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Administrative data

Description of key information

Several studies for repeated dose toxicity are available for the target substance propyl gallate.

In a 13-week study from Speijers et al. (1993) 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. 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. In a chronic study equivalent to OECD TG 451 (supervised by NTP) male and female F344/N rats and B6CF1 mice received daily by feed 0, 6000 and 12000 ppm bw/day propyl gallate diluted in 20% ethanol in water for 103 weeks. LOAEL’s of 475 mg/kg (male rats), 464 mg/kg (female rats), 1305 mg/kg (male mice) and 1714 mg/kg (female mice) can be considered based on the results of this study.

The NOAEL was concluded to be 135 mg/kg body weight based on adverse effects in the hematopoietic system (Speijers 1993).

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:
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
Reference:
Composition 0
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 information:
Composition 1
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%
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
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
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
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.
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.
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
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
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
135 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Comparable to guideline study
System:
haematopoietic
Organ:
blood

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

Mode of Action Analysis / Human Relevance Framework

Additional information

Oral route:

Several oral repeated dose studies are available for propyl gallate.

There are studies available which were conducted within the US NTP programme (NTP 1982). They do not fully comply with the current respective guidelines due to the lack of clinical chemistry data, hematology data, and urinalysis data. Instead, clinical signs, body weight development and gross and histopathology examinations were performed. The studies were assessed as of high quality, since they were peer-reviewed by a panel nominated by the US NTP.

In the 13-week dose finding study male and female mice received daily 0, 800, 1500, 3000, 6000, 12,500 ppm propyl gallate by feed ad libitum. All mice survived, and no microscopic pathologic effects were observed. Weight gain could not be evaluated due to a malfunction in the automatic watering system. Based on the results obtained in this study, it can be stated that the test item was well tolerated up to 1200 ppm.

The respective 13-week dose-finding study in male and female rats revealed weight gain depressions of 10% or more when compared with weight gains for controls, and feed consumption generally increased as the dose increased. Based on the gastrointestinal effects in rats fed diets containing 25,000 ppm propyl gallate, doses of 6,000 and 12,000 ppm were selected for rats in the 2-year study.

In the consecutive chronic studies (studies on carcinogenicity; NTP 1982), rats received by diet (ad libitum) 0, 6000 and 12000 ppm bw/day propyl gallate for 103 weeks. Lower mean body weights were observed in the mid dose (5% in males and 11% in females) as well as in the high dose (8% in males and 11% in females). Thus, 6000 ppm can be considered as a LOAEL from this study, which is equal to 1305 mg/kg bw for males and 1714 mg/kg bw for females based on average food consumption and mean body weights in the mid dose group. In the chronic rats study, male and female F344/N rats received daily by feed 0, 6000 and 12000 ppm bw/day propyl gallate diluted in 20% ethanol in water for 103 weeks.

Lower mean body weights were observed in the mid dose (4% in males and 11% in females) as well as in the high dose (8% in males and 18% in females). Further, in both doses a hepatic cytoplasmic vacuolisation and a suppurative inflammation of the prostate was observed. Thus, 6000 ppm can be considered as a LOAEL from this study, which is equal to 475 mg/kg bw for males and 464 mg/kg bw for females based on average food consumption and mean body weights in the mid dose group.

A more guideline-compliant oral subchronic study was conducted by Speijers et al. (1993). In contrast to the NTP studies, clinical chemistry, hematology, and urinalysis was evaluated in addition to clinical signs, body weight and gross and histopathology examinations. 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.

Several other studies conducted with different doses of propyl gallate did not reveal any significant toxic effects (Johnson 1961, Stierum et al. 2008). In a one-week study from Feuer et al. (1965), rats (7-8 animals/group) were administered five doses of 0 to 500 mg propyl gallate/kg bw/day by gavage for 1 week. Animals were killed 24 h after the final dosing. Four additional groups of six rats each were maintained at the high dose (500 mg/kg per day) and killed 14 and 28 days after the last dosing. Histopathological examination and biochemical analyses were performed on the liver of all animals. Effects were seen in 500 mg/kg bw propyl gallate in rats. At this, extensive fatty change was observed 24 h after the final dosing, but the severity decreased significantly after 14 days of recovery. By 28 days, the livers of most animals had returned to normal. Propyl gallate also significantly increased the number of abnormal mitotic figures in hepatocytes. At the highest dose tested, this effect persisted throughout the first 14 days of the recovery period but had disappeared by the 28th day post treatment. In a study from Dacre (1974), albino mice (University Animal Breeding Station closed strain colony) (25 animals/sex/group), were maintained on diets containing 745, or 1490 mg propyl gallate /kg bw/day for 21 months. Body weights, feed consumption, and hematological parameters were monitored. All surviving mice were killed at 21 months and necropsied. Water intake, food consumption and growth of treated test animals were comparable to controls. In this study, Dacre concluded a NEL of 1 % equivalent to an intake of 1490 mg/kg bw/day for propyl gallate. These studies support the NOAEL determined based on the NTP studies as well as from the study from Speijers (1993).

The study from Speijers et al. (1993) can be considered as more reliable as the NTP chronic studies and based on the findings in this study, a NOAEL of 135 mg/kg body weight can be concluded based on the findings in the hematopoetic system in rats.

 

Dermal route: No studies available.

 

Inhalation route: No studies available.

Justification for classification or non-classification

Based on the available data, the target substance does not warrant classification for specific target organ toxicity in accordance to CLP regulation (EC) No 1272/2008.