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Description of key information

Repeated dose toxicity tests have been peformed by oral route in 2 publications for 28 and 90 days with determination of

  • NOAEL of 1000 mg/kg bw/d in 28-day test and
  • NOAEL of 119 and 128 mg/kg bw/d, respectively for males and females, in 90-day test

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
Gallic acid was obtained from Danippon Pharmaceutical Co. Ltd (Osaka, Japan), as a yellowish-white crystalline powder with a purity over 98%.
The preparation had little odor, and was astringent and somewhat acid in taste.
Species:
rat
Strain:
other: F344/DuCrj
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: 5 weeks of age
- Housing: housed three to four rats per polycarbonate cage, with sterilized softwood chips as bedding, in a barrier-sustained animal room
- Diet (e.g. ad libitum): basal pelleted diet (CRF-1: Oriental Yeast Co.)
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: 6 days of acclimation


ENVIRONMENTAL CONDITIONS
- Temperature (°C): air conditioned at 23-25 °C
- Humidity (%): 50-60%
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Animals received either 0 (control), 0.2, 0.6, 1.7 or 5% gallic acid in the diet for a treatment period of 13 weeks.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
An assessment of the sability of gallic acid at concentrations of 0.2 and 0.5% in basal diet was conducted and no decomposition was apparent within a period of 4 weeks at 4°C and following 1 week at room temperature.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Dose / conc.:
0.2 other: %
Dose / conc.:
0.6 other: %
Dose / conc.:
1.7 other: %
Dose / conc.:
5 other: %
Dose / conc.:
0 other: %
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent no treatment
Details on study design:
Animals were randomly divided into five groups, each consisting of 10 males and 10 females.
Positive control:
None
Observations and examinations performed and frequency:
The animals were observed daily for clinical signs and mortality.
Body weight and food consumption were measured weekly.
Sacrifice and pathology:
At the end of week 13, all animals were anesthetized with ether and blood samples were collected from the abdominal aorta for hematology and blood biochemistry.
Animals were sacrified by exsanguination.
Other examinations:
Hematological examination was performed using a automatic hematology analyzer, M-2000 (Toa Medical Electronics; Hyogo, Japan) and the following parameters were determined:
white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (Hb) concentration, hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet count.
Differential leukocyte counts and reticulocyte counts were obtained with a Microx HEG-120A (Omron Tateishi Electronics Co., Tokyo, Japan).
Parameters for blood chemistry included total protein (TP), albumin (Alb), the alb:globulin ration (A/G), glucose (Glc), total cholesterol (TC), blood urea nitrogen (BUN), creatinine (CRN), sodium (Na), chloride (Cl), potassium (K), calcium (Ca), phosphorus (P), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), direct reacting bilirubin (DBL), indirect reacting bilirubin (IBL), γ-glutamyl transpeptidase (γ-GTP).

The brain, lungs, heart, liver, spleen, adrenal glands, kidneys and testes were weighed.
In addition to these organs, the pituitary, nasal cavity, tongue, trachea, aorta, thyroid gland, thymus, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, urinary bladder, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, male and female mammary gland, mesenteric and submandibular lymph nodes, mandibular salivary glands, sternum, femur, sciatic nerve, trigeminal nerve, spinal cord (cervical, thoracic and lumbar cords), eyes and sampls of skin and skeletal muscle were fixed in 10% buffered formalin for all animals. Tissues sections of all organs/tissues were routinely processed for parafin-embedding and sections were prepared ans stained with hematoxilin and eosin.
Histopathological assessment was performed on all tissues in the control and 5%-GA treated animals. In addition, liver, spleen and kidneys were also examined in other GA-treated animals, because histopathological changes were found in these organs of the 5% GA-treated animals. Berlin blue staining was additionally performed for kidney, spleen and liver sections in all animals.

Statistics:
Variance in data for body weight, food consumption, hematology, blood biochemistry and organ weights (both absolute and relative weights) was checked for homogeneity by Bartlett's procedure. If the variance was homogeneous, the data were assessed by one-ways analysis of variance (ANOVA). If not, 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. As to histopathologic changes, incidences were compared by use of Fisher's exact probability test and severity was analyzed with the mann-Whitney U test. P values less than 0.05 were considered to be significant in all cases.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no obvious clinical signs in all animals throughout the feeding period.
Mortality:
no mortality observed
Description (incidence):
All rats survived to the end of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of both male and female rats in the 5 % group were significantly reduced from week 1 as compared to those in the control group.
The amount of rats of the 5% group at week 13 was 16 and 14%, respectively, as compared to those in the corresponding control animals.
Male rats in the 1.7% group showed significantly lower body weights than those of the control group at weeks 7 or 8, which persisted thereafter.
From week 9 to the end of the study, female rats in the 1.7% group also showed significant reduction of body weight compared to those of the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no significant variation in food consumption between control and treatment groups.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In males, decreases of Hb and Ht were observed with 0.6% of GA treatment and above.
In addition, a decrease of RBC in the 0.5 and 5% groups, and decrease of MCV and MCH in the 5 % groupe were also found.
In female rats, a decrease of MCV in the 1.7 % group and decreases of RBC, Hb, Ht and MCH in the 5% group were observed.
Males and females receiving 5% GA also exhibited significant increase of blood reticulocytes.

Among parameters in blood biochemistry, a slight increase of BUN at 5% GA and a decrease of CRN from 1.7% GA were noted in both sexes.
In males, a slight increase of ALP was also found at 5%. No other parameters demonstrated fluctuation in association with the dose of GA.

The severity of anemia was weak even at 5% GA in the diet, because the histopathological lesions were limited in the spleen and hemosiderin deposition was not noted in other organs such as the liver and kidneys.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No color change in the urinary content of GA-treated animals may support the severity of anemia.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
An increase in the relative liver weight was observed in both sexes of GA-treated animals from 1.7%.
In addition, increases of both absolute and relative spleen weights were apparent in both sexes with 5% GA treatment.
Other organs did not show any dose-related weight changes.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
GA treatment-related effects were noteed in the liver, spleen and kidney of male and female rats.
In the livers of male and female rats, dose-dependent centrilobular liver cell hypertrophy was observed from 1.7%.
The spleens of rats administrated 5% GA showed increased incidences of extramedullary hematopoiesis and Berlin blue-positive hemosiderin deposition, although their severity was only slight. Minimal congestion was also noted in these 5% GA-treated animals.
In the kidneys, deposition of a fine-granular brown pigment in proximal tubular cells was observed in animals of both sexes administrated 5% GA. The pigments were not stained with Berlin blue.
There were no other gross or histopathologic lesions that appeared to be associate with GA treatment.
Details on results:
Summary :
Toxic effects in both sexes included reduction of Hb, Ht and RBC and increase in reticulocytes, suggesting the development of anemia.
Histopathological assessment revealed centrilobular liver cell hypertrophy, increased extramedullary hematopoiesis associated with increased hemosiderin deposition and congestion in the spleen, and Berlin blue-negative brown pigment deposition in the proximal renal tubular epithelial cells.
However, the severity of anemia was weak even at 5% GA in the diet, because the histopathological lesions were limited in the spleen and hemosiderin deposition was not noted in other organs such as the liver and kidneys.
Key result
Dose descriptor:
NOAEL
Effect level:
119 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
128 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
0.6 other: %
System:
other: Hematology/blood biochemistry
Organ:
not specified
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 other: %
System:
immune system
Organ:
spleen
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1.7 other: %
System:
gastrointestinal tract
Organ:
liver
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
5 other: %
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Although the effects of repeated administration of GA on the induction of liver enzymes are not known, induction of O-methyltransferase and some other enzymes may be responsible for the dose-dependent increase of liver weight and centrilobular liver cell hypertrophy in the present study.

Conclusions:
Orally administrated GA for 13 weeks in F344 rats caused anemia, of probable hemolytic origin and liver cell hypertrophy.

Based on the hematological changes observed from the 0.6% dose in males, the no-observed-adverse-effect-level NOAEL of GA was estimated to be 0.2% in diet under the present experimental conditions in rats.
It was translated into 119 and 128 mg/kg/day, respectively for males and females by calculation using mean values of body weight and food intake in the 0.2% group.
Executive summary:

Subchronic toxicity of gallic acid (GA) was investigated in F344 rats by feeding diet containing 0, 0.2, 0.6, 1.7 and 5% GA for 13 weeks.

Each group consisted of 10 rats of each sex. Toxicological parameters included clinical signs, body weight, food consumption, hematology, blood biochemistry, organ weights and histopathological assessment.

Body weight gain in the 5% GA-treated animals of both sexes from week 1 to the end of the experiment was significantly lower than that of the untreated controls. Toxic effects following administration of 0.6% or more in males and 5% in females included reduction of hemoglobin concentration, hematocrit and red blood cell counts and increase in reticulocytes. Histopathologically, extramedullary hematopoiesis, hemosiderin deposition and congestion appeared in the spleens of 5 % GA-treated animals, suggesting developement of hemolytic anemia. In addition, centrilobular liver cell hypertrophy, reflected in increase in liver weight, was observed in animals of both sexes from 1.7%. In the kidney, Berlin blue-negative brown pigment deposition in the proximal tuber epithelium was observed at 5% GA. However, the severity of these pathological change was weak.

Based on the present toxicology data, 0.2% was determined to be a no-observed-adverse-effect level (NOAEL) in rats.

This level was translated into 119 and 128 mg/kg/day, respectively for male and female rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
119 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch code 2
System:
other: hematology

Additional information

Justification for classification or non-classification

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