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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no ophtalmological examination and neurobehaviour was investigated. No information on results for gross necropsy
Principles of method if other than guideline:
- Principle of test: A subchronic toxicity experiment with propyl gallate in Wistar RIVM:Tox rats was performed. Groups of 10 female and 10 male rats were fed a semisynthetic diet containing 0, 490, 1910 and 7455 mg Propyl gallate /kg feed.
- Parameters analysed / observed: Body weight gain was recorded weekly and food-intake twice weekly. Other parameters comprised haematology, biochemical determinations in urine, serum anti liver and complete histopathological examinations.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Propyl gallate was purchased from Fluka, Buchs, Switzerland
- Purity: > 98%

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RIVM:Tox
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Weanling SPF-derived Wistar rats RIVM:Tox, Bilthoven, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: no data
- Weight at study initiation: 40-60 g
- Fasting period before study: no
- Housing: The animals were kept, two of equal sex, in stainless steel wire mesh cages.
- Diet (e.g. ad libitum): ad libitum; semi-synthetic feed, SSP-TOX standard (Trouw, Putten, The Netherlands)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 – 26
- Humidity (%): 40 – 60
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were carried out according to the HPLC-method described by Strik et al., 1986. The actual analyzed concentrations in the feed averaged respectively < 5 (lower limit of detection of the analytical method), 460, 1910 and 7435 mg/kg feed.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
ad libitum
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg diet
Remarks:
control
Dose / conc.:
500 mg/kg diet
Remarks:
low-dose
Dose / conc.:
2 000 mg/kg diet
Remarks:
mid-dose
Dose / conc.:
8 000 mg/kg diet
Remarks:
high-dose
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: Animals were observed for general condition.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food intake was determined per cage twice daily

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In week 13, previous to obduction
- Anesthetic used for blood collection: Yes (ether anesthesia)
- Animals fasted: not specified
- How many animals: all animals
- Parameters checked: hemoglobin concentration, packed cell volume, red blood cell and white blood cell concentration, white blood cell differential count, erythrocyte and thrombocyte morphology. The red blood indices: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated.

CLINICAL CHEMISTRY: Yes
First sampling:
- Time schedule for collection of blood: In week 8 blood samples were collected from the retro-orbital plexus
- Anesthetic used for blood collection: ether
- Animals fasted: Yes, for 24 hours
- How many animals: all animals
- Parameters checked: total cholesterol concentration, HDL cholesterol, triglyceride concentrations, lipoproteins

Second sampling:
- Time schedule for collection of blood: In week 12 after 17 hours urine sampling
- Anesthetic used for blood collection: ether
- Animals fasted: no drinking water was provided
- How many animals: all animals
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, creatinine

Third sampling:
Previous to obduction, blood was collected from the retro-orbital plexus under ether anasthesia and hemoglobin concentration (Hb), packed cell volume (PCV), red blood cell (RBC) and white blood cell concentration (WBC), white blood cell differential count and morpholgy of erythrocytes and thrombocytes were determined. The red blood blood indices: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated.


ANALYSIS following AUTOPSY:
At autopsy, ether anaesthetized animals were exsanguinated from the abdonimal aorta. The first portion of the collected blood was used to prepare serum for analyses of IgG, IgM, IgA and urea concentrations. Following exsanguination, a liver sample was homogenized in 4 volumes of 0.1 mol/L phospahte buffer (pH 7.4) and centrifuged for 10 minutes at 100 kN/kg. The supernatant was centrifuged for 30 min at 1500 kN/kg (0-4 °C).Cytosol and microsomes were collected separately and stored at -90 °C until analysis. In the microsomal fraction the activity of aminoppyrin-N-demethylase (APDM) was determined. The concentration of formaldehyde, the product of incubation with aminopyrin was measured as indication of the amino-N-demethylase activity. Ethoxy-resorufin-O-deethylase (EROD) activity was determined using the fluorometric method of Burke and Mayer, 1974. Glucuronyltransferase was measured according to Mulder and Van Doorn, 1975. In liver cytosol gluthatione S-transferase was determined and the concentration of cytochrome P-450 was also recorded. Protein analyses were carried out with bovine serum albumin as a standard (Lowry et al., 1951).

URINALYSIS: Yes
- Time schedule for collection of urine: In week 12 during 17 hours
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No drinking water was provided
- Parameters checked: volume, osmolarity, protein and creatinine concentration, pH, glucose, ketones, bilirobin

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Yes
- Time schedule for examinations: In week 13 at autopsy
- How many animals: all animals
- Dose groups that were examined: all dose groups
- Parameters checked: The first portion of the collected blood was used to prepare serum for analyses of IgG, IgM, IgA and urea concentrations.
Sacrifice and pathology:
Gross- and histopathology:
Following macroscopical inspection, brain, liver, kidneys, spleen,pituitary, thymus, thyroid, pancreas, adrenals, ovaries or testes, uterus, popliteal and mesenteric lymph nodes were weighed. These organs and prostate, stomach, duodenum, jejunum, ileum, coecum, colon, urinary bladder, spinal cord, ischic nerve, quadriceps muscle and femur were fixed in 10% phosphate buffered formalin, except the pituitary which was fixed in formalin sublimate. Intestine was fixed using the Swiss roll-technique (Moolenbeek and Ruitenberg, 1981). For histopathological investigations, 5 µm thick Paraplast sections were stained with hematoxylin-eosin (H&E). Of the kidneys 1 µm sections in glycolmethacrylate were stained with the PASM and the Giemsa method. Additional slides of the liver were stained with oil red O for detection of fat. A portion periportal and centrolobular samples of the liver were fixed for ultramicroscopic examination.
Statistics:
The data from this experiment were analyzed by means of the statistical software Stata (Computing Resource Center, Santa Maria, California, 90401) and Genstat 5 (NAG, Oxford, UK). The data or the log transformed data were analyzed. with variance analyses (ANOVA) in combination with the t-test of Dunnett. The statistical analysis of the growth was performed by means of a logistic growth curve.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of the male animals of the highest dose group was significantly lower than in the other groups. Further, a statistic significant retarded growth was observed in the male animals at the highest dose.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant differences in foos intake were recorded.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the haematological investigation significant changes between the groups were seen in Hb, PCV and RBC. The ad-hoc test of Dunnett revelaed only a significant lower HB value in the high dose group when compared to the control group. No effect on the immunoglobulins were recorded
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The determination of lipoprotein in the serum revealed significant difference in the levels between groups for low density lipoprotein and very low-density lipoprotein. However only LDL was significantly higher than the control in the serum of male rats of the high dose group.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
A significant decreased protein concentration and protein/creatinin ratio in the high dose group compared to the controls was observed.
Behaviour (functional findings):
not specified
Immunological findings:
no effects observed
Description (incidence and severity):
No effect on the investigated immunoglobulins were recorded.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The only organ weight significantly changed was the adrenal of male rats of the high dose group. When expressed relatively as % of the body weight the relative adrenal weight still was significantly lower in three male rats. No consistent changes in the weights of other organs were observed.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In the corticomedullary region of the kidneys from the control females, nephrocalcinosis was seen with a normal incidence for the rat strain and the semi-synthetic diet used. In the high dose females this mineralization of that region had disappeared. Extramedullary hematopoiesis was slightly increased in the females of the high dose group and slightly decreased in the high dose males, judged from the H&E-stained sections of the spleen.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
For the phase 1 oxidative and reductive liver enzymes no differences in P450, APDM and an activity due to the treatment with Propyl gallate were noticed between the groups. For the EROD activity significant difference in the levels between the groups were recorded. A significant lower EROD value in both female and male rats of the high dose group (8 g/kg feed) when compared to the control group was observed.
For the phase 2 (conjugative) liver enzymes glucuronyl-transferase and the (cytosolor) glutathione-S-transferase significant differences in levels were noticed. The glutathione concentration in the liver revealed also significantly different levels between the test groups.
The results of the ad hoc test revealed significantly higher values in the females than the control for glucuronyltransferase and GSH in the high dose group and for gluthathion-S-transferase in the mid and high dose group. In the males significant higher values for gluthathion-S-transferase and GSH in the high dose group were seen and for gluthathion-S-transferase and GSH in the high dose group and for glucuronyltransferase in the mid and high dose group.
Details on results:
Although retarded growth in the males of the high dose group mwas found, no effects on the food intake were observed. The only effect of Propyl gallate on the organ weights was noticed in the adrenals of males fed the high dose diet. The increased protein concentration also relative to creatinin elevated in the males of the high dose group, could be related to reduced kidney function. No effects of Propyl gallate were seen on the oxidative microsomal liver enzymes. The histopathological observation that Propyl gallate reduces the incidence of nephrocalcinosis, which is normally observed in female rats on a semisynthetic diet, is not considered as an effect relevant to the human health situation. Since for these rats no effects were found on the food-intake (or body weight), this phenomenon is not likely to be the consequence of general toxicity, but may be attributable to a more specific effect of Propyl gallate on the regulation of calcium and phosphate metabolism in the rat. The increase of conjugative enzymes might be considered as a stimulation of a detoxication route.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
135 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
8 000 mg/kg diet
System:
haematopoietic
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results obtained from this study, the NOAEL can be considered to be 1910 mg Propyl gallate/kg feed corresponding with 135 mg/kg body weight.
Executive summary:

In a sub-chronic toxicity study conducted similar to OECD 408, propyl gallate (> 98% purity) was administered to 10 male and female Wistar rats per dose by feed at dose levels of 0, 490, 1910 and 7455 mg/kg diet. Adverse effects of Propyl gallate observed in the high dose group were effects on the hematopoietic system reflected in a reduction of haemoglobin concentration, packed cell volume and red blood cell concentration content and the morphological changes in the spleen. The other effects observed comprised decreased incidence of the nephrocalcinosis in female rats, the increased activity of EROD in the high dose group and increased activity of the conjugating enzymes; glucorynyl- transferase and glutathione-s-transferase in the mid and high dose group of Propyl gallate. The effects on the nephrocalcinosis and on the conjugating enzymes may be considered as not adverse. Based on the results obtained from this study, the NOAEL can be considered to be 1910 mg Propyl gallate/kg feed corresponding with 135 mg/kg body weight.

 

This sub-chronic toxicity study in the rat is acceptable and satisfies the requirement for a sub-chronic oral study in rats.