Ashes (residues), coal

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Jan 2008 - 02 Apr 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted on 27th July, 1995

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues), coal

EC Number:

931-322-8

Cas Number:

68131-74-8

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Ashes (residues), coal

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 6-7 weeks
- Weight at study initiation: males ca. 188 g, females ca. 138 g
- Housing: 2-3 rats of the same sex in one plastic cage (40x25x20 cm)
- Diet (e.g. ad libitum): complete pelleted diet for rats and mice in SPF breeding (ST 1 BERGMAN) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 19 Feb 2008 To: 20 Mar 2008

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose in water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing suspensions were prepared daily prior to administration. The test material was suspended in 0.5% methylcellulose in water.

VEHICLE
- Concentration in vehicle: The test material concentration in vehicle was adjusted accounting for body weight in order to achieve a constant administration volume of 1 mL/100 g bw.
- Amount of vehicle (if gavage): 1 mL/100 g bw

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a dose-range finding study, groups of 5 animals per sex per dose were given 250, 500, 750 and 1000 mg/kg bw of the test material daily for 14 days. No mortalities occurred. An increase in body weight gain was observed in males, especially at 500 mg/kg bw. No signs of toxicity and no impact on basic haematological parameters were noted. Macroscopic changes were seen in liver (marked structure, changed colour), kidneys (changed colour), stomach (haemorrhage on mucosa, change of mucosa), and uterus (dilatation) of some animals, the incidence of which showed, however, no dose-response.
On the basis of these results, three dose levels (250, 500, 1000 mg/kg bw were chosen for the main 28-day study.
- Rationale for selecting satellite groups: Two groups of 5 animals per sex per group were treated with vehicle or the highest dose.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

GENERAL CLINICAL OBSERVATION: Yes
- Time schedule: daily
This observation was made in order to record possible clinical effects after application and changes in animal behaviour.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first administration and weekly thereafter.
Observation of animal behaviour in the cages: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypes or bizarre behaviour.
Observation of animals during removal from the cages: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT: Yes
- Time schedule for examinations: before the first administration, weekly thereafter and at termination
The body weight was recorded on automatic balances with a group average computing module. The weight increment was computed as an average per group per day.

FOOD CONSUMPTION: Yes
- Time schedule: weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The food consumption per animal per day was calculated from the weekly average values of each group.

FOOD EFFICIENCY: Yes
- Calculation of food conversion in %: weight increment/food consumption x 100.

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice a week
Average values (water consumption per animal per day) were calculated for each week of the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On days 29 (main groups) and 43 (satellite groups)
- Anaesthetic used for blood collection: Yes, light ether narcosis
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On days 29 (main groups) and 43 (satellite groups)
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: On days 28 (main groups) and 42 (satellite groups)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table No.3 were examined.

MORTALITY CONTROL: Yes
- Time schedule: daily

FUNCTIONAL OBSERVATION: Yes
- Time schedule: in the last week of the administration period (main groups) and in last week of the recovery period (satellite groups)
During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using dynamometer. Measurements were made on: 1) pectoral legs, 2) pelvis legs, 3) all four legs. Grip power was expressed in Newtons.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. After gross necropsy of the cranial, thoracic and abdominal cavities, the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes, ovaries, epididymidis, uterus, thymus, spleen, brain, pituitary gland and heart were recorded. The relative organ weights were calculated thereafter. Organs for subsequent histopathological examination were removed and stored in fixative (neutral 4% formaldehyde).

HISTOPATHOLOGY: Yes (see table No.4). Tissue were processed by routine paraffin technique and stained by hematoxyline-eosine. Cryotome sections of liver and kidneys were stained by oil red for neutral lipids.

Statistics:

The Analysis of Variance (ANOVA) test (QC.Expert 2.5) was used for the statistical analysis (significance level 0.05). This statistical analysis was used for the results of haematology, blood chemistry, urinalysis, biometry of organs and body weight. Treated groups were compared to vehicle control group, the treated satellite group was compared to the satellite vehicle control group.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortalities occurred and no signs of toxicity were observed in any group.
One male treated with 500 mg/kg bw showed red staining around the snout on the day 16 of the study. In the absence of a dose-related response, this was considered to be incidental.
The activity (poise, gait, reaction to handling) of all animals was similar during the study and unchanged compared to the activity of animals in the control groups.
No changes were seen at any dose level during the examinations of skin, hair, eyes, lacrimation, visible mucous membrane, secretion, excretion and respiration.

BODY WEIGHT AND WEIGHT GAIN
No effects on the average body weight at any dose level in the main and satellite groups.
In males, a slightly lower body weight gains were recorded at 500 mg/kg bw in the first week and at 1000 mg/kg bw in the second week of administration. A mild increase in body weight gain measured at 250 mg/kg bw in the third week of study. In satellite males, a slightly higher body weight gain of the treated animals was seen in the fifth week of study. In females, only in the third week of study the body weight gain was slightly increased at 500 mg/kg bw. No effects on the body weight gain of satellite females was observed.

FOOD CONSUMPTION
Food consumption was slightly increased in males at 250 mg/kg bw in the third week. A slight decrease was observed in females of the 250 mg/kg bw group in the fourth week. No effects were observed in the satellite groups.

FOOD EFFICIENCY
Food conversion was slightly decreased in males at 1000 mg/kg bw in the second week and at 500 mg/kg bw in the first week. A slight increase was recorded at 250 mg/kg bw in the third week. In females, a slight increase was recorded at the all treated groups in the third week. A decrease was observed at 250 and 500 mg/kg bw in the fourth week. In satellite males, a slight increase in food conversion was recorded in the treated group only in the third and fifth week of study. In satellite treated females, a slight increase was observed in the fourth week and a slight decrease in the sixth week.

WATER CONSUMPTION
In males, a slight decrease in water consumption was observed at 1000 mg/kg bw in the first and second week, and a slight increase was noted at 250 mg/kg bw in the third and fourth week of study. In females, a slight decrease was observed at 250 and 1000 mg/kg bw in the second week and at 250 mg/kg bw also in the fourth week, while a slight increase was seen at 500 mg/kg bw in the fourth week. In satellite treated males, a slight decrease was recorded only the first week of study. In satellite treated females, a slight increase was recorded only in the fourth and fifth week of the administration period.

HAEMATOLOGY
Males
Prothrombin time and fibrinogen were increased in a dose-dependent manner (with statistical significance at 500 and 1000 mg/kg bw). A statistically significantly increased platelet count was recorded at 250 mg/kg bw. A decrease in erythrocyte count and haemoglobin value without statistical significance was noted at 500 mg/kg bw. Changes in differential leucocyte count were recorded at the middle and highest dose level (without statistical significance). Granulocytes value was increased and lymphocytes value was decreased. All other parameters were similar to the control group. All parameters were within physiologic limits.

Satellite males
A statistically significantly decreased erythrocyte count and an increase in the mean corpuscular volume were recorded in the treated group. Also prothrombin time was statistically significantly increased compared to the control group. A slightly decreased volume of haemoglobin and changes in differential leukocyte count were measured in the treated group. All other parameters were similar to the control group. All parameters were within physiologic limits.

Females
A slight decrease in total erythrocyte count and haemoglobin value was observed at 500 mg/kg bw. Platelet count was slightly increased and the total leukocyte count was slightly decreased in animals of the 1000 mg/kg bw group. An increased granulocyte value was recorded at 1000 mg/kg bw. Statistical analysis of the data revealed no significant intergroup differences. All other parameters were similar to the control group. All parameters were within physiologic limits.

Satellite females
Statistically significant changes in differential leukocyte count were recorded in the treated group. Prothrombin time was slightly increased.

CLINICAL CHEMISTRY
Males
All parameters were similar to the control group. Only a slightly increased value of creatinine was recorded at 250 mg/kg bw. Statistical analysis of the data revealed no significant intergroup differences. All observed parameters were within physiologic limits.

Satellite males
An increase in glucose without statistical significance was measured in the treated group. All other parameters were similar to the control group. Statistical analysis of the data revealed no significant intergroup differences. All observed parameters were within physiologic limits.

Females
All measured parameters were similar to the control group. Statistical analysis of the data revealed no significant intergroup differences. All observed parameters were within physiologic limits.

Satellite females
A satistically significant increase in ALP activity was recorded in the treated group. The glucose value was also significantly increased. A slightly decreased value of creatinine without statistical significance was recorded in the treated group. All other parameters were similar to the control group. All observed parameters were within physiologic limits.


URINALYSIS
Males
A statistically significant decrease in urine volume was recorded at 1000 mg/kg bw. At this dose level protein, urobilinogen and blood were observed in the urine of one animal and leucocytes were found in 2 animals. The urine of one control animal and one animal at the highest dose level showed a white cloud.

Satellite males
There were no treatment-related changes in urinary parameters. The urine of two treated animals and two control animals showed a yellow or white cloud. Statistical analysis of the data revealed no significant intergroup differences.

Females
There were no treatment-related changes in urinary parameters. Statistical analysis of the data revealed no significant intergroup differences.

Satellite females
There were no treatment-related changes in urinary parameters. Statistical analysis of the data revealed no significant intergroup differences.

NEUROBEHAVIOUR
Reaction to approximation, contact point, reaction to noise, reaction to pain and pupillary reflex of animals in all treated groups were the same as in the control groups. The numbers of up standings, emiction and defecation in the treated groups were similar to the control groups. No changes were observed in the values of grip strength of pectoral legs and pelvis legs. All inter and intra group differences in scores were considered to be a result of normal variation for rats of the strain and age used, and they were of no toxicological importance.

ABSOLUTE ORGAN WEIGHTS
Males
A statistically significant increase in thymus weight was recorded at 250 mg/kg bw. All treated groups showed a decrease in pituitary gland weight with statistical significance at 500 and 1000 mg/kg bw.

Satellite males
Thymus weight was significantly decreased in the treated group compared to the control group.

Females
Animals in all treated groups showed a decrease in pituitary gland weight with statistical significance at 250 and 1000 mg/kg bw. The weight of ovaries was decreased in all treated groups (without statistical significance). A Slightly decreased weight of adrenal glands was recorded at 1000 mg/kg bw.

Satellite females
The weight of adrenal glands was significantly decreased in the treated group.

RELATIVE ORGAN WEIGHTS
Males
Animals in all treated groups showed a decreased pituitary gland weight with statistical significance at 500 mg/kg bw. A slightly increased weight of thymus was recorded at 250 mg/kg bw.

Satellite males
No effects

Females
Animals in all treated groups showed a decreased pituitary gland weight with statistical significance at 250 and 1000 mg/kg bw. The weight of ovaries was decreased at all treated groups (without statistical significance).

Satellite females
The relative weight of adrenal glands in the treated group was significantly decreased compared to the control group. A slightly decreased weight of the pituitary gland was also recorded.

GROSS PATHOLOGY
Males
In 2/5 control animals, 3/5 animals in the 250 mg/kg bw group, 3/5 animals in the 500 mg/kg bw group and 1/5 animals in the 1000 mg/kg bw group, no macroscopic changes were observed.
In the thoracic cavity of one animal (1000 mg/kg bw) focal changes in the lungs (dark red foci) were diagnosed. In the abdominal cavity of 1 control animal and 3 animals in the 1000 mg/kg bw group, irregular colour or marked structure of the liver were seen. Irregular colour of kidneys was seen in 1 control animal, 2 and 3 animals in the 250 and 1000 mg/kg bw, respectively. Macroscopic changes were more often found in the stomach: mucous membrane congested (1 control animal, 2 and 1 animals in the 500 and 1000 mg/kg bw groups, respectively), focal changes of mucosa (punctiform stratum) in 1 animal at each of the 3 dose levels, and haemorrhage in 1 animal at 1000 mg/kg bw. Marked Peyer´s patches were recorded in 1 control animal. Reduced seminal vesicle in 1 control animal was diagnosed. In the cranial cavity no changes were diagnosed.

Satellite males
In 2/5 control and 2/5 treated animals no macroscopic changes were observed.
In the thoracic cavity of 2 control and 1 treated animal focal changes in the lungs (red stratum or grey colour of lobe) were diagnosed. In the abdominal cavity of 2 control and 1 treated animal irregular colour or marked structure of the liver were seen. Irregular colour of kidneys was noted in 2 treated animals and irregular colour of adrenal glands was observed 1 treated animal. Macroscopic changes were recorded in the stomach: focal changes of mucosa (red strata) in 2 control animals, and changed mucosa without plicas in 1 treated animal. In the cranial cavity no changes were diagnosed.

Females
In 3/5 control animals, 1/5 animals in the 250 mg/kg bw group, 2/5 animals in the 500 mg/kg bw group and 4/5 animals in the 1000 mg/kg bw no macroscopic changes were observed.
In the thoracic cavity 1 control animal and 1 animal in the 500 mg/kg bw group, focal changes in the lungs (red strata) and increase of salivary glands of were diagnosed. In the abdominal cavity 1 control animal and 3 animals in the 250 mg/kg bw group, irregular colour or marked structure of liver was observed. Irregular colour of kidneys was seen in 1 animal in the 250 mg/kg bw group, and focal changes in the kidneys (red strata) were noted in 1 animal in the 500 mg/kg bw group. Macroscopic changes were also recorded in the stomach: mucous membrane congested 1 animal at 500 mg/kg bw and focal changes of mucosa (such as red or russet strata or ulceration) in 2 animals in the 250 mg/kg bw group and 1 animal at each of the higher dose levels. Uterus dilatation with fluid was diagnosed in 3 control animals, 1 animal at 250 mg/kg bw, 4 animals at 500 mg/kg bw, and 2 animals at 1000 mg/kg bw. In the cranial cavity no changes were diagnosed.

Satellite females
In 2/5 control and 2/5 treated animals no macroscopic changes were observed.
In the thoracic cavity of 1 animal in each group, petechiae (punctiform haemorrhage) on thymus were diagnosed. In the abdominal cavity of 3 control and 1 treated animal, irregular colour or marked structure of liver was and adhesion of the adrenal glands were noted. Changed mucosa in the stomach was recorded in 1 treated animal. Uterus dilatation with fluid was diagnosed in 1 control and 2 treated animals. In the cranial cavity no changes were diagnosed.

HISTOPATHOLOGY: NON-NEOPLASTIC
Complete histological examination (i.e. examination of all collected organs) was performed only at the dose level of 1000 mg/kg bw and in the control group. At 250 and 500 mg/kg bw, examination of selected organs (organs showing changes in the control and the highest groups) was performed. Forestomach, stomach, liver, small intestine, kidneys, lungs, heart, trachea, thymus, spleen, adrenal glands, pituitary gland, brain, uterus, ovaries, testes, prostate glands and salivary glands were included in this examination.

Males
Stomach, small intestine and liver were affected. Erosion of stomach mucosa – focal damage of mucosa, which did not involve the full thickness of the mucous membrane, was described in 1 animal at 500 and 1000 mg/kg bw, respectively. Inflammation (mainly eosinophilous infiltration of stomach mucosa and/or submucosa) was diagnosed in 2 animals at 500 mg/kg bw and 1 animal at 1000 mg/kg bw. Desquamation of the epithelium was observed in 1 animal at 250 mg/kg bw and 2 animals at 1000 mg/kg bw. Focal inflammation in the liver (mononuclear infiltration) was detected in 1 control animal, 2 animals at each 250 and 500 mg/kg bw, and 3 animals at 1000 mg/kg bw. Foci of extramedullary haemopoiesis (presence of myeloid cells) were seen in 1 animal at 500 and 1000 mg/kg bw, respectively, and haemorrhage in 1 animal in the 1000 mg/kg bw group. Areactive necrosis of the mucosa was diagnosed in the small intestine of 2 animals at each 250 and 500 mg/kg bw and 1 animal at 1000 mg/kg bw. Changes in the salivary glands (basophil nodule) were observed in 1 animal at 1000 mg/kg bw.
In the haematopoietic and lymphatic systems, histological changes were diagnosed in thymus. Tubular structures in 3 control animals, 2 animals at 500 mg/kg bw and 1 animal at 1000 mg/kg bw; involution (proliferation of epithelial cells in medulla) 1 animal in the 500mg/kg bw group; and focal haemorrhages in the medulla of 1 animal at 250 mg/kg bw. Extramedullary haematopoiesis was found in the spleen of 3 control animals, 5 animals at 250 mg/kg bw and 3 animals at each 500 and 1000 mg/kg bw. The intensity of haematopoiesis was similar in all groups.
Only sporadic changes were seen in the urinary system: dystrophic changes of tubules (foci with basophil tubules and reinforcement of basal membrane) in 1 animal at 250 mg/kg bw and 2 animals at 1000 mg/kg bw; proliferation of fibrous tissue in 1 animal at 250 mg/kg bw; and pycnosis of nuclei of tubular epithelial cells in cortico-medullar zone or petty haemorrhage in 1 animal of the 1000 mg/kg bw group.
Interstitial inflammation in the prostate gland (with mononuclear infiltration) was diagnosed in 2 control animals, 1 animal at 500 mg/kg bw and 2 animals at 1000 mg/kg bw. Edema of interstitium was seen in 2 animals at 250 mg/kg bw, 3 animals at 500 mg/kg bw and 2 animals at 1000 mg/kg bw. The incidence of other pathological changes in the male genital tract was sporadic.
The presence of cysts or pseudocysts in the adenohypophysis was recorded in the pituitary gland of 2 control animals, 2 animals at 250 mg/kg bw, 3 animals at 500 mg/kg bw and 2 animals at 1000 mg/kg bw.
Inflammation of myocardium in was noted in 2 control animals and 1 animal at each 500 and 1000 mg/kg bw. Cartilaginous metaplasia was seen in the heart of 1 animal in the 500 mg/kg bw group.

Satellite males
Focal inflammation in the liver (mononuclear infiltration) was detected in 1 control and 3 treated animals. Extramedullary haemopoiesis (presence of myeloid cells) was noted in 1 treated animal, and haemorrhage in 1 control animal. Focal vacuolar dystrophy of the exocrine tissue was diagnosed in the pancreas of 2 control animals. Areactive necrosis of the mucosa in the small intestine was found out in 1 animal of each group. Desquamation of the epithelium or inflammation (eosinophilous infiltration) or erosion was described only in 1 treated animal.
In the haematopoietic and lymphatic systems, extramedullary haematopoiesus was diagnosed in the spleen of 3 control and all treated animals. The intensity of haematopoiesis was similar in both groups. Tubular structures in thymus were diagnosed only in 1 control animal.
Sporadic changes were noted in the urinary system: dystrophic changes of tubules (foci with basophil tubules and reinforcement of basal membrane) in 1 control and 2 treated animals and haematoma in cortex in 1 treated animal.
In the genital tract only chronic inflammation of the prostate gland in 2 animals of each group and oedema in the interstitium of the prostate gland in 1 animal of each group was described. Testes were not histologically affected.
The presence of cysts or pseudocyst in the adenohypophysis was recorded in the pituitary gland of 2 control and 3 treated animals.
Cartilaginous metaplasia in the heart of 2 treated animals and focal monocytolysis in 1 treated animal were noted.

Females
In the stomach, erosion of mucosa (focal damage of mucosa, which did not involve the full thickness of the mucous membrane) was seen in 1 control animal and 2 animals at 500 mg/kg bw. Desquamation of the epithelium was observed in 1 animal at 250 mg/kg bw and 2 animals at 1000 mg/kg bw. Inflammation was recorded in 1 animal at 500 mg/kg bw. Focal inflammation in the liver (mononuclear infiltration) was detected in 1 control animal, 2 animals at 250 mg/kg bw, and 3 animals at each 500 and 1000 mg/kg bw. Foci of extramedullary haematopiesis were recorded in 1 control animal and 1 animal in the 250 mg/kg bw group.
In the haematopoietic and lymphatic systems, histological changes were diagnosed in the spleen. Extramedullary haematopoiesis was seen in 5 control animals, 4 animals at 250 mg/kg bw, and 5 animals at each 500 and 1000 mg/kg bw. Pigmentation (increased amount of rust-brown pigmentation in red pulp) was detected in 2 control animals and 1 animal in the 250 mg/kg bw group. The intensity of haematopoiesis was similar in all groups. Tubular structures were observed in the thymus of 2 control animals, 3 animals at 250 mg/kg bw, and 2 animals at each 500 and 1000 mg/kg bw. No histological changes were observed in kidneys.
Hydrometra (dilatation of uterus with pellucid liquid, different degree) was detected in 1 control animal, 1 animal at 250 mg/kg bw, 3 animals at 500 mg/kg bw, and 2 animals at 1000 mg/kg bw.
The presence of pseudocysts in adenohypophysis was recorded in the pituitary gland of 2 animals at 250 mg/kg bw, 1 animal at 500 mg/kg bw and 4 animals at 1000 mg/kg bw.

Satellite females
In the liver of satellite animals inflammation was seen in 3 control and 2 treated animals. Only atrophy of phundal gland was recorded in the stomach of 1 animal in each group.
In the haematopoietic and lymphatic systems, extramedullary haematopoiesis was diagnosed in the spleen of all control and 4 treated animals. The intensity of haematopoiesis was similar in both groups. Pigmentation (increased amount of rust-brown pigmentation in red pulp) was found in 2 animals of each group. Tubular structures in the thymus were diagnosed in 3 control and 1 treated animals. Hydrometra was observed in the uterus of 1 control and 2 treated animals. Cysts in the pituitary gland were recorded in 3 control animals.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: overall effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The oral administration of the test substance Ashes (residues) to rats by gavage for a period of twenty-eight consecutive days at dose levels of 250, 500 and 1000 mg/kg/day produced no toxicologically significant changes in the parameters measured. No major functional changes in any organ systems or severe organ dysfunction were detected. Consistent changes in clinical biochemistry, haematology and urinalysis parameters indicating organ dysfunction were not recorded in any dose level. Histopathological examination showed slight pathological changes in both control and treated males (oedema in prostate glands).

Based on the results of laboratory investigations in clinical biochemistry, haematology and urinalysis and histopathological examination, the NOAEL (No-Observed-Adverse-Effect-Level) was considered to be 1000 mg/kg bw/day for both male and female rats.