Key result
Dose descriptor:
NOAEL
Effect level:
4.56 other: % in diet
Based on:
other: Read-across from an analogue
Sex:
male/female
Basis for effect level:
other: no significant effects observed
Remarks on result:
other: read-across from an analogue for which NOAEL = 5% in diet (equivalent to 7500 mg/kg bw)
Key result
Critical effects observed:
no
Conclusions:
Based on read across from the analogue methyl hesperidin , the substance extract of fava d'anta has a NOAEL in mice of 4.56% in diet (equivalent to 6833.36 mg/kg bw).
Executive summary:

A subchronic repeated dose toxicity study was conducted with the test item in B6C3F1 mice. The administration of the test item was achieved orally, by mixing it with the diet in five different concentrations : 0.3, 0.6, 1.25, 2.5 and 5.0 % in diet. Also, a control group was monitored. 10 males and 10 females were distributed per group and were administered the test item on a daily basis for 13 weeks.


Survival, haematological and clinical-chemistry examinations were performed as well as monitorisation of body weight and food consumption changes, with no relevant adverse effects observed, except in clinical chemistry. Thus, females fed 2.5% and 5.0% test item demonstrated significant decreases in glucose level. However, the lack of any clear dose-relation or adverse effects on body weight, organ weight, gross pathological and histopathological appearance indicated an absence of toxicity. Therefore, the no-observed-adverse-effect level (NOAEL) of the test item in mice was 5.0% (7500 mg/kg bw) in diet for both males and females (conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives).


Based on these results, the read-across approach was applied and the substance extract of fava d'anta was determined to have a NOAEL of 4.56% in diet (equivalent to 6833.36 mg/kg bw) in mice.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1993
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 five different concentrations : 0.3, 0.6, 1.25, 2.5 and 5.0 %. Also, a control group was monitored. 10 males and 10 females were distributed per group and were administered the test item on a daily basis for 13 weeks.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) of test material: Hamari Pharmaceutical Ind., Ltd.,Osaka, Japan.
- Purity: > 95.5%
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Inc., Kanagawa, Japan.
- Age at study initiation: 6 weeks old
- Housing: They were housed five to a plastic cage on hardwood chips.
- Diet: powdered diet (Oriental MF, Oriental yeast Co., Ltd., Tokyo, Japan) ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2ºC
- Humidity (%): 50±10%
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle
Route of administration:
oral: feed
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Dose / conc.:
0 other: % diet
Dose / conc.:
0.3 other: % diet
Dose / conc.:
0.6 other: % diet
Dose / conc.:
1.25 other: % diet
Dose / conc.:
2.5 other: % diet
Dose / conc.:
5 other: % diet
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, plain diet
Details on study design:
Groups of ten males and ten females were fed powdered diet containing 0, 0.3, 0.6, 1.25, 2.5 and 5.0% of test item for 13 weeks.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: erythrocyte and leukocyte counts, hematocrit and hemoglobin concentration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: glutamate-
oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), alkaline phosphatase (ALP), total bilirubin (T-BIL), urea nitrogen (BUN), total cholesterol (T-CHO), glucose and total protein (TP).

PLASMA/SERUM HORMONES/LIPIDS: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A gross pathological examination was made on each animal during necropsy. The brain, heart, liver, spleen, kidneys, ovaries and testes were weighed before fixation along with other tissues in 10% buffered formalin.

HISTOPATHOLOGY: Yes
Histopathological examinations were performed on routinely prepared sections of heart, lymph nodes, bone marrow, thymus, spleen, pituitary, thyroid, adrenals, trachea, lungs, salivary glands, esophagus, stomach, small and large intestines, pancreas, liver, gall bladder, kidneys, urinary bladder, testes, prostate, seminal vesicles, mammary gland, ovaries, uterus, bone, skin, eyes, Harderian glands, brain, spinal cord, sciatic nerve and all gross lesions in the 0% and 5.0% groups, and heart, spleen, pancreas, liver, kidneys, lungs, brain and all gross lesions in the other groups.
Statistics:
The data were subjected to analysis of variance and differences between means were tested by Student’s t-test. The incidences of histopathologic lesions were analyzed by the two-sided Fisher’s exact probability test.
Clinical signs:
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1 death observed in each 1.25 and 3% groups. The cause of death in each case showed no relationship to the methyl hesperidin treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Significant differences in body weight gains of males given 0.3% test item were observed at weeks 5 and 7 as compared with the control group. In the female groups, the mice receiving 0.3% and 0.6% test item demonstrated increased body weights at 7 weeks and thereafter. These changes were, however, not considered as effects of test item treatment.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Females fed 2.5% and 5.0% test item demonstrated significant decreases in glucose level. However, there was no clear dose-dependence and no chemical effects on any of the other clinical chemistry parameters at any dosage level were noted.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Significant differences in the absolute and relative weights of the heart and kidneys were sporadically observed, but this was not dosedependent and there were no related histopathological findings.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
5 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 7500 mg/kg bw
Key result
Critical effects observed:
no

Table 1. Hematology data for mice fed test item for 13 weeks.



















































































































Conc. (%)No. of miceErythrocytes (x1000)Leukocytes (x100)Hemoglobin (g/dl)Hematocrit (%)
Female
0101012±46a73.4±29.317.0±1.147.5±2.0
0.3101013±9960.7±9.917.1±1.648.0±5.2
0.6101032±5665.7±18.117.3±1.548.4±2.6
1.25101048±7680.1±32.517.3±1.748.6±3.8
2.5101023±9091.5±42.417.1±1.547.8±3.6
5.0101015±4964.6±18.317.2±1.747.5±2.5
Male
0101023±101156.5±175.717.3±1.548.0±4.3
0.391005±105113.7±77.216.8±2.245.5±5.1
0.610979±146188.7±175.716.5±2.446.1±5.6
1.2591037±74110.4±47.317.2±2.048.9±3.8
2.5101031±90110.3±70.117.4±1.648.1±3.9
5.0101030±4788.0±29.017.5±1.848.4±2.8

a Data are mean ± SD values.


 


Table 2. Clinical chemistry data for mice fed test item for 13 weeks.







































































































































































Conc. (%)No. of miceGOT (KU)GPT (KU)ALP (KU)T.BIL (mg/dl)BUN (mg/dl)T.CHO (mg/dl)Glucose (mg/dl)TP (mg/dl)
Female
010106.0±47.2a24.8±12.68.3±1.00.20±0.11c28.3±3.5c79.2±6.7c190.5±26.2c5.2±0.1c
0.31095.2±32.531.7±24.68.2±1.1b0.20±0.1423.0±2.5b**82.0±13.9c182.4±17.6d5.3±0.2b
0.61081.9±15.019.7±14.18.1±0.80.20±0.13b26.7±3.581.1±6.1174.1±17.8b5.2±0.3
1.2510102.3±22.235.3±23.39.1±0.6b0.20±0.15d27.0±7.4d86.6±5.5c*171.7±11.8c5.0±0.6c
2.51096.9±16.322.8±7.48.3±0.60.20±0.14c28.8±6.379.2±12.6c157.0±21.0c*5.1±0.2c
5.010109.3±34.338.2±19.58.8±0.80.13±0.0428.8±6.888.5±7.2*157.7±24.2*5.2±0.1
Male
010105.2±22.832.0±18.26.2±0.90.23±0.25b33.6±3.8108.7±11.8157.7±21.1b5.5±0.3
0.3989.0±18.037.7±20.56.4±1.40.15±0.07b30.7±4.3109.2±8.3b160.7±31.6b5.3±0.2b
0.61093.9±28.624.9±11.25.3±1.30.23±0.2029.2±7.4110.0±11.3142.2±28.85.3±0.2
1.25980.8±10.4*23.8±4.56.1±0.90.16±0.1331.1±6.8106.7±8.8146.7±25.15.4±0.2
2.51093.9±30.126.1±14.85.8±1.10.20±0.15b29.6±8.9b109.0±8.7b182.0±43.4b5.3±0.3b
5.010104.2±35.142,1±27.76.2±2.0c0.13±0.05c32.6±8.9b113.6±13.7b164.0±32.1d5.4±0.3b

*,** Significantly different from control group value at P < 0.05, 0.01, respectively.
a Data are mean + SD values.
b Sample volume from one animal was insufficient for complete analysis.
c Sample volume from two animals was insufficient for complete analysis.
d Sample volume from three animals was insufficient for complete analysis.


 


Table 3. Mean final body and absolute organ weights in mice fed test item for 13 weeks.






























































































































































Conc. (%)

No. of mice

Final body weight
Absolute organ weights (g)
BrainHeartLiverSpleenKidneysOvaries/testes
Female
01024.6±1.0a0.49±0.020.13±0.011.02±0.070.09±0.010.33±0.030.021±0.005
0.31025.6±1.30.49±0.020.13±0.011.07±0.100.09±0.020.35±0.020.023±0.005
0.61025.5±1.40.49±0.020.14±0.011.07±0.070.09±0.010.35±0.020.022±0.005
1.251025.2±1.60.50±0.010.13±0.021.08±0.100.09±0.010.36±0.02*0.023±0.004
2.51024.4±0.90.50±0.030.13±0.011.04±0.060.09±0.010.36±0.02*0.035±0.035
5.01024.4±1.40.49±0.020.12±0.011.07±0.070.09±0.010.35±0.020.022±0.005
Male
01030.0±2.30.47±0.010.16±0.011.27±0.170.11±0.030.49±0.050.23±0.01
0.3929.8±3.60.46±0.020.15±0.021.22±0.160.10±0.030.47±0.050.23±0.02
0.61030.1±1.80.47±0.020.15±0.011.29±0.120.14±0.050.47±0.040.23±0.01
1.25929.0 f 1.30.47±0.010.14±0.01*1.19±0.090.11±0.030.48±0.040.23±0.01
2.51029.4±2.10.46±0.010.16±0.021.26±0.130.11±0.040.46±0.040.23±0.02
5.01029.0±2.30.47±0.010.15±0.011.22±0.160.09±0.020.45±0.040.23±0.02

a Data are mean + SD values.


*,** Significantly different from control group value at P < 0.05.


 


Table 4. Mean relative organ weights in mice fed test item for 13 weeks.

















































































































































Conc. (%)

No. of mice
Absolute organ weights (g)
BrainHeartLiverSpleenKidneysOvaries/testes
Female
0101.98±0.11a0.53±0.054.16±0.200.36±0.051.35±0.060.086±0.020
0.3101.90±0.110.50±0.064.20±0.230.35±0.041.38±0.080.091±0.020
0.6101.92±0.140.55±0.064.20±0.320.35±0.041.39±0.100.087±0.019
1.25101.98±0.140.52±0.094.30±0.290.36±0.031.43±0.09*0.092±0.019
2.5102.05±0.120.52±0.064.27±0.200.37±0.041.46±0.04**0.142±0.147
5.0101.99±0.110.49±0.044.37±0.240.39±0.041.42±0.08*0.091±0.020
Male
0101.57±0.140.53±0.034.22±0.310.37±0.081.62±0.080.78±0.05
0.391.58±0.170.50±0.074.11±0.440.34±0.121.59±0.120.78±0.07
0.6101.57±0.080.50±0.02*4.27±0.360.47±0.171.55±0.120.75±0.04
1.2591.61±0.060.50±0.044.10±0.220.37±0.091.67±0.120.80±0.04
2.5101.58±0.110.53±0.044.29±0.320.37±0.141.56±0.100.79±0.05
5.0101.63±0.130.53±0.064.20±0.380.31±0.061.54±0.07*0.81±0.07

a Data are mean + SD values.


*,** Significantly different from control group value at P < 0.05.

Conclusions:
The no-observed-adverse-effect level (NOAEL) of the test item in mice was was 5.0% (7500 mg/kg bw) in diet for both males and females.
Executive summary:

A subchronic repeated dose toxicity study was conducted with the test item in B6C3F1 mice. The administration of the test item was achieved orally, by mixing it with the diet in five different concentrations : 0.3, 0.6, 1.25, 2.5 and 5.0 % in diet. Also, a control group was monitored. 10 males and 10 females were distributed per group and were administered the test item on a daily basis for 13 weeks.


Survival, haematological and clinical-chemistry examinations were performed as well as monitorisation of body weight and food consumption changes, with no relevant adverse effects observed, except in clinical chemistry. Thus, females fed 2.5% and 5.0% test item demonstrated significant decreases in glucose level. However, the lack of any clear dose-relation or adverse effects on body weight, organ weight, gross pathological and histopathological appearance indicated an absence of toxicity. Therefore, the no-observed-adverse-effect level (NOAEL) of the test item in rat was 5.0% (7500 mg/kg bw) in diet for both males and females (conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives).

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
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
Limit test:
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 acic (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 oral 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
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 groups had reduced body weight gain in comparison to controls during the last half of the study.
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.
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:
yes
Relevant for humans:
not specified

Table 1. Survival, body weight, feed consumption and estimated chemical consumed in rats in the 2-year study of the test item












































































































































































Dose (ppm)Initial6 months15 months24 monthsAverage food consumption (g)Estimated chemical consumed (mg/kg/day)
No. survivingMean body wt (g) +SENo. surviving Mean body wt (g) +SENo. surviving Mean body wt (g) +SENo. surviving Mean body wt (g) +SE1-13 weeks14-52 weeks53-104 weeks1-13 weeks14-52 weeks53-104 weeks
Male rats
070161.8±2.560433.8±8.046485.6±5.526464.4±7.918.519.219.5---
100070159.6±2.260437.9±3.549493.2±5.029451.0±6.018.519.319.4734440
1000070165.4±2.160430.3±8.148489.9±5.325439.8±5.9*19.020.219.1736467404
4000070155.5±2.260413.2±3.5*47457.8±4.5**25403.3±6.4**19.120.020.6298119341872
Female rats
070138.2±1.460255.2±2.249342.9±3.530357.4±6.313.114.214.4---
100070140.8±1.260257.4±1.850348.5±3.828360.2±6.613.314.314.7725442
1000070139.2±1.556236.8±1.5**48330.5±3.3*35349.3±4.812.513.113.8704537416
4000070140.7±1.360220.3±4.0**48291.0±4.3**28311.3±7.1**12.312.314.3281821701936

*Significantly different from the controls (p≤0.05)


**Significantly different from the controls (p≤0.01)


 


Table 2a. Males. Organ Weights, Organ-to-Body Weights Ratios, Hematology, and Clinical Chemistry Data for Rats at the 15-Month Interim Termination Period.











































































































































































 


 



Dose (ppm)



0



1,000



10,000



40,000



Necropsy body wt 



460±12



466±8



459±14



456±15



Brain a



Absolute



2.07±0.03



2.06±0.02



2.04±0.03



2.05±0.03



Relative



4.53±0.09



4.44±0.08



4.48±0.14



4.53±0.11



Right kidney



Absolute



 1.44±0.05



1.51±0.06


1.44±0.04

1.59±0.05



Relative 



3.15±0.09



3.27±0.17



3.16±0.07



3.49±0.06*



Liver



Absolute



15.66±0.65



15.12±0.63



15.23±0.48



17.40±0.75



Relative


34.0±1.0

32.4±0.9



33.2±0.5


38.1±1.0**

Hematologyb



Erythrocytes ( 106/µl)



9.44±0.23



9.65±0.21



9.65±0.14



9.43±0.24



Leukocytes ( 103/gl)



5.08±0.26



5.33±0.30



4.88±0.21



4.99±0.33



Segmented neutrophils (103/µl)



2.03±0.24



1.67±0.11



1.56±0.16



1.89±0.19



Lymphocytes ( 103/µl])



2.72±0.19



3.38±0.22



3.02±0.17



2.87±0.18



Monocytes ( 103/µl)



0.24±0.04



0.20±0.03



0.20±0.03



0.19±0.03



Eosinophils ( 103/µl)



0.10±0.02



0.08±0.03



0.10±0.03



0.05±0.02



Nucleated erythrocytes (103/µl) 



0.03±0.02



0.04±0.02



0.06±0.02



0.03±0.02



Clinical chemistry b



BUN (mg/dl)


17.8±1.0

32.4±9.4



18.3±1.0


17.8±1.4

Creatinine (mg/dl)



0.49±0.05



0.72±0.16



0.44±0.02



0.58±0.04



Sodium (meq/liter)



146±0



147±1



146±0



146±0



Potassium (meq/liter)



3.61±0.08



3.72±0.11



3.54±0.08



3.78±0.06


Chloride (meq/Iiter)

110±1



108±1



109±1



107±1*



SDH (IU/liter)



816±114b



621±70b



708±73b



345±34**



Organ weights are given in grams ± SE; organ weight-to-body weight ratios are given as mg organ weight/g body wt (mean ± SE)


Mean ± SE. BUN, blood urea nitrogen; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SDH, sorbitol dehydrogenase.


*Significantly different from the controls (p≤0.05)


**Significantly different from the controls (p≤0.01)


 


Table 2b. Females. Organ Weights, Organ-to-Body Weights Ratios, Hematology, and Clinical Chemistry Data for Rats at the 15-Month Interim Termination Period.

























































































































































































 


 



Dose (ppm)



0



1,000



10,000



40,000



Necropsy body wt 



324±9



337±8



307±6



287±6**



Brain



Absolute



1.90±0.03



1.90±0.02



1.89±0.02



1.90±0.02



Relative



5.88±0.12



5.65±0.13



6.20±0.13



6.65±0.15**



Right kidney



Absolute



 0.89±0.03



0.93±0.02


0.87±0.02

1.59±0.05



Relative 



2.74±0.06



2.77±0.07



2.85±0.04



3.08±0.09**



Liver



Absolute



9.21±0.21



9.44±0.31



8.90±0.28



9.53±0.34



Relative


28.5±0.6

27.9±0.4



29.1±0.8


33.2±0.8**

Hematologyb



Erythrocytes ( 106/µl)



8.62±0.11



8.48±0.12



8.56±0.10



8.15±0.11**



Leukocytes ( 103/gl)



3.12±0.16



3.01±0.12



3.10±0.17



3.33±0.23



Segmented neutrophils (103/µl)



1.05±0.06



0.89±0.04



0.95±0.07



0.91±0.10



Lymphocytes ( 103/µl])



1.90±0.11



1.93±0.12



1.98±0.14



2.23±0.15



Monocytes ( 103/µl)



0.13±0.02



0.16±0.02



0.15±0.02



0.16±0.04



Eosinophils ( 103/µl)



0.04±0.01



0.03±0.01



0.03±0.01



0.03±0.01



Nucleated erythrocytes (103/µl) 



0.04±0.01



0.04±0.02



0.05±0.01



0.04±0.01



Clinical chemistry b



BUN (mg/dl)


16.5±1.2

14.0±0.6b



15.8±0.9


17.1±1.7

Creatinine (mg/dl)



0.55±0.04



0.62±0.05



0.54±0.02



0.59±0.04



Sodium (meq/liter)



147±0



147±1



146±1



146±0



Potassium (meq/liter)



3.17±0.07



3.32±0.08



3.30±0.08



3.27±0.09


Chloride (meq/Iiter)

110±0



111±1



111±1



111±1


ALT (IU/liter)

29±2



28±2b



29±2



33±3


AST (IU/liter)

63±4



63±2b



67±5



61±3



SDH (IU/liter)



816±114b



621±70b



708±73b



345±34**



Organ weights are given in grams ± SE; organ weight-to-body weight ratios are given as mg organ weight/g body wt (mean ± SE)


Mean ± SE. BUN, blood urea nitrogen; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SDH, sorbitol dehydrogenase.


*Significantly different from the controls (p≤0.05)


**Significantly different from the controls (p≤0.01)


 


Table 3. Kidney lesions of the renal tubules in rats in the 2-year feed study of the test item.










































































































































 


 



Dose (ppm)



0



1,000



10,000



40,000



Male rats



Initial evaluation



Hyperplasia



1/50



2/50



3/50



4/50



Adenoma



0/50



0/50



0/50



3/50*



Adenoma or adenocarcinoma 



0/50



0/50



0/50



4/50*



Step sectionevaluation



Hyperplasia



2/50



1/50



5/50



7/50*



Adenoma



1/50



2/50



7/50



5/50*



Single and step section combined



Hyperplasia



3/50



3/50



8/50



11/50*



Adenoma



1/50



2/50



7/50



8/50*



Adenoma or adenocarcinomab



1/50



2/30



7/50



9/50*



Female rats



Initial evaluation



Hyperplasia



1/49



1/49



3/50



1/50



Adenoma



0/49



0/49



1/50



0/50



Step section a evaluation



Hyperplasia



1/49



-



-



3/50



Adenoma



0/49



-



-



0/50



Single and step section combined



Hyperplasia



2/49



-



-



4/50



Adenoma



1/49



-



-



0/50



aAdditional animals identified with kidney lesions.
b NTP historical incidence for renal tubule adenoma or adenocarcinoma control male F344/N rats: 3/99 (3.0%, range 0-6%) at this laboratory; 8/499 (1.6%, range 0-6%) for all laboratories. -, not evaluated.
* Trend test p < 0.05 (significant by all tests used); tumors randomly distributed among animal cages.

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 (1900 mg/kg bw) in diet for females.
Executive summary:

A chronic repeated dose toxicity 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 (40 mg/kg bw), 10000 (410 mg/kg bw) and 40000 ppm (1900 mg/kg bw). 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 (1900 mg/kg bw) in diet for females.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by Zoster S.A. (Raiguero, S/N, Zeneta-Murcia, Spain), lot no.1999
- Purity: > 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The sample was stored in sealed plastic bags in a cool and dry place.
- Stability under test conditions: NHDC was found to be stable in the diet under the experimental conditions applied.
Species:
rat
Strain:
Wistar
Remarks:
(Cpb: WU; Wistar random)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: TNO Central Institute for the Breeding of Laboratory Animals, Zeist, The Netherlands.
- Weight at study initiation: males weighed around 84 g and females around 79 g.
- Housing: The rats were housed in groups of five of the same sex and treatment group in suspended stainless-steel wire-screen cages.
- Diet: cereal-based, open-formula diet (TNO-C1VO Toxicology and Nutrition Institute, the Netherlands). Food was provided ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The Institute's basal diet is analysed for both nutrients and contaminants twice a year. Typical results of analysis for calcium, phosphorus and magnesium are: Ca, 0.7-0.9%; P, about 0.6%; Mg, about 0.17%.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 40-80%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh batches of the test diets were prepared five times in the course of the study.
- Mixing appropriate amounts with (Type of food): NHDC was mixed into the Institute's cereal-based, open-formula diet, the composition (%) of which was as follows: soybean oil meal 11; fish meal 7; meat and bone scraps 4; whole ground wheat 36; whole ground maize 29.7, grass meal 3; whey powder 2; defatted bone meal 0.4; soybean oil 3; trace elements in salt 0.5; brewers' yeast 3; vitamin B preparation 0.1; vitamin ADEK mixture 0.3. Typical results of analysis for calcium, phosphorus and magnesium are: Ca, 0.7-0.9%; P, about 0.6%; Mg about 0.17%.
- Storage temperature of food: The diets were stored at about 15ºC.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Diet samples were analysed for NHDC content by HPLC, according to Fisher (1977), using an M&N 25cm x 4.6mm i.d. reverse-phase Poligosil 60-5-CI8 column. The adequacy of the mixing procedure was investigated by analysing five samples of each test diet from the first batch. The stability of NHDC in this batch of diets was examined after storage for 4 wk at room temperature. In addition, the NHDC content of the test diets was analysed monthly.
- Throughout the study, the actual levels of NHDC in the low-, intermediate- and high-dose diet were 95%, 97-99% and 94-105% of the intended levels, respectively. The quintuplicate analyses of the first batch of each of the NHDC-containing diets showed coefficients of variation of less than 5%, indicating satisfactory homogeneity.
Duration of treatment / exposure:
13 consecutive weeks.
Frequency of treatment:
Daily.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
0.2 other: % in diet
Dose / conc.:
1 other: % in diet
Dose / conc.:
5 other: % in diet
No. of animals per sex per dose:
20 rats/sex/group.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Groups of 20 male and 20 female rats were fed diets containing 0, 0.2, 1.0 or 5.0% NHDC for 13 consecutive weeks. No further details.
- Owing to the normal decrease in food intake per unit body weight with increasing age of rats, the intake of NHDC per kg body weight became lower in the course of the study. The overall actual intakes of NHDC were roughly 150, 750 and 4000 mg/kg body weight/day for the low-, intermediate- and high-dose groups, respectively.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The rats were observed daily for external signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: The rats were weighed weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. Food intake was measured over 1-wk periods.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (not a drinking water study): Yes
- Time schedule for examinations: water intake was recorded in wk 7 and 12.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of the study and at wk 13.
- Dose groups that were examined: control group and high-dose group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: early in week 13.
- Anaesthetic used for blood collection: No
- Animals fasted: Not specified.
- How many animals: 10 rats/sex/group
- Parameters examined: haemoglobin concentration, packed cell volume, and counts of thrombocytes and red and white blood cells (including a differential white cell count).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in wk 13.
- Animals fasted: Yes
- How many animals: 10 rats/sex/group
- Parameters examined: glucose (Boehringer Glucoquant No. 245-178; Boehringer Mannheim GmbH Mannheim, FRG); alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, r-glutamyltransferase, total protein, albumin, total bilirubin, urea, cholesterol, creatinine and calcium (Cobas-Bio Centrifugal Analyzer), ornithine-carbamyl transferase (Spectro-fotometric), inorganic phosphate (Boehringer Kit No. 124-974, Boehringer Mannheim GmbH), chloride (Chloro Counter, Marius), and sodium and potassium (Electrolyte-2-Analyzer, Beckman).

URINALYSIS: Yes
- Time schedule for collection of urine: urine was collected from ten rats/sex/group during the last 16 hr of a 24-hr period of deprivation of food and water in wk 13.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined: volume (calibrated tubes), density (Bellingham & Stanley refractometer), for protein, glucose, occult blood, ketones, bilirubin and urobilinogen (pooled samples; Combur test strips, Boehringer Mannheim GmbH), and microscopy of the sediment (pooled samples).

OTHER: In week 13, pH was determined in urine samples collected over 3 hr early in the morning from ten non-fasted rats/sex/group (Philips P.W. 9410 pH meter).

NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Early in wk 14, the rats were killed by exsanguination from the abdominal aorta, under light ether anaesthesia, and subjected to a thorough post-mortem examination. Any abnormalities were recorded and the adrenals, brain, caecum (with and without contents), gonads, heart, kidneys, liver, spleen, thymus and thyroid (with parathyroids) were weighed. Samples of these organs and of the aorta, axillary lymph nodes, colon, duodenum, epididymides, eyes, ileum, jejunum, lungs, mammary glands (both sexes), mesenteric lymph nodes, oesophagus, pancreas, pituitary, prostate, rectum, sciatic nerve, skeletal muscle, spinal cord, sternum, stomach (glandular and non-glandular), salivary glands (parotid, sublingual and submaxillary), trachea, urinary bladder and uterus (with cervix) were preserved in 10% phosphate buffered formaldehyde.

HISTOPATHOLOGY: Yes
- Wax-embedded 5-11 m sections of the preserved tissues from all animals in the control and high-dose group were stained with haematoxylin and eosin and examined microscopically. One female of the intermediate-dose group that died intercurrently was subjected to histopathological examination as well.
Statistics:
Data on body weights were evaluated by one-way analysis of covariance, followed by Dunnett's multiple-comparison tests. The laboratory determinations and organ weights were evaluated by one-way analysis of variance, followed by Dunnett's multiple-comparison tests, except for the differential white blood cell counts and urinary pH values which were analysed by the Mann Whitney U test. Data on food and liquid intake was evaluated by analysis of variance, followed by least significant difference tests (experimental unit: the cage). Fisher's exact probability test (two sided) was used for results of ophthalmoscopy and for pathology data.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed in the low- and intermediate-dose groups. In the high-dose group, both males and females showed soft stools in wk 2 and 3.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female rat in intermediate dose group was found dead at day 30 but, as no additional mortality was found in any dose groups and macroscopic and microscopic examination of the animal found dead revealed renal failure not seen in any other dose groups, this death is considered a fortuitous finding.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The low and medium dose groups showed no effects. At the high-dose level, growth was depressed in males throughout the study and in females in the first 2 weeks and, accordingly, food intake slightly decreased.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The low and medium dose group showed no effects. At the high-dose level in males throughout the study and in females in the first 2 weeks food intake was slightly decreased.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
The low and medium dose group showed no effects. At the high-dose level a transient decrease in food-conversion efficiency was observed.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was not significantly affected by the administration of NHDC at any dose level.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examinations did not reveal any treatment related effects.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no significant changes in haematology among the groups.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Biochemical changes that might have been due to treatment, were observed in the high-dose group only: in females, plasma alkaline phosphatase activity and bilirubin concentration were increased; total plasma protein concentration was decreased in males; plasma urea concentration was relatively low in both sexes.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinary pH was decreased in both sexes of high-dose group.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- The organ-weight data showed marked enlargement of the caecum in the high-dose group in both sexes. Slight, though statistically significant increases in caecal weight occured also in males of the low- and intermediate-dose group but there was no dose-response relationship.
- The relative weights of the brain and testicles were increased in males of the high-dose group. The mean absolute weights of these organs were, however, very similar to those of the controls. This change is not ascribed to a direct effect of the test item. The other organ weights did not show treatment-related changes in either sex.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross examination at autopsy did not reveal treatment-related changes in any of the experimental groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination of the organs and tissues from all rats of the control and high-dose group did not reveal any treatment-related changes.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Details on results:
- Intake of NHDC: Owing to the normal decrease in food intake per unit body weight with increasing age of rats, the intake of NHDC per kg body weight became lower in the course of the study. The overall actual intakes of NHDC were roughly 150, 750 and 4000mg/kg body weight/day for the low-, intermediate- and high-dose groups, respectively.
- In the present study feeding Wistar rats with diets containing up to 5% NHDC was associated with several changes of little, if any, toxicological significance.
Key result
Dose descriptor:
NOEL
Effect level:
1 other: % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
5 other: % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effects were observed.
Key result
Critical effects observed:
no

Table 1. Body weights, food consumption and test item intakes of rats fed diets containing 0-5% NHDC for 13 wk.








































































































































































Dose


(% in diet)



Body weight (g) at week



Food consumption (g/rat/wk)



Mean intake**


(mg/kg bw/d)



Males



0



1



2



3



4



8



13



1



2



3



4



8



12



0



84±0.9



125±1.4



166±2.4



214±3.7



255±4.4



343±7.0



392±8.3



105



128



135



149



148



147



0



0.2



84±0.9



125±1.7



166±2.8



212±3.9



254±4.4



341±9.1



391±11.0



106



128



133



148



142



146



150



1.0



84±1.1



125±1.8



164±2.6



210±3.8



249±4.1



338±5.6



387±6.5



106



126



134



147



146



146



757



5.0



84±0.9



125±2.2*



146±3.3*



192±3.4*



232±3.8*



310±6.5*



355±7.4



93*



111*



132



148



142



146



4011



Females



 



0



79±0.9



110±1.2



133±1.6



156±2.4



172±2.4



210±4.3



230±5.1



92



100



103



106



111



109



0



0.2



79±0.8



110±1.1



135±1.6



159±2.3



174±2.7



213±3.7



234±4.2



91



101



100



105



112



109



166



1.0



79±1.2



109±1.5



132±2.0



154±2.8



169±2.8



206±4.6



232±4.5



91



102



98



106



111



111



848



5.0



79±0.9



104±1.6*



126±2.1*



153±2.7



170±2.9



206±4.1



227±4.2



87



100



102



108



111



113



4334



**Overall means of weekly calculations from data on body weight, food intake and nominal levels of NHDC.


Body-weight values are means ± SEM for groups of 20 rats. Food consumption values are the means for four cages of five animals. Although growth and food consumption were recorded weekly, only values obtained during the first 4 weeks and subsequendly monthly are presented. The values marked with * differ significantly (body weight; Dunnett’s test, and food consumption; least significant difference test) from the control value (**P < 0.01).


 


Table 2. Plasma biochemical and urinary parameters of rats fed diets containing 0-5% NHDC for 13 wk.


 








































































































Parameter



% NHDC in diet



0



0.2



1.0



5.0



MALES



Plasma



Alkaline phosphatase (U/L)



144.9±7.0



141.0±5.8



163.9±7.2



150.3±4.0



Total protein (g/L)



63.4±0.9



61.3±0.4



61.7±0.5



59.6±0.6**



Bilirubin (μmol/L)



0.30±0.13



0.20±0.08



0.02±0.01



0.61±0.24



Urea (mmol/L)



7.08±0.29



7.68±0.55



7.76±0.34



6.50±0.14



Urine



pH



8.0



7.5



7.8



7.2**



FEMALES



Plasma



Alkaline phosphatase (U/L)



129.1±4.7



124.8±8.9



142.8±5.9



154.4±5.1*



Total protein (g/L)



61.7±0.6



61.0±0.5



60.9±0.8



61.6±0.7



Bilirubin (μmol/L)



0.32±0.13



0.25±0.09



0.62±0.10



1.04±0.17**



Urea (mmol/L)



8.06±0.75



8.52±0.71



7.78±0.50



6.34±0.33



Urine



pH



8.0



7.9



7.9



7.3**



Values are means ± SEM for groups of ten rats; those marked with asterisks differ significantly [Dunnett’s test or Mann-Whitney U-test (for pH values)] from the corresponding control value (*P < 0.05; **P < 0.01). No other blood, plasma or urinary analysis values differed significantly from the corresponding control value.


 


Table 3. Relative organ weights of rats fed diets containing 0-5% NHDC for 13 wk.















































































































































































OrganRelative organ weights (g/kg bw)
00.21.05.0
MALES
Adrenals0.132±0.0030.140±0.0040.145±0.0040.142±0.005
Brain5.13±0.125.04±0.145.12±0.075.55±0.13*
Caecum (full)11.6±0.614.8±0.6**14.5±0.6**20.4±0.9**
Caecum (empty)2.3±0.12.8±0.1*2.6±0.13.5±0.2**
Heart3.21±0.063.32±0.073.18±0.063.28±0.06
Kidneys6.29±0.136.31±0.116.28±0.126.33±0.08
Liver36.0±0.636.2±0.735.4±0.534.1±0.5
Spleen1.60±0.041.71±0.051.64±0.051.60±0.04
Testes8.55±0.208.54±0.288.65±0.239.49±0.23*
Thymus0.92±0.050.99±0.050.85±0.040.88±0.04
Thyroid0.063±0.0030.066±0.0030.062±0.0040.067±0.003
FEMALES
Adrenals0.286±0.0100.285±0.0100.285±0.090.309±0.009
Brain7.90±0.187.71±0.127.78±0.157.94±0.13
Caecum (full)15.5±0.614.7 ± 0.716.6±0.722.5±0.8**
Caecum (empty)3.3±0.23.3±0.23.5±0.14.6±0.2**
Heart3.85±0.073.85 ± 0.063.87±0.093.90 ±0.07
Kidneys6.71±0.096.58 ± 0.136.62±0.106.85 ±0.09
Liver33.4±0.333.3 ± 0.533.0±0.533.9 ± 0.5
Spleen1.86 ± 0.051.95±0.051.84±0.041.95 ±0.04
Ovaries0.363±0.0130.372±0.0140.377±0.0130.347±0.010
Thymus1.27±0.051.31±0.041.29±0.041.36±0.05
Thyroid0.092±0.0030.082±0.040.089±0.0040.094±0.003

Values are means ± SEM for groups of 20 rats. Those marked with asterisks differ significantly (Dunnett’s test) from the corresponding control value (*P < 0.05; **P < 0.01).


 


Table 4. Major histopathological changes in rats fed NHDC for 13 wk.


 
















































































































































































































Organ and lesion Dose(%):No. of rats affected
MalesFemales
05.005.0
Adrenals
- Cortical vacuolation7400
Epididymis
- Reduced spermatogenesis10  
Heart
- Cartilaginous metaplasia0101
Kidneys
- Nephrosis131135
- Calcareous depositsin the cortico-medullary layer011411
- Calcareous deposits in pelvis0040
- Urothelial hyperplasia in pelvis0030
- Hydronephrosis0101
Large intestines
- Parasites2113
Liver
- Aggregates of RES cells3455
Lung
- Perivascular lymphocytic infiltrates1193
Pituitary
- Cysts1112
- Tubular hyperplasia in pars neuralis0001
Spleen
- Hematopoiesis0101
Stomach
- Fundic glandular dilatation0030
Sub-lingual salivary glands
- Demi-lune cell proliferation0002
Testes
- Atrophy10  
- Tubular calcareus deposits20  
Urinary bladder
- Urothelial hyperplasia0001
Uterus
- Polyp  01
- Endometritis  02

The tissues of 20 animals were examined microscopically in all cases except that the pituitaries of only 19 control males were examined.

Conclusions:
The test item has a sub-chronic oral NOAEL of 5% in diet (4000 mg/kg bw) in rats, and a NOEL of 1% in diet (750 mg/kg bw), based on clinical findings and biochemistry.
Executive summary:

A study on the sub-chronic toxicity of the test item was performed by a method similar to OECD 408, without GLP. The test item was administered to groups of 20 male and 20 female Wistar rats at dietary levels of 0 (control), 0.2, 1.0 and 5.0% for 91 days. No treatment-related ophthalmoscopical, haematological or histopathological effects were observed. In the high-dose group, a marked caecal enlargement occurred in both sexes, accompanied by soft stools in the early stages of the study, somewhat lower plasma urea concentrations and increased plasma alkaline phosphatase activity and a decreased urinary pH. This group also showed slight growth depression accompanied by transient reduction in food intake; in males the body weights remained relatively low throughout the experimental period. Furthermore, bilirubin level was increased in females and total protein level was decreased in males of the high-dose group. The above changes were considered adaptive responses or chance effects rather than manifestations of clear toxicity. Therefore the NOAEL could be set at 5% in diet (4000 mg/kg bw). The low- and intermediate-dose groups did not show any compound-related untoward effect, so it was concluded that the 1% group (750 mg/kg bw), was the NOEL. (conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2013.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: ‘‘Technical Guideline for Long Term Toxicity Test of chemical drugs’’ (SFDA, 2005 - PR China)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
6-month study, animals dosed 6 days/week.
GLP compliance:
yes
Remarks:
No certificate included in publication.
Limit test:
no
Specific details on test material used for the study:
Naringin was extracted from Citrus grandis ‘Tomentosa’ by water and purified by recrystallization as described previously (Li et al., 2013c). The HPLC purity of naringin was determined to be >98.3% by external standard method.
Identification was performed by ultraviolet–visible spectroscopy (UV/Vis), electron spray ionization–mass spectrometry, proton nuclear magnetic resonance (1H NMR) and carbon-13 nuclear magnetic resonance (13C NMR) spectroscopy. The purity analysis was performed on a Shimadzu (HPLC) LC-6A instrument (Shimadzu Corp., Kyoto, Japan) with a Dionex C18 column (5 µm, 4.6 mm 250 mm, USA) and a TL9000 Chromatographic Station. The mobile phase was prepared by a 45/55 (v/v) mixture of methanol/water and the pH was adjusted to 3.0 with acetic acid. The injection volume was 20 ll. The UV detector was set at a wavelength of 283 nm. The HPLC purity of naringin was determined to be >98.3% by external standard method.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: male and female SD rats, certified specific pathogen-free, were purchased in good health from Slack Shanghai Laboratory Animal Co., Ltd.(Shanghai, China) under the license number SCXK (HU) 2007-0005.
- Age at study initiation: 4 to 6-week-old rats.
- Weight at study initiation: At initial of dosing, the animal weights ranged from 109.6 to 142.2 grams (mean, 127.5 ± 6.9) for males and 116.3 to 158.9 grams (mean, 140.2 ± 9.9) for females.
- Fasting period before study: overnight
- Housing: Animals were housed in labelled polypropylene cages (four rats per cage) with feed and water available ad libitum.
- Acclimation period: 1 week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr):
- Photoperiod: 12 hrs dark / 12 hrs light.
- Air change: 10-12 cycles/h.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
10mL/kg bw.
Details on oral exposure:
Since naringin is a flavonoid with slight solubility (1.1 g/L) in water (Lauro et al., 2007), suspension of naringin in sterile water was made up fresh each day just before it was administrated to the animals by using oral gavage at a dosing volume of 10 ml/kg body weight. During administration, suspension of naringin was always shaken well with suspended magnetic stir, thereby ensuring its homogeneity and stability.

PREPARATION OF DOSING SOLUTIONS: suspension of naringin in sterile water was made up fresh each day just before it was administrated to the animals.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Amount of vehicle (if gavage): 10 ml/kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The HPLC purity of naringin was determined to be >98.3% by external standard method. Dosing solutions were prepared fresh every day.
Duration of treatment / exposure:
6 months.
Frequency of treatment:
6 days/week.
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
8 males and 8 females for the 6-month study, plus 4 males and 4 females for the recovery period (1 month).
Control animals:
yes
Details on study design:
One hundred and seventy-six rats were randomly assigned to control and three test groups, each consisting of 22 males and 22 females. 8 animals were assigned to the recovery subgroup; recovery period was designated as one month. Control animals received sterile water alone.
- Rationale for selecting satellite groups: one group was kept to observe the recovery after administration.
- Post-exposure recovery period in satellite groups: 1 month
Positive control:
No.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily (morning and afternoon; first at the time of dose administration and approximately 1–2 h following dose administration) for signs of clinical toxicity and mortality.
- Observations included, but were not limited to, changes in skin, fur, eyes, appearance, genital organ, glandular secretion (salivary gland secretions), oral mucosa, faecal characteristics, respiration, circulation and behaviour.

BODY WEIGHT: Yes
- Body weight was recorded twice per week in the first 4 weeks after dosing and then once a week thereafter.

FOOD EFFICIENCY:
- The 24 h feed intake pattern was measured on rats weekly. Mean daily food consumption was calculated once a week by subtracting the weight of the remaining food from the weight of the supplied food.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: not specified.
- Dose groups that were examined: all.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during euthanasia procedure.
- Anaesthetic used for blood collection: phenobarbital sodium (60 mg/kg bw.)
- Animals fasted: Yes (night fasting)
- How many animals: 12
- Parameters: WBC (white blood cell count and differential), NEU, LYM, MONO, EOS, BASO, RBC (erythrocyte count), HGB (haemoglobin concentration), HCT (haematocrit), MCV (mean corpuscular volume), MCH (mean corpuscular haemoglobin), MCHC (mean corpuscular haemoglobin concentration), RET (reticulocyte count), PLT (platelet count), PT (prothrombin time), APTT (activated partial thromboplastin time). PT and APTT were measured using blood plasma, while other haematology parameters used EDTA-anticoagulated whole blood. Serum from non-anticoagulated whole blood collected in separator tubes was analysed for changes in biochemistry parameters.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during euthanasia procedure.
- Animals fasted: No data
- How many animals: 12
- Parameters: ALP (alkaline phosphatase), ALT (serum alanine aminotransferase), AST (serum aspartate aminotransferase), CK (creatine kinase), Urea (urea nitrogen), Crea (creatinine), TP (total serum protein), ALB (albumin), A/G (albumin/globulin ratio), GLU (blood glucose), TBIL (total bilirubin), CHOL (total cholesterol), TG (triglycerides), HDL-c (high-density lipoprotein cholesterol), LDL-c (low density lipoprotein-cholesterol), K+ (potassium), Na+ (sodium), Cl- (chloride). Serum sex hormone levels, including double hydrogen testosterone (DHT, a male sex hormone) and estradiol (E2, the primary ovarian estrogen), were determined by radioimmune analysis using commercially available kits obtained from Diagnostics Systems Laboratories, Inc., Corporate Headquarters, USA and Union Medical & Pharmaceutical Technology Co Ltd., Tianjin, China, respectively.
Sacrifice and pathology:
GROSS PATHOLOGY: Necropsies included examination of the visceral organs, external surface, all orifices, as well as the cranial, thoracic, abdominal and pelvic cavities including viscera. Gross lesions were examined from all animals in all groups. The vital organs from each rat, such as brain, pituitary, heart, liver, spleen, lungs, kidneys, adrenal glands, thymus, testis, epididymides, bladder, ovaries and uterus, were removed and weighed. Paired organs were weighed together. Organ-to-final-body-weight and organ-to-brain-weight ratios were calculated.

HISTOPATHOLOGY: At the time of necropsy, the following tissues and organs were collected: brain, spinal cord (cervical, thoracic and lumbar), gastrointestinal tract (oesophagus, submaxillary gland, stomach, duodenum, jejunum, ileum, cecum, colon and rectum), pancreas, heart, liver, spleen, lungs, kidneys, ureter, prostate, seminal vesicle, epididymis, testis, ovaries, uterus, mammary gland, sciatic nerve, bladder, pituitary gland, adrenal glands, thyroids, parathyroid glands, thymus, trachea, aorta, bone marrow, lymph node (mesenteric lymphoid node, submandibular lymph node). Epididymis and testis were fixed in modified Davidson’s fluid, while others in 10% neutral buffered formalin. After fixation, the collected tissue samples were processed into paraffin blocks. The labelled paraffin blocks were sectioned at 4–8 µm and the paraffin ribbons of the sectioned tissue were placed on clean glass microscope slides.
Upon completion of staining with hematoxylin and eosin, microscopic examinations were first performed on all tissues from all animals in the control and 1250 mg/kg naringin treatment groups euthanized at the scheduled necropsy. If treatment-related changes were noted in a particular organ or tissue in 1250 mg/kg naringin treatment group, extended examination was conducted on the corresponding organs or tissues from lower dose treated groups. In addition, all tissues from rats dying or killed during the study at unscheduled times and all tissues showing macroscopic abnormalities were also examined by histopathologic examination.
Statistics:
The values are expressed as the mean ± SD. Body weight, organ weight (both absolute and relative weights), food consumption, hematology, serum biochemistry and serum sex hormone analyses were tested by conducting One-Way Analysis of Variance (ANOVA) using SPSS 13.0 statistical software. If statistically significant differences were indicated, the least significant difference (LSD) test was employed for comparisons between groups. Levene’s test was used to assess the homogeneity of variances in data. If the variance was not homogeneous, the Kruskal–Wallis test was applied. When statistically significant differences were indicated, the Mann–Whitney U test was employed for inter-group comparison. Statistically significant differences between groups were defined as p < 0.05.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs related to the administration of naringin were observed. From the second month to the sixth month after administration, hair loss was noted on some skin areas including the necks in three female rats from control group, the necks in one female and one male rats from 50 mg/kg group, the necks, ears and/or around the eyes in four female and one male rats from 250 mg/kg of naringin group, the backs, necks, chests, abdomens and femoral regions in three female and two male rats from 1250 mg/kg group. Histopathological examination showed that no any histological changes occurred. Microorganism examination showed that no any pathogenic microorganisms were found on these skin areas. After cessation of naringin administration, hair loss gradually returned to normal and completely disappeared at the end of recovery period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality and clinical signs related to the administration of naringin were observed except that one female rat in 50 mg/kg group and one female rat in 1250 mg/kg group died at days 76 and 24 after dosing, respectively, due to improper intragastric administration on basis of macroscopic and histopathologic examination.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical analysis of mean weekly bw showed consecutive and/or isolated periods of significant decrease in some treatment groups (p < 0.05). However, mean weekly body weights of female rats in 50 mg/kg group on weeks 28–30 were significantly increased (p < 0.05) compared with the corresponding control values.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At weeks 5, 13, 15–16, 21–22, 24, food consumption in 1250 mg/kg group females was less than the corresponding control values (p < 0.05), while more than the control value at week 18.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Any changes observed were neither continuous nor dose-related.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmological abnormalities were identified in the eyes of any study animals over the course of the study.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few significant changes were noted higher HGB level in 50 mg/kg female group, lower NEU percentage in 1250 mg/kg male group, higher RBC, HGB and HCT levels in 250 and 1250 mg/kg male groups at the end of 6-month treatment period.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
After 6 months, the levels of Crea, TP, ALB and TBIL of female rats and the levels of Urea, GLU and Cl of male rats from 1250 mg/kg group were significantly decreased compared with corresponding control values (p < 0.05).
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
All functional behavioral results of the naringin treatment groups of male and female rats were considered comparable to the control groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Liver and spleen-to-body weight ratios were significantly elevated in 250 mg/kg female group (p < 0.05) after 6 months.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of recovery period, some effects were noted in individuals from various dose groups. All of those pathological changes were sporadically detected in control and naringin treatment groups. There existed no pathological lesions in any other tissues. These results indicated that there were no pathological changes in the organs or tissues that could be attributed to naringin treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Various lesions, both after 6 months treatment and after recovery period, in minimal or slight severity; but also found in control group. There were no pathological changes in the organs or tissues that could be attributed to naringin treatment.
After 6 months, the lesions mainly included accumulation of foam cells in pulmonary alveoli and local pneumopleuritis, vacuolar degeneration and focal necrosis of liver cells, chronic progressive nephropathy, renal tubular and/or interstitial calcification, focal myocardial degeneration necrosis and inflammatory cell infiltration in heart. At the end of recovery period, the lesions mainly included accumulation of foam cells in pulmonary alveoli, chronic progressive, focal myocardial degeneration necrosis and inflammatory cell infiltration, and cystic dilatation of gastric glands. The severities of all of these lesions are minimal and were sporadically detected both in control and naringin treatment groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
In present toxicity study, no naringin-related deaths or abnormalities in clinical signs were noted except that reversible and non-pathological hair loss was identified.
In conclusion, daily doses of 50, 250, and 1250 mg/kg of naringin for six months were well tolerated and did not cause either lethality or toxic clinical symptoms and changes in both sexes of rats except for slight body weight decrease and non-pathological and reversible hair loss, which returned to normal after 4 weeks recovery. The NOAEL of naringin is proposed to be greater than 1250 mg/kg/day following daily oral administrations to SD rats for six months. Using the body surface area normalization method, a dose of 1250 mg/kg naringin in rat corresponds to 200 mg/kg in humans, or 12 g for a 60 kg human.
Key result
Dose descriptor:
NOAEL
Effect level:
1 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant effects observed.
Key result
Critical effects observed:
no

Table 1. Histopathological findings in rats treated orally with naringin for 13 weeks. Number of animals with lesions.























































































































































































































































































































 


Organs


 



 


Lesions


 



Administration period



Recovery period



0



1250



0



50



250



1250



Female n=8



Male  n= 8



Female n=8



Male n=8



Female n=4



Male n=4



Male n=1



Male n=1



Female n= 4



Male n=4



 


    Lungs                 


 



Accumulation of foam cells in pulmonary alveoli



1



0



1



1



0



0







1



0



Local pneumopleuritis



0



0



1



0



0



0







0



0



Liver


 


 


 


 



Vacuolar degeneration of liver cells



1



6



0



0



0



4





1



0



2



Focal necrosis of liver cells accompanied with or without haemorrhage



1



1



1



1



0



0





1



1



0



Periportal chronic inflammatory infiltrate



0



1



0



0



0



0





0



0



0



Proliferation of intrahepatic bile-duct



1



0



0



0



0



0





0



0



0



Wegener



0



6



0



0



0



1





0



1



0



Spleen


 


 


 


 



Necrosis, hemorrhage accompanied with fibrotic focus or neutrophil infiltration



0



2



0



0



0



0





0



0



0



Focal episplenitis



0



1



0



0



0



0





0



0



0



Extramedullary hematopoiesis



0



1



0



0



0



0





0



0



0



Splenic capsule cyst



0



1



0



0



0



0





0



0



0



Blood stasis



0



0



0



0



0



0





1



0



0



Kidneys


 


 


 


 



Chronic progressive nephropathy



0



1



0



2



0



3







0



1



Renal tubular and/or interstitial calcification



3



0



0



1



0



0







0



0



Foreign body granuloma accompanied with calcification in renal pelvis



0



0



1



0



0



0







0



0



Interstitial chronic inflammatory infiltrate



0



1



0



0



0



1







0



0



Localized interstitial amyloidosis



0



0



0



1



0



0







0



0



Heart


 


 



Focal myocardial degeneration necrosis and inflammatory cell infiltration accompanied with or without connective tissue



0



6



0



2



0



3



1





0



2



Localized endocarditis



0



0



0



0



0



0



0





0



1



Focal myocardial fibrosis



0



0



0



0



0



1



0





0



0



Stomach



Cystic dilatation of gastric glands



0



0



0



0



0



1







1



2



– Not examined.

Conclusions:
The test item has a NOAEL > 1250 mg/kg bw/d in rats, after 6 months oral administration.
Executive summary:

The subchronic toxicity of naringin was studied on rats for 6 months, by a method according to "Technical Guideline for Long Term Toxicity Test of chemical drugs" (SFDA, 2005), similar to OECD TG 408, with GLP (no certificate available). 8 male and 8 female Sprague Dawley rats were administered the test substance 6 days a week by gavage at doses of 0 (control), 50, 250, or 1250 mg/kg for the 6 -months study, plus 4 males and 4 females that were kept for a 1 -month recovery period (same treatment). During the 6-month treatment period and one month recovery period, no mortality and toxicologically significant changes in clinical signs, opthalmoscopic examination, hematology, clinical biochemistry, serum sex hormone, macroscopic findings, organ weights and histopathological examination were noted and attributed to naringin administration. Although consecutive and/or isolated periods of significant body weights and food consumption decreases were relevant to naringin administration, they were not considered toxicologically significant. In addition, slight, non-pathological and reversible hair loss was noted during the 6-month treatment period and considered as a kind of change possibly relevant to naringin administration; however, it was not considered adverse change and to be of toxicological significance. Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) of naringin in rats is greater than 1250 mg/kg/day when administered orally for 6 consecutive months.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: ‘‘Technical Guideline for Long Term Toxicity Test of chemical drugs’’ (SFDA, 2005 - PR China)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
Animals dosed 6 days/week. Doses tested 50, 250, 1250 mg/kg.
GLP compliance:
yes
Remarks:
Good Laboratory Practice (GLP) Regulations of the State Food and Drug Administration of China.
Limit test:
no
Specific details on test material used for the study:
Naringin (batch No. 20080203) was extracted and purified in the laboratory. Prepared from pulverized Citrus grandis 'Tormentosa' by the following procedures: extracted with water, precipitated by ethanol, and filtered; and then collected and further concentrated the filtrate; the filtrate, on standing, deposited crystals; the precipitate was separated and recrystallized from mixtures of ethanol and water at different ratios; the recrystallized precipitate was dried at 110 C.
Naringin was obtained, and identification was performed by ultraviolet–visible spectroscopy (UV/Vis), electron spray ionization–mass spectrometry, proton nuclear magnetic resonance (1H NMR) and carbon-13 nuclear magnetic resonance (13C NMR) spectroscopy. The purity analysis was performed on a Shimadzu (HPLC) LC-6A instrument (Shimadzu Corp., Kyoto, Japan) with a Dionex C18 column (5 µm, 4.6 mm 250 mm, USA) and a TL9000 Chromatographic Station. The mobile phase was prepared by a 45/55 (v/v) mixture of methanol/water and the pH was adjusted to 3.0 with acetic acid. The injection volume was 20 ll. The UV detector was set at a wavelength of 283 nm. The HPLC purity of naringin was determined to be >98.3% by external standard method.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Male and female Sprague-Dawley (SD) rats, certified specific pathogen-free, were purchased from Slack Shanghai Laboratory Animal Co., Ltd. (Shanghai, China) under the license number SCXK(HU) 2007-0005.
- Age at study initiation: 6 weeks
- Weight at study initiation: 158.2 – 167.4 g for males and 138.4 – 156.4 g for females.
- Housing: Animals were housed in suspended plastic cages with feed and water available ad libitum. 4 animals per cage.
- Acclimation period: 1 week.
- Age: One hundred and seventy-six six-week-old rats.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25ºC
- Humidity (%): 55+-15%
- Air changes (per hr):
- Photoperiod: 12 hrs dark / 12 hrs light.
- Air change: 10-12 cycles/h.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
10mL/kg bw.
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Naringin was dissolved in sterile water for injection and orally administered at a gavage volume of 10 ml/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The purity analysis was performed on a Shimadzu (HPLC) LC-6A instrument (Shimadzu Corp., Kyoto, Japan) with a Dionex C18 column (5 µm, 4.6 mm 250 mm, USA) and a TL9000 Chromatographic Station. The mobile phase was prepared by a 45/55 (v/v) mixture of methanol/water and the pH was adjusted to 3.0 with acetic acid. The injection volume was 20 ll. The UV detector was set at a wavelength of 283 nm. The HPLC purity of naringin was determined to be >98.3% by external standard method.
Duration of treatment / exposure:
13 weeks.
Frequency of treatment:
6 days/week
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 males and 6 females per dose for the 13-weeks study; plus 4 males and 4 females kept for the recovery study, 8 males and 8 females kept for a 6 months study, and 4 males and 4 females kept for the 6-month recovery study.
Control animals:
yes, concurrent vehicle
Details on study design:
3 test groups (44 rats/group, m/f 1:1), subdivided: 12, 8, 16, and 8 animals for 13-week subchronic toxicity, subchronic toxicity recovery, 6-month chronic toxicity and chronic toxicity recovery, respectively.
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (first at the time of dose administration and approximately 1–2 h following dose administration)
- Observations included, but were not limited to, changes in skin, fur, eyes, appearance, genital organ, glandular secretion (salivary gland secretions), oral mucosa, faecal characteristics, respiration, circulation and behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week in the first 4 weeks and weekly thereafter.

FOOD EFFICIENCY:
- Mean daily food consumption was calculated once a week by subtracting the weight of the remaining food from the weight of the supplied food. The 24 h feed intake pattern was measured on rats weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: not specified.
- Dose groups that were examined: all.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during euthanasia procedure.
- Anaesthetic used for blood collection: phenobarbital sodium (60 mg/kg bw.)
- Animals fasted: Yes (night fasting)
- How many animals: 12
- Parameters: WBC (white blood cell count and differential), NEU, LYM, MONO, EOS, BASO, RBC (erythrocyte count), HGB (haemoglobin concentration), HCT (haematocrit), MCV (mean corpuscular volume), MCH (mean corpuscular haemoglobin), MCHC (mean corpuscular haemoglobin concentration), RET (reticulocyte count), PLT (platelet count), PT (prothrombin time), APTT (activated partial thromboplastin time).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during euthanasia procedure.
- Animals fasted: No data
- How many animals: 12
- Parameters: ALP (alkaline phosphatase), ALT (serum alanine aminotransferase), AST (serum aspartate aminotransferase), CK (creatine kinase), Urea (urea nitrogen), Crea (creatinine), TP (total serum protein), ALB (albumin), A/G (albumin/globulin ratio), GLU (blood glucose), TBIL (total bilirubin), CHOL (total cholesterol), TG (triglycerides), HDL-c (high-density lipoprotein cholesterol), LDL-c (low density lipoprotein-cholesterol), K+ (potassium), Na+ (sodium), Cl- (chloride).
Sacrifice and pathology:
GROSS PATHOLOGY: Necropsies included examination of the visceral organs, external surface, all orifices, as well as the cranial, thoracic, abdominal and pelvic cavities including viscera. Gross lesions were examined from all animals in all groups. The vital organs from each rat, such as brain, pituitary, heart, liver, spleen, lungs, kidneys, adrenal glands, thymus, testis, epididymides, bladder, ovaries and uterus, were removed and weighed. Paired organs were weighed together. Organ-to-final-body-weight and organ-to-brain-weight ratios were calculated.

HISTOPATHOLOGY: At the time of necropsy, the following tissues and organs were collected: brain, spinal cord (cervical, thoracic and lumbar), gastrointestinal tract (oesophagus, submaxillary gland, stomach, duodenum, jejunum, ileum, cecum, colon and rectum), pancreas, heart, liver, spleen, lungs, kidneys, ureter, prostate, seminal vesicle, epididymis, testis, ovaries, uterus, mammary gland, sciatic nerve, bladder, pituitary gland, adrenal glands, thyroids, parathyroid glands, thymus, trachea, aorta, bone marrow, lymph node (mesenteric lymphoid node, submandibular lymph node). Epididymis and testis were fixed in modified Davidson’s fluid, while others in 10% neutral buffered formalin. After fixation, the collected tissue samples were processed into paraffin blocks. The labelled paraffin blocks were sectioned at 4–8 µm and the paraffin ribbons of the sectioned tissue were placed on clean glass microscope slides. Upon completion of staining with hematoxylin and eosin, microscopic examinations were first performed on all tissues from all animals in the control and 1250 mg/kg naringin treatment groups euthanized at the scheduled necropsy. If treatment-related changes were noted in a particular organ or tissue in 1250 mg/kg naringin treatment group, extended examination was conducted on the corresponding organs or tissues from lower dose treated groups.
Statistics:
One-Way ANOVA using SPSS 13.0 statistical software. When statistically significant differences were indicated, the least significant difference (LSD) test was employed for comparisons between groups. Levene’s test was used to assess the homogeneity of variances in data. If the variance was not homogeneous, the Kruskal–Wallis test was applied. When statistically significant differences were indicated, the Mann–Whitney U test was employed for inter-group comparison. Statistically significant differences between groups were defined as p < 0.05.
Clinical signs:
no effects observed
Description (incidence and severity):
No mortality or abnormal clinical signs related to the administration of naringin were observed.
Mortality:
no mortality observed
Description (incidence):
No mortality or abnormal clinical signs related to the administration of naringin were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights in female and male rats at high dose were less than control values after weeks 6 and 11. Compared with control group, the decrease in mean body weight in females were significant (p < 0.05) at weeks 7, 8, 12, 13. The ability of the test item to regulate fatty acid, cholesterol and glucose metabolism may be considered as a possible explanation of these alterations. The body weight loss was not associated with other clinical signs, and no related indication of pathological abnormality was observed.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Any changes observed were neither continuous nor dose-related.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Any changes observed were neither continuous nor dose-related.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmological abnormalities were observed in any of naringin treatment or control animals over the course of the study.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Lymphocytes(%) increased at high dose, other parameters unchanged. The alterations in hematology and clinical biochemistry analyses were assumed to be toxicologically irrelevant because they were within normal physiological ranges and were not dose-related or reflected by any changes in other related parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Level of urea decreased at high dose, TBIL levels of all groups were significantly decreased; the other parameters remained unchanged. The alterations in hematology and clinical biochemistry analyses were assumed to be toxicologically irrelevant because they were within normal physiological ranges and were not dose-related or reflected by any changes in other related parameters.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional behavioral results of the naringin treatment groups of male and female rats were considered comparable to control groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute heart and lung weights were significantly decreased in females at high dose, ovary weight and ovary-to-bw ratio significantly increased in female at 250 mg/kg. Heart-to-brain and lung-to-brain weight ratios in the female 1250 mg/kg decreased. However, these changes were not sex or dose-related, were within the normal laboratory ranges, and/or were not supported by any other consistent or toxicologically significant changes in hematological and blood biochemistry analyses and histopathological examination. Therefore, these differences were considered incidental in nature without toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Dirty red spots of 0.1–0.2 cm in diameter scattered irregularly on the surface of liver lobes and isabeling particles of 0.2 cm in diameter were observed at the juncture of left and right middle lobes in one male rat (250mg/kg). No similar signs were observed in any other animal.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
see table 1. Various lesions (minimal severity) for the high dose group. In the histopathological examination, the lesions in the lungs, liver, spleen, kidneys, duodenum, heart and stomach were noted. However, all of those pathological changes were sporadically detected in controls and the rats administrated with 1250 mg/kg of naringin, and no consistent histopathological changes were observed in either sex. Therefore, these lesions could be considered to be spontaneous and/or incidental in nature but not relevant to the treatment
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
No other pathological lesions were found in brain, spinal cord (cervical, thoracic and lumbar cords), gastrointestinal tract (oesophagus, submaxillary gland, jejunum, ileum, cecum, colon and rectum), pancreas, ureter, prostate, seminal vesicle, epididymis, testis (males only), ovary and uterus (females only), mammary gland, sciatic nerve, bladder, pituitary gland, adrenal glands, thyroids, parathyroid glands, thymus, trachea, aorta, bone marrow and lymph node (mesenteric lymphoid node and submandibular lymph node). These results indicated that there were no pathological changes in the organs or tissues that could be attributed to naringin treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
1 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant effects observed
Key result
Critical effects observed:
no

Table 1. Histopathological findings in rats treated orally with test item for 13 weeks.




























































































































Number of animals with lesionControlTest item (1250 mg/kg)
OrgansLesionsFemale (n = 6)Male (n = 6)Female (n = 6)Male (n = 6)
LungsHaemorrhage0010
Interstitial chronic inflammatory infiltrate1111
Interstitial inflammation around thickened wall blood vessels0100
LiverHaemorrhage0001
Vacuolar degeneration of liver cells2510
Wegener1112
SpleenFocal necrosis accompanied with haemorrhage0100
KidneysChronic progressive nephropathy2502
Renal tubular and/or interstitial calcification1000
Renal tubular cystic disease2210
DuodenumFocal chronic inflammatory cell infiltration of intestinal villus0001
StomachCystic dilatation of gastric glands1000
Focal haemorrhage of gastric mucosa0100
HeartFocal myocardial degradation and necrosis accompanied with or without fibrous tissue hyperplasia and inflammatory cell infiltration1103


 


 


Table 2. Absolute and relative weights of organs of female rats treated with test item for 13 weeks.


 




























































































































































































































































ParametersDose (mg/kg/day)
0502501250
Body weight (g)315.5±30.0307.4±21.1286.3±38.2271.6±9.9*
Brain1.92±0.121.98±0.031.98±0.081.89±0.11
Heart1.01±0.111.09±0.080.92±0.090.87±0.11*
Liver8.21±0.427.88±0.557.81±1.097.50±0.46
Spleen0.65±0.090.65±0.040.66±0.160.61±0.09
Lungs1.42±0.191.38±0.071.34±0.101.18±0.04*a
Thymus0.27±0.070.24±0.090.24±0.060.23±0.07
Kidneys1.93±0.191.94±0.171.92±0.341.74±0.15
Adrenals0.064±0.0060.058±0.0070.068±0.0150.055±0.006
Ovaries0.083±0.0120.109±0.0330.115±0.018*0.087±0.018
Uterus0.74±0.180.65±0.270.85±0.200.67±0.29
Pituitary0.015±0.0040.015±0.0020.014±0.0030.015±0.003
Organ-to-body weight ratio (%)
Brain0.61±0.060.64±0.030.70±0.07*0.70±0.05*
Heart0.32±0.020.36±0.040.32±0.030.32±0.03
Liver2.62±0.232.56±0.092.73±0.132.76±0.13
Spleen0.21±0.020.21±0.030.23±0.050.22±0.03
Lungs0.45±0.050.45±0.020.47±0.030.44±0.02
Thymus0.08±0.020.08±0.030.09±0.030.09±0.02
Kidneys0.61±0.040.63±0.030.67±0.040.64±0.04
Adrenals0.021±0.0030.019±0.0030.024±0.0050.020±0.003
Ovaries0.026±0.0020.036±0.0120.041±0.010*0.032±0.007
Uterus0.24±0.070.21±0.100.30±0.090.24±0.10
Pituitary0.0047±0.00160.0047±0.00080.0050±0.00040.0054±0.0009
Organ-to-brain weight ratio (g/g)
Heart0.53±0.070.55±0.040.47±0.040.46±0.05*
Liver4.29±0.163.99±0.233.94±0.483.97±0.31
Spleen0.34±0.030.33±0.030.33±0.080.32±0.03
Lungs0.74±0.070.70±0.030.68±0.04*0.63±0.02*
Thymus0.14±0.040.12±0.040.12±0.030.12±0.04
Kidneys1.01±0.060.98±0.070.97±0.150.92±0.08
Adrenals0.034±0.0040.029±0.0040.035±0.0080.029±0.003
Ovaries0.043±0.0060.055±0.0170.059±0.0110.046±0.010
Uterus0.39±0.090.33±0.140.43±0.120.35±0.15
Pituitary0.0077±0.00210.0073±0.00120.0072±0.00110.0078±0.0014

Values are mean ± SD for 6 rats in each group.
a Data from 5 rats.
* Statistically significant compared to control (p < 0.05).


 


 


Table 3. Haematological values of rats treated orally with test item for 13 weeks.


 



































































































































































 


Parameters



Dosage (mg/kg /day)



0



50



250



1250



WBC (109/L)



4.21 ± 1.96



3.17 ± 2.05



3.26 ± 1.87



3.70 ± 1.95



NEU (109/L)



1.075 ± 1.148



0.453 ± 0.291



0.450 ± 0.256



0.491 ± 0.334



LYM (109/L)



2.86 ± 1.58



2.53 ± 1.69



2.65 ± 1.61



3.05 ± 1.61



MONO (109/L)



0.173 ± 0.177



0.109 ± 0.073



0.086 ± 0.034



0.080 ± 0.072



EOS (109/L)



0.087 ± 0.033



0.060 ± 0.031



0.062 ± 0.032



0.071 ± 0.034



BASO (109/L)



0.014 ± 0.010



0.012 ± 0.008



0.009 ± 0.006



0.011 ± 0.010



NEU% (%)



24.1 ± 15.2



14.6 ± 4.5



14.4 ± 4.1



12.9 ± 4.5



LYM% (%)



69.5 ± 16.9



79.4 ± 4.6



79.8 ± 5.4



82.7 ± 4.8*



MONO% (%)



3.81 ± 2.60



3.49 ± 0.75



3.32 ± 1.77



2.05 ± 1.20



EOS% (%)



2.22 ± 0.68



2.11 ± 0.73



2.20 ± 0.86



2.04 ± 0.53



BASO% (%)



0.356 ± 0.260



0.410 ± 0.243



0.284 ± 0.194



0.288 ± 0.216



RBC (1012/L)



8.08 ± 0.60



7.83 ± 0.48



8.04 ± 0.51



7.98 ± 0.40



HGB (g/L)



145 ± 8



145 ± 6



145 ± 6



146 ± 6



HCT (%)



43.2 ± 2.5



43.0 ± 1.6



43.3 ± 1.9



43.5 ± 1.5



MCV (fL)



53.6 ± 1.7



55.0 ± 1.9



54.0 ± 2.1



54.5 ± 1.6



MCH (pg)



18.0 ± 0.7



18.5 ± 0.6



18.0 ± 0.7



18.3 ± 0.6



MCHC (g/L)



336 ± 4



336 ± 3



335 ± 3



336 ± 3



RET (%)



2.01 ± 0.39



2.03 ± 0.36



2.09 ± 0.24



1.84 ± 0.26



PLT (109/L)



1035 ± 200



940 ± 127



1010 ± 99



960 ± 90



PT (s)



7.2 ± 0.9



7.0 ± 0.8



7.2 ± 0.9



7.5 ± 1.0



APTT (s)



15.6 ± 2.4



15.6 ± 1.9



16.0 ± 1.2



16.0 ± 2.1



Values are mean ± SD for 12 rats in each group except 11 for the control group. Blood sample from one rat in 1250 mg/kg naringin treatment group coagulated. The hematological data were not determined.
* Statistically significant compared to control group (p < 0.05).


 


Table 4. Serum biochemistry of rats treated orally with test item for 13 weeks


 





















































































































































 


Parameters



Dosage (mg/kg /day)



0



50



250



1250



ALP (U/L)



56.4 ± 22.7



52.9 ± 20.8



58.9 ± 20.9



58.8 ± 16.4


ALT (U/L)29.5 ± 6.831.0 ± 9.631.1 ± 10.326.1 ± 7.1
AST (U/L)99.2 ± 27.297.2 ± 23.997.1 ± 17.790.6 ± 27.8
CK (U/L)313 ± 134324 ± 144305 ± 93333 ± 179
LDH (U/L)958 ± 525871 ± 486927 ± 321918 ± 584
Urea (mmol/L)7.54 ± 1.427.57 ± 1.186.66 ± 1.305.77 ± 0.80*
Crea (µmol/L)58.2 ± 13.653.1 ± 9.551.4 ± 8.147.4 ± 8.2
TP (g/L)67.2 ± 5.166.5 ± 5.368.2 ± 5.965.5 ± 2.1
A/G42.9 ± 6.943.8 ± 5.244.1 ± 5.543.7 ± 3.1
ALB (g/L)1.83 ± 0.421.95 ± 0.301.83 ± 0.282.02 ± 0.32
GLU(mmol/L)6.28 ± 1.036.41 ± 0.696.48 ± 0.885.83 ± 0.69
TBIL (µmol/L)3.2 ± 0.72.4 ± 0.6*2.5 ± 0.7*2.2 ± 0.6*
CHOL (mmol/L)1.57 ± 0.471.40 ± 0.261.47 ± 0.451.52 ± 0.38
TG (mmol/L)0.94 ± 0.330.86 ± 0.491.10 ± 0.530.82 ± 0.34
HDL-c (mmol/L)1.48 ± 0.421.38 ± 0.221.37 ± 0.371.49 ± 0.35
LDL-c (mmol/L)0.12 ± 0.060.09 ± 0.040.10 ± 0.050.14 ± 0.09
K+ (mmol/L)3.99 ± 0.373.91 ± 0.213.93 ± 0.253.75 ± 0.33
Na+ (mmol/L)142.1 ± 1.2142.0 ± 1.6142.8 ± 1.3143.0 ± 1.5
Cl- (mmol/L)104.0 ± 1.8103.7 ± 0.8104.5 ± 1.2103.3 ± 0.9

Values are mean ± SD for 12 rats in each group.
* Statistically significant compared to control (p < 0.05).

Conclusions:
The test item has a NOAEL > 1250 mg/kg bw/d in rats, after 90-day oral administration.

Executive summary:

The 90d repeated oral dose toxicity was studied on Sprague-Dawley rats according to the ‘‘Technical Guideline for Acute Toxicity Test of chemical drugs’’(SFDA, 2004, China), similar to OECD 408 (GLP study, certificate not available). The test item was orally administered to 22 male and 22 female Sprague-Dawley rats, at doses of 0 (control), 50, 250 or 1250 mg/kg bw/d. All animals were thoroughly observed immediately after administration for the onset of any toxic signs and once daily thereafter. Survival, feed intake, and body weight were monitored. During the subchronic oral toxicity study, no mortality and toxicologically significant changes in clinical signs, food consumption, opthalmoscopic examination, hematology, clinical biochemistry, serum sex hormone, macroscopic findings, organ weights and histopathological examination except for slight body weight decrease were noted and attributed to naringin administration. Under test conditions, the test item was found to have a NOAEL of 1250 mg/kg in rats.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
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: Clinical sign, urinalysis, haematological and clinical-chemistry examinations were performed as well as monitorisation of body weight and food consumption changes. Gross and histopathological examinations were also carried out.
GLP compliance:
no
Limit test:
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 oral 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
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
daily
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
Details on study design:
MAIN STUDY:
Groups of 50 male and 50 female rats were given diet containing 0, 1.25 and 5.0% test item for 104 weeks and then normal diet for a further 8 weeks. The selection of the highest dose was based on previous reports and also obeyed the guidelines for oncogenicity studies, which recommend 5% as the maximum dietary level.
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 cumulative mortality and 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
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):
Statistically significant increases of relative organ weights of brain in both sexes at 5%, and of the kidneys at 5% in the male group, 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 hyperplasic 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.
Details on results:
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 were 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. Final body (g) and relative organ weights (% body weight) of F344 rats given test item in the diet (Mean±SD).







































































SexGroupNo. of ratsFinal body weight (g)BrainLiverSpleenKidneys
Male0%28424±490.50±0.082.88±0.410.33±0.110.67±0.10
1.25%33420±570.51±0.093.05±0.440.43±0.310.68±0.10
5.0%34387±43**0.55±0.072.78±0.530.47±0.530.73±0.10*
Female0%32304±340.63±0.072.42±0.390.28±0.220.68±0.19
1.25%31292±310.66±0.082.40±0.260.37±0.440.64±0.08
5.0%36284±320.68±0.08*2.60±0.510.50±0.790.66±0.08

*,** Significantly different from the control group values at P<0.05 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 tumorMalesFemales
0%1.25%5.0%0%1.25%5.0%
Effective no.of rats505050505050
Small intestine
Adenocarcinoma1(2)1(2)0000
Leiomyoma1(2)00000
Cecum
Hyperplastic polyp0011(22)a002(4)
Inflammatory polyp000001(2)
Adenoma001(2)000
Adenocarcinoma002(4)000
Sarcoma, NOS001(2)000
Colon
Adenoma000002(4)
Liver
Focus (area) of cellular alteration19(38)21(42)14(28)25(50)22(44)10(20)b
Hyperplastic (neoplastic) nodule2(4)2(4)3(6)2(4)00
Bile duct hyperplasia47(94)44(88)0a1(2)00
Urinary bladder
Transitional cell hyperplasia01(2)1(2)003(6)
Papillomatosis001(2)001(2)
Transitional cell papilloma1(2)0001(2)0
Leiomyoma1(2)00000

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


 


Table 3. 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 tumorMalesFemales
0%1.25%5.0%0%1.25%5.0%
Effective no. of rats505050505050
Heart
Sarcoma, NOS00001(2)0
Spleen
Lymphoma0001(2)00
Fibroma01(2)0000
Hemangiosarcoma001(2)000
Leiomyosarcoma001(2)000
Pituitary
Adenoma, pars distalis14(28)12(24)9(18)20(40)19(38)17(34)
Adenoma, pars intermedia1(2)1(2)1(2)01(2)0
Craniopharyngioma01(2)0000
Carcinoma, pars distalis02(4)1(2)2(4)4(8)0
Thyroid
Follicular cell adenoma1(2)00000
C-cell adenoma5(10)8(16)3(6)3(6)02(4)
Follicular cell carcinoma001(2)000
C-cell carcinoma03(6)1(2)1(2)1(2)1(2)
Parathyroid
Adenoma1(2)0001(2)0
Adrenals
Pheochromocytoma4(8)8(16)6(12)2(4)1(2)0
Ganglioneuroma2(4)00000
Cortical carcinoma0002(4)00
Malignant pheochromocytoma2(4)1(2)2(4)1(2)00
Malig. ganglioneuroma-pheochromocytoma0002(4)00
Lungs
Adenoma1(2)2(4)01(2)00
Adenocarcinoma1(2)01(2)000
Esophagus
Fibroma00001(2)0
Stomach
Squamous cell papilloma00001(2)0
Squamous cell carcinoma0001(2)00
Pancreas
Acinar cell adenoma001(2)000
Islet-cell adenoma2(4)1(2)2(4)000
Kidneys
Adenoma1(2)00000
Adenocarcinoma01(2)0000
Transtional cell carcinoma01(2)0000
Nephroblastoma1(2)00000
Testes
Interstitial cell tumor48(96)47(94)47(94)---
Mlignant interstitial cell  tumor01(2)1(2)---
Prostate
Adenoma9(18)12(24)6(12)---
Adenocarcinoma3(6)1(2)1(2)---
Preputial/clitoral gland
Adenoma2(4)4(8)3(6)1(2)2(4)4(8)
Adenocarcinoma1(2)1(2)2(4)000
Mammary gland
Adenoma1(2)001(2)00
Fibroadenoma6(12)11(22)9(18)15(30)8(16)4(8)a
Adenocarcinoma001(2)02(4)0
Ovaries
Granulosa/Theca cell tumor---1(2)00
Uterus
Adenoma---1(2)00
Endometrial stromal polyp---8(16)10(20)12(24)
Adenomatous polyp---01(2)0
Adenocarcinoma---1(2)1(2)3(6)
Endometrial stromal sarcoma---2(4)1(2)1(2)
Leiomyosarcoma---2(4)00
Fibrosarcoma---1(2)00
Sarcoma, NOS---01(2)0
Skin/subcutis
Squamous cell papilloma3(6)2(4)3(6)000
Fibroma11(22)9(18)10(20)3(6)2(4)4(8)
Lipoma3(6)00000
Lymphangioma01(2)0000
Squamous cell carcinoma1(2)00000
Basal cell carcinoma01(2)1(2)001(2)
Sebaceous carcinoma1(2)00000
Fibrosarcoma1(2)01(2)000
Malignant fibrous histiocytoma2(4)1(2)01(2)1(2)1(2)
Liposarcoma01(2)0000
Sarcoma, NOS001(2)000
Malignant schwannoma0001(2)00
Brain
Granular cell tumor001(2)000
Astrocytoma1(2)00000
Mixed glioma000001(2)
Spinal cord
Astrocytoma01(2)0000
Peripheral nerve
Malignant schwannoma01(2)0000
Thoracic cavity
Mesothelioma1(2)00000
Abdominal cavity
Lipoma002(4)000
Liposarcoma00001(2)0
Mesothelioma5(10)3(6)0000
Malignant fibrous histiocytoma000001(2)
All sites
Malignant lymphoma/leukemia6(12)18(36)a14(28)8(16)14(28)17(34)
Site unknown
Malignant fibrous histiocytoma01(2)0000
Others
Odontoma01(2)0000
Lipoma001(2)000
Osteosarcoma0001(2)00

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

Conclusions:
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.
Executive summary:

A chronic repeated dose toxicity 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 in 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.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: Designation of Food Additives and for Revision of Standards for Use of Food Additives of Japan (1996).
Deviations:
no
GLP compliance:
not specified
Remarks:
The paper does not specify if the test is conducted under GLP conditions.
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): Enzymatically decomposed rutin obtained from San-Ei Gen F. F. I. (Osaka, Japan)
- Purity: 95% isoquercitrin
Species:
rat
Strain:
Wistar
Details on species / strain selection:
[BrlHan: WIST@Jcl (GALAS)]
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: CLEA Japan Inc. (Shizuoka, Japan)
- Age at study initiation: 4 weeks old
- Weight at study initiation: 144.6-162.4 g (males) and 112.5-126.8 g (females)
- Fasting period before study: no
- Housing: individually in stainless steel cages
- Diet: ad libitum
- Water: tap water ad libitum
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: powdered basal diet CE-2 (CLEA Japan Inc., Tokyo,
Japan) which was then pelleted and sterilized by c-ray irradiation at 10 kGy (CLEA
Japan Inc., Tokyo, Japan)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1–24.3°C
- Humidity (%): 39.3–73.2%
- Air changes (per hr): 10–25 times/h
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle
Route of administration:
oral: feed
Details on route of administration:
The test item was mixed at concentrations of 0%, 0.04%, 0.2%, 1% and 5% into the diet
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): The pelleted diet containing the test substance was replaced at least once every 2 weeks.
- Mixing appropriate amounts with (Type of food): powdered basal diet CE-2
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For confirmation of concentration and homogeneity in each dose level of the diet, three samples (upper, middle and lower part) each in the mixed powder diets of the first batches were analyzed and it was confirmed that their concentration and homogeneity were within acceptable range (the average recovery rates from the 0.04%, 0.2%, 1% and 5% diet were 94.4%, 95.4%, 98.0% and 99.0%, respectively). In addition, the stability of the test item in this pellet diet was analyzed and it was also confirmed that their contents were within an acceptable range (the average recovery rates from the 0.04%, 0.2%, 1% and 5% diet were 79.6%, 82.2%, 85.5% and 91.4%, respectively).
Duration of treatment / exposure:
52 weeks
Frequency of treatment:
The pelleted diet containing the test item was replaced at least once every 2 weeks.
Dose / conc.:
0.04 other: % in diet
Dose / conc.:
0.2 other: % in diet
Dose / conc.:
1 other: % in diet
Dose / conc.:
5 other: % in diet
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dietary levels of enzymatically decomposed rutin in the diet were determined to be 0%, 0.04%, 0.2%, 1% or 5%, based on the results of the previous 13-week dietary administration study.
- Fasting period before blood sampling for clinical biochemistry: no
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: weekly until week 13 and every 4 weeks thereafter, and the body weights at week 51 and at the end of the experiment were also recorded.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule: Food consumption was measured weekly until week 14 and every 4 weeks thereafter, and then the consumption during the terminal week (at week 52) were recorded.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of the experiment
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: all of them
- Parameters checked: red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (Plt), white blood cell count (WBC), reticulocyte percentage and differential leukocyte counts (neutrophil, lymphocyte, monocyte, eosinophil, basophil and large unstained cells).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of the experiment
- Animals fasted: No
- How many animals: all of them
- Parameters checked: glucose (Glu), total protein (TP), albumin (Alb), albumin/globulin ratio (A/G), total cholesterol (T-Cho), triglyceride (TG), total bilirubin (TBil), gamma-glutamyltranspeptidase (g-GTP), alanine transaminase (ALT), aspartatetransaminase (AST), alkaline phosphatase (ALP), urea nitrogen (BUN), creatinine (Cre), creatine kinase (CK), calcium (Ca), inorganic-phosphorus (IP), sodium (Na), potassium (K) and chloride (Cl).

URINALYSIS: Yes
- Time schedule for collection of urine: week 50
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked: pH, protein, glucose, occult blood, ketone, bilirubin and urobilinogen.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
PLASMA/SERUM HORMONES/LIPIDS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The following organs were weighed and the weights relative to the final body weight were calculated: brain, thymus, heart, lungs, liver, spleen, adrenals, kidneys, testes, ovaries, pituitary and thyroids (including parathyroid glands). The pituitary and thyroid glands were weighed after being fixed in 10% phosphate-buffered formalin. In addition to these organs, the submandibular glands, sublingual glands, epididymides, prostate, seminal vesicle, uterus, vagina, parotid glands, extraorbital lacrimal glands, submandibular lymph nodes, eye (left), harderian gland (left), urinary bladder, skin, subcutaneous tissue including mammary glands, preputial gland, clitoral gland, sternum (including bone marrow), femurs (including bone marrow), sciatic nerves, skeletal muscle (thigh), esophagus, stomach, duodenum, jejunum, ileum (including Peyer’s patches), cecum, colon, rectum, mesenteric lymph nodes, pancreas, spinal cord, trachea, tongue, aorta, Zymbal’s glands, eyes and masses found in the animals sacrificed in moribund condition were removed and fixed in 10% buffered formalin. Testes and epididymides were fixed in formalin–acetic acid fixative and the eye with Harderian gland was fixed in Davidson’s solution.

HISTOPATHOLOGY: Yes
Paraffin-embedded tissue sections of all organs/tissues mentioned above were routinely prepared and stained with hematoxylin and eosin for histopathological assessment.
Statistics:
For the body weights, food consumption, urinalysis (quantitative analyzes only), hematology, clinical chemistry, and absolute and relative organ weights of animals, homogeneity of variance was examined by Bartlett’s test. When variance between the groups was confirmed to be homogenous, the difference between the control group and each treated group was examined by Dunnett’s test for multiple comparisons. When variance between the groups was not homogenous, Dunnett-type rank test was performed for comparisons between the control and each treated group. The incidences of lesions with grading and the number of animals with lesions were compared by the cumulative chi-squared test and the Fisher’s exact probability, respectively. Significance was inferred at either p < 0.05 or p < 0.01.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Endocrine findings:
not examined
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Significant increase in mean urinary Ca concentration was observed in males of the 5% group. In addition, significant increase in daily urinary Ca excretion was observed in both sexes in the 5% groups. Daily potassium and chloride excretion significantly increased, but to a small degree in males of the 1% and 5% groups.
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):
Mineralization in the renal pelvis significantly increased in males of the 5% group. In addition, the total number of males in the 5% group with findings in the renal pelvis, including inflammatory cell infiltration, inflammatory cell debris, transitional cell hyperplasia or mineralization, was significantly greater than that of the control group.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Two control males sacrificed at the early stage of the treatment were diagnosed as hemangiosarcoma and undifferentiated sarcoma, respectively.
One male in the 5.0% group sacrificed at week 39 was histologically diagnosed as having myeloid leukemia. One female of the 5.0% group was sacrificed and extrahepatic cholangitis, coagulative necrosis of the liver and pancreatic abscess were found. These lesions were incidental and unrelated to the test substance treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
1 other: % in diet
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
urinalysis
Remarks on result:
other: 542.4 mg/kg bw day
Key result
Dose descriptor:
NOAEL
Effect level:
1 other: % in diet
Based on:
test mat.
Sex:
female
Basis for effect level:
urinalysis
Remarks on result:
other: 674 mg/kg bw day
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 other: % in diet
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1. Food consumption and chemical intake in rats fed diet containing the test item for 52 weeks.










































































































 


Dose level (%)



Body weight (g)



 



 



 



 


Initial                                       



Terminal



Food consumption


(g/rat/day)



Intake of test article


(mg/kg/day)



Intake of test article adjusted by the recovery rates in the pellet diet (mg/kg/day)



Male



 


0


153.5±8.9 (20)

535.3 ± 68.7 (18)



22.8 ± 0.9







0.04



154.2±7.9 (20)



550.5 ± 73.4 (20)



23.0 ± 0.9



25.2 ± 0.7



20.1



0.2



153.6±8.6 (20)



553.7 ± 42.9 (20)



23.0 ± 1.1



125.5 ± 1.9



103.2



1



153.5 ± 8.0 (20)



525.4 ± 48.2 (20)



22.3 ± 1.1



634.4 ± 12.5



542.4



5



154.7 ± 7.7 (20)



562.7 ± 53.3 (19)



24.8 ± 1.5



3330.0 ± 52.3



3046.4



Female



 


0



120.3 ± 6.2 (20)



326.8 ± 50.5 (20)



17.6 ± 0.7







0.04



119.5 ± 7.2 (20)



336.4 ± 43.6 (20)



17.7 ± 0.7



31.3 ± 0.7



24.9



0.2



118.4 ± 5.9 (20)



336.0 ± 41.3 (20)



17.7 ± 0.8



157.8 ± 3.9



129.7



1



118.5 ± 6.0 (20)



333.5 ± 37.2 (20)



17.6 ± 0.8



788.3 ± 17.1



674.0



5



120.6 ± 6.2 (20)



326.2 ± 35.2 (19)



18.1 ± 1.0



4066.4 ± 64.7



3716.7



Data are mean ± S.D. values.


Numbers of animals that survived are expressed in parenthesis.


 


Table 2. Histopathological findings in kidney for male rats fed diet containing the test item for 52 weeks.





















































































































































































Organ



Findings



Grade



Dose level (%)



0



0.04



0.2



1



5



No. of animals examined:



18



20



20



20



19



Kidney


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 



Chronic nephropathy



+a



5



5



3



2



4



++



2


0

1



0



0



Total



7


5

4



2



4



Dilatation, tubules



+



1



0



0



0



0



Mineralization, cortico-medullary junction



+



0



0



0



0



0



Dilatation, pelvis



+



0



2



0



0



0



Cell debris, inflammatory, pelvis



+



6



5



3



7



10



++



1



0



0



0



1



Total



7



5



3



7



11



Cellular infiltration, inflammatory, pelvis



+



2



0



2



2



5



++



1



0



1



0



0



Total



3



0



3



2



5



Hyperplasia, transitional cell, pelvis



+



2



4



3



2



8



++ Total



1



0



0



0



0



Mineralization, pelvis



+



0



5



3



4



12



++



1



0



0



0



1



Total



1



5



3



4



13*



No. of animals with findings in pelvis



 



7



8



5



8



15*



a +, Slight; ++, moderate.
* Significantly different from the controls at p < 0.05.


 


Table 3. Histopathological findings in kidney for female rats fed diet containing the test item for 52 weeks.




















































































































































Organ



Findings



Grade



Dose level (%)



0



0.04



0.2



1



5



No. of animals examined:



20



20



20



20



19



Kidney


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 



Chronic nephropathy



+a



1



1



1



0



1



Mineralization, cortico-medullary junction



+



15



13



14



13



19



++



1



1



1



0



0



Total



16



14



15



13



19



Dilatation, pelvis



+



1



0



0



1



0



++



0



0



0



0



1



Total



1



0



0



0



1



Cell debris, inflammatory, pelvis



+



3



2



1



3



2



Cellular infiltration, inflammatory, pelvis



+



0



1



1



1



0



Hyperplasia, transitional cell, pelvis



+



1



1



1



5



1



Mineralization, pelvis



+



11



7



5



8



6



++



0



1



1



1



2



Total



11



8



6



9



8



No. of animals with findings in pelvis



 



11



8



7



11



8



a +, Slight; ++, moderate.

Conclusions:
The no observed adverse effect level (NOAEL) was estimated to be 1% in diet for both sexes (542.4 mg/kg body weight/day in males and 674.0 mg/kg body weight/day in females).
Executive summary:

A 52-week repeated dose toxicity study was conducted with the test item according to the Designation of Food Additives and for Revision of Standards for Use of Food Additives of Japan Guideline (1996). The administration of the test item was conducted by  mixing it with the feed in four different concentrations : 0.04%, 0.2%, 1.0% and 5.0% mg/kg bw/day in diet. Also, a control group was monitored. 20 males and 20 females were distributed per group and were administered the test item on a daily basis for 52 weeks. No significant effects were observed in the clinical sign, haematological and clinical-chemistry examinations. The monitorisation of body weight and food consumption changes was also performed, with no test item-related adverse effects observed. Necropsy was performed for all animals and the relative weights calculated. The hispathological examinations revealed that the test item induced mineralization in the renal pelvis often associated with inflammatory cell debris, inflammatory cell infiltration and/or transitional cell hyperplasia in males of the 5% group. Urinalysis showed an increased daily calcium excretion in both sexes of the 5% group, and an increase in urinary calcium concentration was observed in the male 5% group. Therefore, the no-observed-adverse-effect level (NOAEL) of the test item in Wistar rats were 1% in diet for both sexes (542.4 mg/kg body weight/day in males and 674.0 mg/kg body weight/day in females).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: Designation of Food Additives and for Revision of Standards for Use of Food Additives of Japan (1996).
Deviations:
no
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): Enzymatically decomposed rutin obtained from San-Ei Gen F. F. I. (Osaka, Japan)
- Purity: 95% isoquercitrin
Species:
rat
Strain:
Wistar
Details on species / strain selection:
[BrlHan: WIST@Jcl (GALAS)]
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Crea Japan (Hamamatsu, Japan)
- Age at study initiation: 5 weeks old
- Weight at study initiation: 161.5±5.3 g (0%), 161.9±6.1 g (0.2%), 160.0±2.8 g (1%) and 162.0±4.7 g (5%) (males) and 130.4±5.5 g (0%), 130.8±4.1 g (0.2%), 132.6±5.2 g (1%) and 133.5±4.9 g (5%)(females)
- Fasting period before study: no
- Housing: The animals were housed three to four rats per plastic cage, with sterilized soft wood chips.
- Diet: ad libitum.
- Water: tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2ºC
- Humidity (%): 55±5%
- Air changes (per hr): 18 times/h
- Photoperiod (hrs dark / hrs light): 12h light/dark cycle
Route of administration:
oral: feed
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
The test substance-supplemented diet was replaced at least once every 2 weeks.
Dose / conc.:
0 other: % in diet
Remarks:
Control group
Dose / conc.:
0.2 other: % in diet
Dose / conc.:
1 other: % in diet
Dose / conc.:
5 other: % in diet
No. of animals per sex per dose:
10 males and 10 females per dose and control groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Other:
The animals received the test item at concentrations of 0 (control), 0.2, 1 or 5% in the diet for 13 weeks, based on a previous 2-week palatability study, in which no test substance-related findings other than yellowish urine were observed at the high-dose level of 5%.
Clinical signs and mortality were observed daily. Body weights and food consumption were measured weekly. At the end of the experiment, all animals were fasted overnight and euthanized by exsanguination under ether anesthesia after blood sample collection from the abdominal aorta for hematology and blood biochemistry.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-Time schedule: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of the experiment
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (Plt), white blood cell count (WBC),differential leukocyte counts and reticulocyte count (Ebl)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of the experiment
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: total protein (TP), albumin (Alb), albumin/globulin ratio (A/G), total cholesterol (T-Cho), triglyceride (TG), total bilirubin (T-Bil), g-glutamyl transpeptidase (g-GTP), alanine transaminase (AlT), aspartate transaminase (AsT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (Cre), calcium (Ca), inorganic-phosphorus (IP), sodium (Na), potassium (K) and chloride (Cl).

PLASMA/SERUM HORMONES/LIPIDS: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were subjected to a complete necropsy. Brain, heart, lungs, liver, kidneys, spleen, adrenals and testes were weighed. In addition to these organs, nasal cavity, trachea, aorta, pituitary, thyroids, parathyroids, salivary glands, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, urinary bladder, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, mammary gland, skin, mesenteric and submandibular lymph nodes, thymus, sternum, femur including bone marrow, sciatic nerve, trigeminal nerve, spinal cord (cervical, thoracic and lumber cords), eyes, and thigh muscle were excised.

HISTOPATHOLOGY: Yes
All these organs and tissues were fixed in 10% buffered formalin. Testes of five males each of the control and 5% groups were fixed in Bouin’s solution. Paraffin-embedded tissue sections of all organs/tissues were routinely prepared and stained with hematoxilin and eosin for histopathological assessment. All organs and tissues in the control and 5% group animals were compared.
Statistics:
Variance in data for body weights, hematology, serum biochemistry and organ weights (both absolute and relative weights) was checked for homogeneity by Bartlett’s procedure. When the data were homogeneous, one-way analysis of variance (ANOVA) was used. In the heterogeneous cases, the Kruskal–Wallis test was applied. When statistically significant differences were indicated, Dunnett’s multiple test was employed for comparison between control and treated groups.
Clinical signs:
no effects observed
Description (incidence and severity):
No obvious clinical signs, except for yellowish urine in 5% males and females were found in any animals throughout the experimental period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effects on body weight were observed in females, whereas 5% group males showed slight reduction of body weight gain as compared to the control group (Tables 1 and 2).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
A good correlation was observed between the expected dose of the test substance and the actual daily intake (Table 3).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decrease in erythrocyte parameters, i.e. Hb and Ht, and tendency to decrease in RBC detected in the 5% males.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The 5% males showed significant decrease in TG, considered partly related to inhibition of body weight gain. An increase of BUN and Cl in the 1% males and females, respectively, was noted.
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:
effects observed, treatment-related
Description (incidence and severity):
lungs and testes relative weights in the 5% males were slightly increased.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no obvious macroscopic findings were in any group of animals of either sex
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological observation also revealed no significant findings in control and 5% group males and females.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 other: % in diet
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
Remarks on result:
other: equivalent to 539 mg/kg bw/day
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 3227 mg/kg bw/day
Key result
Critical effects observed:
no

Table 1. Body weight change in male rats fed diet containing test item for 13 weeks






















































































































Dose level (%)



0



0.2



1



5



Week/Animals



10



10



10



10



0



161.5±5.3



161.9±6.1



160.0±2.8



162.0±4.7



1



203.7±9.3



212.5±10.4



210.4±6.6



207.0±9.0



2



249.1±13.9



252.8±14.9



254.0±10.1



255.5±13.3



3



295.2±19.1



302.0±19.3



298.9±15.0



295.3±20.7



4



322.9±21.1



330.1±22.3



326.4±18.5



322.5±23.9



5



352.6±24.7



359.9±27.6



353.5±22.2



347.8±27.0



6



372.1±28.9



380.1±29.9



373.6±25.3



365.4±30.6



7



391.6±31.7



399.8±30.7



393.7±26.9



385.7±33.3



8



406.1±33.8



415.2±32.9



409.3±29.7



399.9±38.5



9



417.3±36.3



422.2±31.3



428.0±32.9



410.0±40.4



10



430.3±38.9



436.9±34.3



430.2±32.3



424.1±42.5



11



441.440.4



444.333.8



441.435.3



428.7±44.4



12



451.5±40.3



452.6±35.8



447.6±35.2



436.3±46.4



13



459.7±42.5



461.6±37.4



456.9±36.2



441.0±43.6



Values are mean S.D.


 


Table 2. Body weight change in female rats diet containing test item for 13 weeks






















































































































Dose level (%)



0



0.2



1



5



Week/Animals



10



10



10



10



0



130.4±5.5



130.8±4.1



132.6±5.2



133.5±4.9



1



152.8±7.3



158.1±7.2



153.4±4.7



149.1±9.9



2



174.8±10.7



181.1±13.0



173.4±5.3



172.3±11.7



3



193.0±12.7



202.2±19.0



192.6±7.3



192.7±14.1



4



206.9±12.4



216.9±16.5



197.3±18.0



201.2±14.0



5



218.5±14.0



229.9±18.8



218.3±9.0



218.3±17.9



6



226.3±14.4



237.7±20.8



225.4±10.1



226.8±17.2



7



235.5±16.4



249.0±19.4



236.6±12.3



235.2±16.5



8



239.5±15.9



257.2±19.8



244.1±12.6



242.0±18.9



9



242.5±16.1



260.8±21.2



247.0±12.4



246.1±19.5



10



245.4±17.4



262.9±22.6



248.4±15.6



250.1±18.4



11



248.7±17.9



264.7±21.7



249.1±13.3



249.7±16.9



12



254.4±18.4



266.8±22.1



254.2±14.4



253.7±18.1



13



255.5±17.7



270.0±22.4



256.8±13.1



260.1±19.5



Values are mean S.D.


 


Table 3. Food consumption and chemical intake in rat fed diet containing test item for 13 weeks











































































Sex



Dose level (%)



No. of Animals



Food consumption g/rat/day



Intake of test item mg/kg/day



Initial



Terminal



Male



0 (Control)



10



10



19.8±1.5*





0.2



10



10



19.7±2.1



108±19 *



1



10



10



19.3±2.4



539±113



5



10



10



19.1±2.7



2694±442



Female



0 (Control)



10



10



13.8±0.7





0.2



10



10



13.8±1.7



118±13



1



10



10



13.7±0.8



627±98



5



10



10



14.2±1.6



3227±396



*Values are mean S.D.


 


Table 4. Hematological changes in male rats fed diet containing test item for 13 weeks
































































































































Dose level (%)



0



0.2



1



5



No. of animals



10



10



10



10



RBC (1010/L)



897±48



886±32



889±62



857±39



Hb (g/dL)



15.5±0.8



15.1±0.4



15.4±0.9



14.6±0.6*



Ht (%)



45.0±2.1



43.7±1.3



44.4±2.5



42.5±1.9*



MCV (fL)



50.2±1.0



49.3±1.1



50.0±1.0



49.6±1.3



MCH (pg)



17.2±0.4



17.1±0.5



17.3±0.3



17.0±0.4



MCHC (g/dL)



34.3±0.3



34.7±0.4



34.6±0.6



34.3±0.4



Plt (1010/L)



94.81±1.1



92.1±9.6



98.2±11.4



92.2±7.7



Ebl count/200 WBC



0.0±0.0



0.0±0.0



0.2±0.6



0.2±0.4



WBC (108/L)



47.4±9.9



43.1±8.3



46.8±11.7



45.1±8.4



Differential cell count(%)



Band



0.1±0.2



0.0±0.0



0.0±0.0



0.0±0.0



Seg



17.7±7.2



18.1±7.0



16.5±5.8



20.6±4.1



Eosino



1.7±1.3



1.9±0.8



1.7±0.9



1.0±0.7



Baso



0.0±0.0



0.0±0.0



0.0±0.0



0.0±0.0



Lympho



79.9±7.3



79.7±7.8



81.7±6.4



78.2±4.6



Mono



0.7±0.7



0.3±0.4



0.2±0.2



0.3±0.4



Values are mean S.D. *Significantly different from the control at P<0.05.


 


Table 5. Hematological changes in female rats fed diet containing test item for 13 weeks
































































































































Dose level (%)



0



0.2



1



5



No. of animals



10



10



10



10



RBC (1010/L)



804±54



786±42



784±37



781.3±46



Hb (g/dL)



14.6±0.7



14.3±0.6



14.4±0.6



14.5±0.6



Ht (%)



41.9±2.5



41.2±1.9



41.2±1.6



41.7±2.0



MCV (fL)



52.2±0.9



52.4±1.4



52.6±1.5



53.4±1.1



MCH (pg)



18.2±0.5



18.3±0.6



18.4±0.6



18.5±0.6



MCHC (g/dL)



34.8±0.8



34.8±0.4



35.0±0.4



34.7±0.6



Plt (1010/L)



90.6±6.6



97.2±7.0



97.6±7.6



95.7±9.8



Ebl count/200 WBC



0.6±0.7



0.6±0.7



0.8±1.1



0.1±0.3



WBC (108/L)



26.9±4.6



27±5.9



29.0±7.3



32.5±7.7



Differential cell count (%)



Band



0.1±0.2



0.1±0.2



0.0±0.0



0.1±0.2



Seg



15.4±5.2



15.6±5.1



14.9±3.5



15.5±5.7



Eosino



2.0±1.2



1.4±0.7



1.4±0.9



1.4±1.2



Baso



0.0±0.0



0.0±0.0



0.0±0.0



0.0±0.0



Lympho



81.7±5.3



82.5±5.0



83.0±3.7



82.7±5.6



Mono



0.9±0.6



0.5±0.2



0.8±0.5



0.4±0.2*



Values are mean S.D. *Significantly different from the control at P<0.05.


 


Table 6. Serum biochemistry findings for male rats fed diet containing
test item for 13 weeks











































































































































Dose level (%)



0



0.2



1



5



No. of animals



10



10



10



10



TP (g/dL)



6.4±0.1



6.3±0.2



6.3±0.2



6.3±0.1



Alb (g/dL)



4.5±0.2



4.4±0.2



4.5±0.2



4.5±0.2



A/G



2.5±0.4



2.4±0.2



2.5±0.3



2.5±0.3



T-Cho (mg/dL)



60.2±14.7



64.4±14.7



69.6±16.1



61.7±8.8



TG (mg/dL)


128.2±25.7102.3±26.0113.2±36.2

88.4±23.9**



T-BiL (mg/dL)


0.15±0.050.12±0.040.11±0.03

0.10±0.00*



g-GTP (IU/L)



<2



<2



<2



<2



AlT (IU/L)



34.9±7.1



32.1±5.3



34.1±6.9



35.0±5.8



AsT (IU/L)



83.5±12.0



80.2±10.6



74.9±10.3



81.7±21.5



ALP (IU/L)



185.0±24.2



167.7±21.6



179.1±37.9



202.6±31.7



BUN (mg/dL)



17.2±1.2



16.6±1.8



19.7±2.7*



16.6±1.1



Cre (mg/dL)



0.28±0.04



0.28±0.04



0.28±0.04



0.28±0.04



Ca (mg/dL)



10.2±0.2



10.0±0.3



10.2±0.2



10.2±0.2



IP (mg/dL)



5.8±0.6



5.5±0.4



5.5±0.6



5.0±0.4**



Na (mEQ/L)



146.7±3.0



145.5±3.5



146.1±2.1



147.7±2.2



K (mEQ/L)



4.7±0.3



4.6±0.2



4.5±0.3



4.6±0.4



Cl (mEQ/L)



109.2±2.9



108.6±3.2



109.3±1.7



110.1±1.3



Values are mean S.D. *Significantly different from the control at P<0.05. **Significantly different from the control at P<0.01.


 


Table 7. Serum biochemistry findings for female rats fed diet containing
test item for 13 weeks











































































































































Dose level (%)



0



0.2



1



5



No. of animals



10



10



10



10



TP (g/dL)



7.0±0.3



6.7±0.3



7.0±0.4



6.7±0.4



Alb (g/dL)



5.4±0.4



5.2±0.2



5.4±0.3



5.4±0.4



A/G



3.5±0.5



3.5±0.7



3.4±0.6



4.0±0.5



T-Cho (mg/dL)



57.0±20.4



60.6±17.6



57.7±12.2



58.5±17.1



TG (mg/dL)



31.5±15.2



31.9±21.3



32.3±28.2



28.0±21.2



T-BiL (mg/dL)



0.16±0.05



0.11±0.03



0.14±0.05



0.12±0.06



g-GTP (IU/L)



<2



<2



<2



<2



AlT (IU/L)



25.8±3.6



22.6±2.5



25.2±5.0



26.9±7.5



AsT (IU/L)



72.4±9.6



73.3±9.8



74.2±15.3



81.3±22.7



ALP (IU/L)



74.2±17.5



73.3±19.1



64.5±17.0



69.9±21.3



BUN (mg/dL)



16.1±2.1



15.1±1.6



17.1±2.8



15.8±1.7



Cre (mg/dL)



0.31±0.03



0.30±0.00



0.31±0.03



0.30±0.00



Ca (mg/dL)



10.3±0.4



10.1±0.2



10.1±0.3



10.1±0.3



IP (mg/dL)



5.7±0.6



5.3±0.4



5.2±0.6



5.2±0.6



Na (mEQ/L)



145.8±1.5



146.0±2.1



148.2±2.3



145.7±3.6



K (mEQ/L)



4.2±0.3



4.4±0.2



4.4±0.3



4.2±0.2



Cl (mEQ/L)



108.6±1.7



110.2±2.2



111.5±1.8*



108.8±3.1



Values are mean S.D. *Significantly different from the control at P<0.05.


 


Table 8. Organ weights for male rats fed diet containing test item for 13 weeks










































































































































Dose level (%)



0



0.2



1



5



No. of animals



10



10



10



10



Body weight (g)



444.0±40.3



445.9±37.5



438.4±35.6



422.1±43.5



Absolute



Brain (g)



2.08±0.13



2.05±0.08



2.08±0.08



2.03±0.09



Heart (g)



1.08±0.07



1.09±0.09



1.13±0.11



1.05±0.09



Lungs (g)



1.31±0.15



1.31±0.16



1.34±0.11



1.36±0.16



Kidneys (g)



2.45±0.26



2.44±0.23



2.46±0.28



2.50±0.24



Spleen (g)



0.70±0.10



0.71±0.15



0.73±0.09



0.74±0.13



Adrenals (mg)



58.3±6.5



58.1±10.2



65.1±10.6



59.8±10.0



 Testes (g)



3.52±0.22



3.50±0.30



3.65±0.28



3.75±0.24



Relative (/100 g B.W.)



Brain  (g%)



0.47±0.02



0.46±0.04



0.48±0.04



0.48±0.05



Heart (g%)



0.24±0.02



0.24±0.01



0.26±0.02



0.25±0.01



Lungs (g%)



0.30±0.03



0.29±0.02



0.31±0.02



0.32±0.02*



Liver (g%) 



2.43±0.10



2.32±0.19



2.53±0.23



2.49±0.12



Kidneys (g%)



0.55±0.04



0.55±0.03



0.56±0.05



0.59±0.04



Spleen (g%)



0.16±0.02



0.16±0.03



0.17±0.02



0.17±0.02



Adrenals   (mg%)   



13.2±1.8



13.1±2.3



14.9±2.2



14.2±2.3



Testes (g%)



0.80±0.08



0.79±0.07



0.83±0.05



0.90±0.09*




Table 9. Organ weights for female rats fed diet containing test item for 13 weeks




























































































































No. of animals



10



10



10



10



Body weight (g)



243.5±18.0



257.2±22.9



243.0±12.9



242.8±17.0



Absolute



Brain (g)



1.94±0.07



1.93±0.05



1.94±0.06



1.91±0.08



Heart (g)



0.73±0.05



0.75±0.04



0.74±0.08



0.77±0.09



Lungs (g)



0.97±0.10



0.98±0.07



0.97±0.07



0.98±0.05



Liver (g)



5.68±0.62



6.02±0.56



5.82±0.54



6.08±0.56



Kidneys (g)



1.52±0.14



1.63±0.12



1.55±0.12



1.58±0.10



Spleen (g)



0.53±0.07



0.51±0.06



0.51±0.05



0.52±0.07



Adrenals (mg)



82.7±10.4



80.5±8.7



81.3±11.8



80.8±9.5



Relative (/100 g B.W)



Brain (g%)



0.80±0.05



0.75±0.06



0.80±0.05



0.79±0.05



Heart (g%)



0.30±0.03



0.30±0.04



0.30±0.03



0.32±0.04



Lungs (g%)



0.40±0.04



0.38±0.03



0.40±0.03



0.40±0.04



Liver (g%)



2.33±0.17



2.34±0.13



2.40±0.16



2.51±0.18



Kidneys (g%)



0.63±0.04



0.63±0.03



0.64±0.04



0.65±0.02



Spleen (g%)



0.22±0.03



0.20±0.03



0.21±0.02



0.21±0.02



Adrenals (mg%)



34.1±5.0a



31.4±3.7



33.4±4.3



33.4±4.3



Values are mean S.D. aData from 9 animalsº

Conclusions:
The no-observed-adverse-effect level (NOAEL) of the test item in Wistar rats were 1% in diet (539 mg/kg/day) in males and 5% in diet (3227 mg/kg/day) in females.
Executive summary:

A 13-week repeated dose toxicity study was conducted with the test item according to the Designation of Food Additives and for Revision of Standards for Use of Food Additives of Japan Guideline (1996). The administration of the test item was conducted by  mixing it with the feed in three different concentrations : 0.2%, 1% and 5% in diet. Also, a control group was monitored. 10 males and 10 females were distributed per group and were administered the test item on a daily basis for 13 weeks.


No significant effects were observed in the clinical sign examinations. The monitorisation of body weight and food consumption changes was also performed, only observing a reduced weight gain in the 5% dosed males. Necropsy was performed for all animals and the relative weights calculated, and the hispathological examinations also confirmed that the test item did not induced any effects, even at the highest concentration. However, haematological and clinical-chemistry examinations determined that test item-related effects were observed for the 5% group in males, which exhibited a decreased RBC, Hb, Ht and triglyceride levels. Therefore, the no-observed-adverse-effect level (NOAEL) of the test item in Wistar rats were 1% in males and 5% in females in diet (539 mg/kg/day for males and 3227 mg/kg/day in females).

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1947
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
no guideline followed
Principles of method if other than guideline:
The concentrations used for this experiment were focused on 5 groups with 6 animals each, which were administered the test item with the feed for 400 days: a control group, two treated groups of 0.25, 0.5 % in for males, and a two treated groups of 1% for males and females
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Albino rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: about 90 g
Route of administration:
oral: feed
Details on route of administration:
The test item was mixed intro finely ground Purina Dog Chow
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
groups of 6 animals each and fed diets containing from 0.25 to 1.0% (w/w) of the test item thoroughly mixed into finely ground Purina Dog Chow.
Duration of treatment / exposure:
150 days por rats in 0.25%, 0.5% and control groups. 400 days for rats in 1% group.
Frequency of treatment:
Daily
Dose / conc.:
0.25 other: % in diet
Dose / conc.:
0.5 other: % in diet
Dose / conc.:
1 other: % in diet
No. of animals per sex per dose:
6 males per each dose and control, 6 females per 1% dose.
Control animals:
yes, concurrent no treatment
Details on study design:
- Other:
There were 5 groups of 6 animals each: control (males), 0.25% (males), 0.5% (males), 1% (males) and 1% (females). Their growth parameters were followed for 150 days and then those animals of the groups the highest dose (1%) were continued on the diet following completion of the growth records. They were mated. Afterwards, they were observed periodically until they had been eating the diet for 400 days.
Tissues from all of the animals (both the 150-day and 400-day rats) were embedded in paraffin and sections were stained with hematoxylin and eosin.
Observations and examinations performed and frequency:
BODY WEIGHT: Yes
-Time schedule for examinations: Daily
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Clinical signs:
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The rate of growth of albino rats was not affected when the diet contained as much as 1% of the test item after 400 days on a 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:
no effects observed
Description (incidence and severity):
The weights of the organs did not deviate from control values to a statistically significant degree, and the gross appearance of all tissues was normal.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The histological examination of the tissues showed no evidence of injury which could be definitely related to the test item administration.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The length of the estrous cycle was the same in rats eating a 1% test item diet as in control animals, reproduction was as good, and the young appeared. to be as healthy.
Key result
Dose descriptor:
NOAEL
Effect level:
1 other: % diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effects observed
Remarks on result:
other: Equivalent to 500 mg/kg bw
Key result
Critical effects observed:
no
Conclusions:
After 400 days of oral administration in rats, the test item has a NOAEL of 1% in diet (500 mg/kg bw for both sexes), being non-toxic.
Executive summary:

A 400-day chronic toxicity oral test was perfomed with the test item, without being in accordance to any guideline. The concentrations used for this experiment were focused on 5 groups with 6 animals each, which were administered  the test item with the feed: a control group, two treated groups of 0.25, 0.5 % for males, and a two treated groups of 1% for males and females . It was determined that the test item has a NOAEL of 1% in diet for both sexes (500 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), thus being non-toxic.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
Arbo, M.D., et al.,2009.Subchronictoxicity of Citrus aurantium L. (Rutaceae) extract and p-synephrine in mice. Regul.Toxicol.Pharmacol.54, 114–117.
Deviations:
not applicable
Qualifier:
according to guideline
Guideline:
other: GB15193.22-2014
Version / remarks:
Chinese national guideline
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
other: Kunming
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Experimental Animal Center of Jilin University (Changchun, China)
- Age at study initiation: 8 weeks old
- Weight at study initiation: 25 to 30 g
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2 ºC
- Humidity (%): 60 ± 10%
- Photoperiod (hrs dark / hrs light): 12h dark / 12h light
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): daily
VEHICLE
- Concentration in vehicle: three doses of 2.50g/kg bw d (Low dose), 5.00g/kg bw d (Mid dose) and 10.00g/kg bw d (High dose).
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
2 500 mg/kg bw/day (nominal)
Dose / conc.:
5 000 mg/kg bw/day (nominal)
Dose / conc.:
10 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females per dose and control groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Other:
Based on previous results on acute oral toxicity studies (test item LD50> 30g/kg in mice), three dose concentrations were chosen: 2.50, 5.00 and 10.00 g/kg bw per day.
Eighty mice were randomly divided into three treatment groups and a control group, and each group had 20 mice with half of the number being males and other half females. FMS was dissolved in distilled water and administered by oral gavage on a daily basis for 28 consecutive days. The control group was orally administered with equivalent volume of distilled water.Food and water were available ad libitum throughout the experiment. All mice were observed twice daily for general clinical signs and mortality and these were recorded. Body weights and food consumption of all mice were measured and recorded weekly.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: weekly

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 29
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: Yes
- How many animals: All of them
- Parameters checked: white blood cells (WBC), neutrophils(Nut), lymphocytes(Lym), monocytes
(Mon), eosinophils (Eos), basophils(Bas) ,redbloodcells(RBC), hemoglobin concentration (HGB) ,hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH),
mean corpuscular hemoglobin concentration (MCHC), reticulocytes (RTC), platelets(PLT), mean platelet volume (MPV), platelet distribution width (PWD), prothrombin times (PT) and activated partial thromboplastin times(APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 29
- Animals fasted: Yes
- How many animals: All of them
- Parameters checked: total protein(TP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), albumin/globulin ratio(A/G ratio), alkaline phosphatase (ALP), total cholesterol (TCHO), triglyceride (TG), blood urea nitrogen (BUN), glucose (GLU), creatinine (CRE).

PLASMA/SERUM HORMONES/LIPIDS: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At study termination, all animals were sacrificed by exsanguination. During necropsy, all the organs were examined and observed carefully, and the macroscopic pathological changes
were recorded.The brain, thyroid, thymus, heart, liver, spleen, kidney, adrenals, stomach, duodenum, colon, pancreas, mesenteric lymph node, bladder and testes/ovaries were collected and
weighed separately. The relative organ weight were calculated based on the fasted animal's body weight (Relative organ weight (%)=organ weight/bodyweight x100).

HISTOPATHOLOGY: Yes
These organs obtained from each animal were fixed in10% neutral buffered formalin for histopathology examination. If any treatment-related changes were observed in the high dose group, the organs obtained from the low and mid dose groups were to be investigated. The fixed-organs were embedded in paraffin, processed to produce 5 mm tissue sections and stained with hematoxylin and eosin.Then the slides were examined under a microscope. Finally, the no-observed-adverse-effect levels(NOAEL) of FMS were determined.
Statistics:
Statistical analysis of the experimental data was conducted using SPSS 19.0 software (SPSS Inc., Chicago, USA).Levene's test was performed to analyze the homogeneity of variances. When
the variances were homogeneous, one-way analysis of variance (ANOVA) was carried out. Dunnett's test was used when the variance was significant. Histopathological findings were subjected to Fisher's exact probability test.All values were expressed as mean±standard deviation. A value of p<0.05 was taken as statistically significant.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Some mice presented an increased locomotor activity during the first 30 min after administration, and returned to normal state within 30 min ,which might have been caused by gavage administration stress.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
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:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
- In this evaluation of subchronic toxicity via a 28-day repeated dose toxicity study, all animals survived until the scheduled necropsy without any obvious general clinical signs or changes.
- No significant changes were observed in body weight and food consumption in test item-treated mice after 28 days of repeated administration.
- Relative organ weight values have been shown to reflect the pathological changes in impaired organs. However, no treatment-related alterations in relative organ weights were found in any groups and no obvious pathological changes were also observed during necropsy in these main organs or tissues.
- Results on the examination of haematological and serum biochemical parameters could demonstrate potential lesions in liver and kidney. No statistically significant differences were observed for each test item-treated group with respect to these parameters after they were sacrificed. However, some parameters for some individual animals were slightly increased or decreased in test item-treated groups.These changes were considered as toxicologically
irrelevant because these changes did not appear in both sexes and no statistically significant differences were observed between each test ite -treated group and control group.
Key result
Dose descriptor:
NOAEL
Effect level:
10 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effects observed
Key result
Critical effects observed:
no

Table 1. Effects of test item on weekly body weights of mice.

















































































Sex



Dose (g/kg bw d)



Initial weight (g)



Week 1 (g)



Week 2 (g)



Week 3 (g)



Week 4 (g)



 


 


 


Male


 


 


 



0



28.38±0.89



30.39±0.92



32.35±0.85



34.51±0.69



36.60±0.85



2.50



28.52±0.84



30.49±0.99



32.48±0.85



34.56±0.90



36.29±0.86



5.00



28.55±0.89



30.79±0.86



32.51±0.80



34.49±0.81



36.41±0.97



10.00



28.45±0.93



30.40±0.88



32.34±0.74



34.10±0.89



36.01±0.79



 


 


Female


 


 



0



26.09±0.61



28.01±0.77



30.29±0.88



32.33±0.83



34.21±0.78



2.50



25.95±0.64



27.76±0.68



29.62±0.57



31.71±0.67



33.63±0.75



5.00



26.15±0.73



28.05±0.80



30.14±0.66



32.00±0.78



34.03±0.74



10.00



26.14±0.64



27.99±0.80



29.95±0.72



31.86±0.73



33.94±0.56



No statistically significant differences were found.


 


Table 2. Effects of test item on weekly feed consumption of mice.








































































Sex



Dose (g/kg bw d)



Week 1 (g)



Week 2 (g)



Week 3 (g)



Week 4 (g)



 


 


 


Male


 


 


 



0



5.16±0.48



6.06±0.44



6.91±0.44



7.82±0.52



2.50



5.20±0.51



5.73±0.45



6.55±0.55



7.33±0.47



5.00



5.06±0.56



5.78±0.53



6.44±0.60



7.21±0.52



10.00



5.25±0.44



5.89±0.47



6.71±0.60



7.45±0.50



 


 


 


Female


 


 


 



0



4.20±0.46



5.39±0.51



6.21±0.45



6.92±0.44



2.50



4.04±0.46



4.98±0.36



5.88±0.61



6.52±0.60



5.00



3.95±0.42



4.87±0.56



6.00±0.56



6.83±0.59



10.00



4.01±0.53



4.93±0.59



5.76±0.51



6.68±0.60



No statistically significant differences were found.


 


Table 3. Effects of test item on haematological parameters of mice.































































































































































































































































































Organ


 



Sex


 



Dose (g/kg bw d)



0 (Control)



2.5 (Low dose)



5.0 (Mid dose)



10.0 (High dose)



WBC ( 109/L)



Male



5.82±1.07



5.76±1.10



5.72±1.13



6.03±1.44



Female



5.52±1.28



5.48±1.46



5.65±1.50



5.54±1.39



Nut (%WBC)


 



Male



2.72±1.10



2.98±0.69



2.56±1.35



2.30±0.98



Female



2.66±1.15



2.32±0.90



2.79±1.16



2.50±0.50



Lym (%WBC)


 



Male



80.26±5.58



76.90±9.43



76.48±4.07



77.51±10.46



Female



75.84±7.73



78.32±4.92



75.33±10.87



79.29±5.29



Mon (%WBC)


 



Male



4.57±0.80



4.85±1.44



4.65±1.09



5.02±1.25



Female



4.64±0.99



4.94±1.08



4.47±1.45



4.63±1.05



Eos (%WBC)


 



Male



0.02±0.01



0.01±0.01



0.02±0.01



0.03±0.01



Female



0.02±0.01



0.02±0.01



0.01±0.01



0.02±0.02



Bas (%WBC)


 



Male



9.4271.46



9.7073.02



10.5875.51



8.9373.03



Female



10.84±3.24



9.21±1.73



9.91±3.54



9.21±4.08



RBC ( 1012/L)



Male



9.33±1.51



9.71±1.26



9.41±1.38



8.96±1.31



Female



9.87±1.42



10.02±1.06



9.50±1.26



10.40±1.35



HGB (g/L)


 



Male



144.46±19.01



138.24±14.65



132.28±18.83



146.26±15.94



Female



151.27±13.36



147.81±10.38



158.37±12.82



150.75±12.97



HCT (%)


 



Male



0.49±0.13



0.46±0.13



0.50±0.11



0.51±0.11



Female



0.47±0.11



0.49±0.12



0.43±0.12



0.50±0.13



MCV (fL)


 



Male



49.52±3.82



49.89±3.14



49.35±4.12



48.86±2.94



Female



52.44±2.74



53.24±2.95



53.80±2.82



51.63±2.80



MCH (pg)


 



Male



15.26±1.71



15.17±1.51



15.38±1.73



14.72±2.00



Female



15.73±2.01



15.92±1.59



15.63±1.21



16.46±1.83



MCHC (g/L)


 



Male



310.52±7.68



311.04±6.45



309.37±6.13



311.32±5.46



Female



319.78±6.49



320.73±6.41



319.75±6.58



321.45±7.16



RTC (%)


 



Male



2.70±0.40



2.60±0.63



2.62±0.68



2.75±0.40



Female



2.23±0.43



2.28±0.46



2.17±0.54



2.38±0.56



PLT ( 109/L)



Male



930.60±163.34



891.96±147.13



994.15±139.37



962.98±168.64



Female



759.97±141.84



720.11±134.71



776.66±139.15



699.08±114.03



MPV (fL)


 



Male



6.33±0.71



6.42±0.64



6.41±0.53



6.28±0.54



Female



6.69±0.69



6.82±0.59



6.49±0.61



6.68±0.40



PWD (fL)


 



Male



7.33±0.94



7.04±1.16



7.23±1.08



7.54±0.80



Female



7.92±0.80



7.46±1.31



7.81±1.03



7.69±1.21



PT (s)


 



Male



12.5±0.6



12.4±0.8



12.0±0.7



12.1±0.8



Female



13.3±0.9



13.6±0.7



13.0±0.8



13.3±0.8



APTT (s)


 



Male



25.0±2.1



24.5±3.5



26.5±2.3



25.5±2.7



Female



19.6±3.1



18.9±2.2



20.9±3.0



20.6±3.3



No statistically significant differences were found.


 


Table 4. Effects of test item on serum biochemical parameters of mice.





































































































































































































Organ


 



Sex


 



Dose (g/kg bw d)



0 (Control)



2.5 (Low dose)



5.0 (Mid dose)



10.0 (High dose)



TP (g/L)


 



Male



47.46±6.23



47.76±6.78



45.58±5.92



49.72±6.56



Female



48.36±4.99



50.83±5.65



51.37±6.41



48.72±5.08



AST (U/L)


 



Male



140.21±13.92



145.31±10.89



137.95±10.16



142.18±8.36



Female



114.33±11.10



118.98±11.69



117.75±16.21



115.27±11.07



ALT (U/L)


 



Male



31.70±3.74



33.54±3.97



31.19±2.29



30.55±4.16



Female



26.25±3.53



24.71±2.72



28.18±3.10



25.14±3.72



AST/ALT ratio


 



Male



4.47±0.62



4.38±0.55



4.45±0.46



4.73±0.69



Female



4.42±0.62



4.87±0.70



4.24±0.79



4.66±0.68



ALB (g/L)


 



Male



27.13±2.89



28.35±2.42



26.62±2.33



27.37±1.92



Female



25.60±2.97



25.37±1.91



25.83±2.62



24.53±2.83



A/G ratio


 



Male



1.15±0.07



1.17±0.06



1.19±0.04



1.14±0.05



Female



1.47±0.03



1.48±0.04



1.46±0.05



1.47±0.05



ALP (U/L)


 



Male



104.07±14.11



103.27±11.48



100.89±9.86



102.36±12.41



Female



125.78±5.38



122.47±6.64



127.19±6.09



123.85±7.11



TCHO (mmol/L)


 



Male



2.45±0.27



2.50±0.33



2.39±0.35



2.53±0.36



Female



2.01±0.28



1.92±0.28



1.96±0.25



2.07±0.22



TG (mmol/L)


 



Male



1.98±0.13



2.00±0.14



1.95±0.11



1.98±0.13



Female



1.82±0.11



1.78±0.09



1.85±0.11



1.86±0.09



BUN (mmol/L)


 



Male



6.48±0.53



6.41±0.67



6.53±0.48



6.56±0.66



Female



6.89±0.61



7.00±0.60



7.04±0.46



6.91±0.52



GLU (mmol/L)


 



Male



4.52±0.57



4.32±0.53



4.61±0.53



4.54±0.67



Female



3.73±0.47



3.89±0.67



4.13±0.51



4.04±0.41



CRE (μmol/L)


 



Male



48.09±6.25



47.18±6.14



46.86±5.91



50.11±6.24



Female



39.40±6.17



37.30±4.62



41.04±5.51



40.66±5.71



No statistically significant differences were found.


 


Table 5. Effects of test item on relative organ weights (%) of mice.


















































































































































































































































Organ


 



Sex


 



Dose (g/kg bw d)



0 (Control)



2.5 (Low dose)



5.0 (Mid dose)



10.0 (High dose)



Brain


 



Male



1.235±0.051



1.228±0.054



1.242±0.065



1.239±0.041



Female



1.427±0.050



1.446±0.075



1.425±0.040



1.431±0.034



Thymus


 



Male



0.332±0.047



0.335±0.046



0.318±0.062



0.341±0.074



Female



0.429±0.052



0.437±0.058



0.428±0.059



0.440±0.066



Thyroid


 



Male



0.059±0.024



0.061±0.026



0.054±0.027



0.059±0.022



Female



0.069±0.021



0.073±0.020



0.072±0.024



0.068±0.020



Heart


 



Male



0.614±0.063



0.600±0.061



0.630±0.055



0.619±0.070



Female



0.518±0.081



0.516±0.077



0.526±0.075



0.510±0.074



Liver


 



Male



5.870±0.624



5.816±0.554



5.834±0.519



5.989±0.554



Female



5.531±0.478



5.582±0.640



5.452±0.530



5.509±0.572



Spleen


 



Male



0.204±0.040



0.203±0.041



0.206±0.047



0.195±0.023



Female



0.280±0.041



0.287±0.023



0.274±0.041



0.277±0.031



Kidneys


 



Male



1.687±0.267



1.724±0.282



1.649±0.219



1.704±0.217



Female



1.167±0.151



1.163±0.187



1.173±0.158



1.154±0.137



Adrenals


 



Male



0.014±0.003



0.013±0.003



0.013±0.002



0.014±0.003



Female



0.020±0.002



0.021±0.001



0.021±0.002



0.019±0.003



Stomach


 



Male



1.226±0.211



1.311±0.248



1.204±0.211



1.297±0.206



Female



1.048±0.151



1.075±0.140



1.027±0.151



1.052±0.134



Duodenum


 



Male



0.534±0.091



0.541±0.086



0.527±0.081



0.538±0.117



Female



0.585±0.077



0.580±0.092



0.595±0.091



0.579±0.110



Colon


 



Male



1.595±0.266



1.576±0.238



1.554±0.201



1.607±0.232



Female



1.672±0.125



1.685±0.147



1.692±0.206



1.680±0.155



Pancreas


 



Male



0.375±0.074



0.383±0.067



0.388±0.072



0.374±0.074



Female



0.410±0.045



0.438±0.056



0.400±0.028



0.412±0.061



Mesenteric lymph node


 



Male



0.609±0.080



0.617±0.084



0.600±0.088



0.613±0.098



Female



0.689±0.071



0.679±0.064



0.686±0.077



0.680±0.069



Bladder


 



Male



0.073±0.013



0.075±0.015



0.072±0.012



0.078±0.014



Female



0.058±0.006



0.060±0.007



0.061±0.005



0.058±0.005



Testis/Ovary


 



Male



0.602±0.043



0.625±0.052



0.599±0.056



0.622±0.050



Female



0.071±0.008



0.074±0.007



0.069±0.009



0.072±0.009



No statistically significant differences were found.

Conclusions:
The no-observed-adverse-effect level (NOAEL) of the test item in mice was 10.00g/kg bw/day
Executive summary:

A  repeated dose toxicity study was conducted with the test item according to OECD 407 and GB15193.22-2014 Guidelines (GLP study). The administration of the test item was achieved via oral gavage and three different concentrations were tested: 2.5, 5.0 and 10.0 g/kg bw per day, based on the results of previous studies on acute oral toxicity studies. Also,a control group was monitored. 10 males and 10 females were distributed per group and  were administered the test item on a daily basis for 28 days. Clinical sign, 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 animals and the relative weights calculated, and the hispathological examinations also confirmed that the test item did not induced any effects, even at the highest concentration. Therefore, the no-observed-adverse-effect level (NOAEL) of the test item in mice was 10.00g/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
410 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
A weight of evidence approach has been applied. Several experimental studies are available with a Klimisch score of 2.
System:
urinary
Organ:
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Weight of evidence from experimental results with individual components and analogue substances:


 


Madys stigma (28 days, mouse, oral):


A 28-d repeated dose toxicity study was conducted with the test item Madys stigma (10.5% flavonoids including rutoside and quercetin) according to OECD TG 407 (GLP study) at doses of 2.5, 5.0 and 10.0 g/kg bw per day in mice. The test item did not induce any effects, even at the highest concentration. Therefore, the NOAEL was determined to be 10.0 g/kg bw/day.


Rutin (400 days, rat, diet):


A 400-day chronic toxicity oral test was performed with rutin in rats. Animals were fed with up to 1% in diet. It was determined that the test item has a NOAEL of 500 mg/kg bw in diet (1 % 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), thus being non-toxic.


Isoquercitrin (90 days, rat, diet):


A 13-week repeated dose toxicity study was conducted with the test item in Wistar rats in concentrations of 0.2%, 1% and 5% in diet. Haematological and clinical-chemistry examinations determined that test item-related effects were observed for the 5% group in males, which exhibited a decreased RBC, Hb, Ht and triglyceride levels. Therefore, NOAEL of the test item were determined to be 539 mg/kg/day in males and 3227 mg/kg/day in females (1% for males and 5% in females, conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives).


Isoquercitrin (52 weeks, rat, diet):


A 52-week repeated dose toxicity study was conducted with the test item in Wistar rats in concentrations of 0.04%, 0.2%, 1.0% and 5.0% in diet. The histopathological examinations revealed that the test item induced mineralization in the renal pelvis often associated with inflammatory cell debris, inflammatory cell infiltration and/or transitional cell hyperplasia in males of the 5% group. Urinalysis showed an increased daily calcium excretion in both sexes of the 5% group, and an increase in urinary calcium concentration was observed in the male 5% group. Therefore, NOAEL of the test item were determined to be 542.4 mg/kg bw/day and 674.0 mg/kg bw/day in females (1% 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).


Quercetin (104 weeks, rat, diet):


A chronic repeated dose toxicity study was conducted with the test item in Fischer 344/DuCrj rats in concentrations of 1.25 and 5.0 % in diet. Gross pathological examinations revealed a higher incidence of raised areas of the cecum, which occurred as single lesions, in the 5.0% males when compared to the controls. Therefore, NOAEL of the test item in rat were determined to be 427 mg/kg bw (1.25%) in diet for males and 2372 mg/kg bw (5%) 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).


Quercetin (104 weeks, rat, diet):


A chronic repeated dose toxicity study was conducted with the test item in Fischer 344/N rats in concentrations of 1000 (40 mg/kg bw), 10000 (410 mg/kg bw) and 40000 ppm (1900 mg/kg bw). After two years, histopathological examinations revealed neoplastic lesions in the kidneys of male rats, including increased severity of chronic nephropathy, hyperplasia, and neoplasia of the renal tubular epithelium. Also, the test item showed carcinogenic activity in the kidney of the male rat, causing primarily benign tumors of the renal tubular epithelium. Therefore, NOAEL of the test item in rat were determined to be 410 mg/kg bw in diet for males and 1900 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).


Naringin (90 day, rat, oral):


The 90d repeated oral dose toxicity was studied on Sprague-Dawley rats according to a method similar to OECD 408 (GLP study). The test item was orally administered at doses of 50, 250 and 1250 mg/kg bw/d. During the study no mortality and toxicologically significant changes in clinical signs, food consumption, opthalmoscopic examination, hematology, clinical biochemistry, serum sex hormone, macroscopic findings, organ weights and histopathological examination except for slight body weight decrease were noted and attributed to naringin administration. Under test conditions, the test item was found to have a NOAEL of 1250 mg/kg in rats.


Naringin (6 months, rat, oral):


The subchronic toxicity of naringin was studied on rats for 6 months, by a method similar to OECD TG 408, with GLP. The test item was orally administered at doses of 50, 250 and 1250 mg/kg bw/d. Some effects were observed during the study but they were not considered adverse change and toxicologically significant. Therefore, the NOAEL of naringin in rats was considered to be greater than 1250 mg/kg/day when administered orally for 6 consecutive months.


Neohesperidin dihydrochalcone (90 days, rat, diet):


A study on the sub-chronic toxicity of the test item was performed by a method similar to OECD 408, without GLP. The test item was administered to groups of 20 male and 20 female Wistar rats at dietary levels of 0 (control), 0.2, 1.0 and 5.0% for 91 days. Some effects were observed in the high dose however they were considered adaptive responses or chance effects rather than manifestations of clear toxicity. Therefore, the NOAEL could be set at 5% in diet (ca. 4000 mg/kg bw) and the NOEL was set at the mid dose (1%) of about 750 mg/kg bw/d.


Methyl hesperidin (90 days, mouse, diet):


A subchronic repeated dose toxicity study was conducted with the test item in B6C3F1 mice at doses of 0.3, 0.6, 1.25, 2.5 and 5.0 % in diet (450, 900, 1875, 3750 and 7500 mg/kg bw). Females fed 2.5% and 5.0% test item demonstrated significant decreases in glucose level. However, the lack of any clear dose-relation or adverse effects on body weight, organ weight, gross pathological and histopathological appearance indicated an absence of toxicity. Therefore, NOAEL of the test item in rat was 7500 mg/kg bw (5.0%) in diet for both males and females (conversion performed according to the guidelines for the preparation of working papers for the joint FAO/WHO expert committee on food additives).

Justification for classification or non-classification

Based on the available data, the substance is not classified for specific target organ toxicity by repeated exposure (STOT-RE) according to CLP Regulation (EC) no 1272/2008.