Reaction mass of Dicerium trisulfide and Dilanthanum trisulfide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 15 January 1997 to 20 November 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: the requirements of the Ministry of Health and Welfare for Japan - Part II of the Chemical Substance Control Law (1986)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dicerium trisulphide

EC Number:

234-603-7

EC Name:

Dicerium trisulphide

Cas Number:

12014-93-6

Molecular formula:

Ce2S3

IUPAC Name:

Dicerium trisulphide

Test material form:

solid: particulate/powder

Remarks:

powder

Details on test material:

- Name of test material (as cited in study report): Cerium sulphide

Test animals

Species:

rat

Strain:

other: CD strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: 34-41 days
- Weight at study initiation: on the first day of treatment, the males had a mean body weight range of 161 g to 170 g and the females had a mean body weight range of 150 g to 156 g. At the begining of the treatment the weight variation was checked to ensure that it did not exceed ± 20% of the overall mean vakues for the appropriate sex.
- Fasting period before study:
- Housing: Animals were housed five of one sex per cage. Cages used were type TR 18 (Modular Systems and Developments Company Limited, Hereford, England), which were made of a stainless steel body measuring 54 x 38 x 20 cm with a stainless steel mesh lid and floor. The cages were suspended above absorbent crêpe paper which was changed at appropriate intervals. Cages, cage-trays, food hoppers and water bottles were changed at appopriate intervals.
- Diet: An expanded rodent diet, RM1(E) SQC (Special Diets Services Ltd., Witham, Essex, England), was freely available, except when urine was being collected and overnight before routine blood sampling. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Weighted amounts of diet were provided at intervals during each week to each cage. At the end of each treatment and reversibility week, the weight of uneaten food was recorded. The uneaten diet may have been included in that returned to the cage, after appropriate measurement.
Each batch of diet was routinely analysed by the supplier for various nutrional components and chemical and microbiological contaminants. At approximately 6 months intervals, the diet was sampled so that the potential contaminants investigated by te suplier could be analysed by a laboratory independant of the supplier.
- Water: water taken from the public supply (Essex and Suffolk Water plc, Chelmsford, Essex, England), was freely available, via polycarbonate bottles fitted with sipper tubes, except when urine was being collected.
The quality of the water supply is governed by regulations published by the Department of the Environment. Certificates of analysis were routinely reveived from the supplier. At approximately 6 months inervals, water was routinely sampled for analysis, by a laboratory independant of the supplier, for selected chlorinated and organophosphorus pesticides, polychlorinated biphenyls and lead and cadmium contaminants. It was also examined for coliform bacteria.

No other specific contaminants that were likely to have been present in the diet or water were analysed, as none that have interferedwith or prejudiced the outcome of the study was known.
- Acclimation period: 13 days
- Allocation to treatment groups: Following initial health status checks, animals were non-selectively allocated to study cages. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Cage labels, identifying the occupants by experiment, animal number, sex and treatment group, were colour-coded.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. Periodic checks were made on the number of air changes in the animal rooms. Temperatures and humidity were monitored daily.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
A stand-by electricity supply was available to be automatically brought into operation in case the public supply fails.

IN-LIFE DATES: From: 15 January 1997 (date of arrival) To: 13 March 1997 (necropsy of the reversibility study animals)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

maize oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared for administration as a series of graded concentrations in maize oil. It was pre-weighted into a suitable container. Maize oil was added slowly in small amounts until the required amount of product was attained. The final product was magnetically stirred for approximately one minute to ensure that the test material was fully dispersed in the maize oil. All formulations were prepared freshly each day and were administred as soon as possible aftre preparation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Acceptable homogeneity of the formulation was demonstrated (method of analysis provided by the Sponsor and modified at the Testing laboratory).
- Concentration in vehicle: 3, 30 and 200 mg/mL, respectively for the dose levels of 15, 150 and 1000 mg/kg bw/day
- Amount of vehicle (if gavage): 5 ml/kg bodyweight. The volume administred to each animal was calculated from the bodyweight measured immediately before administration.
- Lot/batch no. (if required): no data
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity of distribution of the test substance in maize oil was assessed analytically in a trial preparation at concentrayions of 3 and 200 mg/ml, prepared before the commencement of treatment. Six unit dose samples taken from positions evenly spaced throughout the bulk preparation were assayed to determine the homogeneity of the formulation. The method of analysis was provided by the Sponsor and modified at the Testing Laboratory (Gravidic determination of the test material in maize oil by Spectophotometry). Acceptable homogeneity of the formulation was demonstrated for both concentrations in maize oil. The concentrations of the analysed preparations from Week 1 and 4 treatment were satisfactory.

Duration of treatment / exposure:

All animals were treated for at least 28 days. Treatment and recording of serial observations continued for all main study animals throughout the main study necropsy period (therefore main study animals were treated for 30 days).
The reversibility phase commenced on Day 29 and lasted for 16 days. The recording of serial observations continued throughout the reversibility necropsy period.

Frequency of treatment:

One each day, seven days per week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- Main study: 5 males and 5 females per dose-level group (0, 15, 150 and 1000 mg/kg/day dosage groups)
- Reversibility study: 5 males and 5 females per dose-level group (0 and 1000 mg/kg/day dosage groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages of 15, 150 and 1000 mg/kg/day were selected on the basis of a 7-day preliminary study (Huntingdon Life Sciences Report No. RNP503/970031) which indicated that dosages of up to 1000 mg/kg/day were well tolerated with only minor indications of non-specific toxicity evident.
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

Not applicable (not required)

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were insepected at least twice daily for evidence of reaction to treatment or ill-health. Any deviations from normal were recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: individual observations of all animals were recorded before and after dosing on each day of treatment. The timing of these observations were performed to estabilish and confirm any pattern of signs. The schedule was:
Week 1: dailly
Weeks 2-4: twice weekly (middle and end of each week
- The detailed observations were performed at the following times during the above specified days:
a) Pre-dose observation
b) As each animal was returned ti its home cage
c) At the end of dosing each group
d) Between 1 and 2 hours after completion of dosing all groups
e) As late as possible in the working day

- In addition, a more detailed weekly examination, which included palpation, was performed on each animal. Cage and cage-trays were inspected daily for evidence of a nimal ill-health, such as blood or loose faeces.

- During the acclimatation and reversibility periods, observations of the animals and their cages were recorded at least once per day.

BODY WEIGHT: Yes
- Time schedule for examinations: each animal was weighted during the acclimatation period, on the day that treatment started, at weekly intervals throughout the treatment and reversibility periods and before necropsy.

FOOD CONSUMPTION: YES
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for each week throughout the treatment and reversibility periods. From these records, the mean weekly consumption per animal was calculated for each cage.

FOOD CONVERSION EFFICIENCY: YES
Group mean food conversion efficiencies were calculated for each week of the treatment and reversibility periods.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY, PERIPHERAL BLOOD: Yes
- Time schedule for collection of blood: Blood samples were withdrawn from the retro-orbital sinus of animals under anaesthesia
1) on day 30 of treatment (before dosing) of the main study
2) on day 16 of the reversibility phase
- Anaesthetic used for blood collection: Yes, halothane/nitrous oxide anaesthesia
- Animals fasted: Yes, overnight
- How many animals: all main study animals and all the reversibility study animals
- Parameters evaluated using a Technicon H 1 haematology analyser:
1) For samples collected into EDTA as anticoagulant: Packed cell volume (PCV), Haemoglobin concentration (HB), Erythrocyte count (RBC), Mean cell haemoglobin concentration (MCHC), Mean cell haemoglobin (MCH), Mean cell volume (MCV), Total and differential leucocyte count (WBC)* and Platelet count (PLAT).
-Blood film-Romanowsky stain, examined by light microscopy for abnormal and unusual cell types, including normoblasts*.
* Performed only on animals of the main study
2) For samples collected into Citrate as anticoagulant: Prothrombin time (PT) ans Activated partial thromboplastin time (PTTK).

BLOOD CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained from all animals into lithium heparin as anticoagulant
1) on day 30 of treatment (before dosing) of the main study
2) on day 16 of the reversibility phase
Samples were then analysed for the appropriate parameters after separation of the plasma.
- Animals fasted: Yes, overnight
- How many animals: all main study animals and all the reversibility study animals
- Parameters checked:
1) Main study animals: Alkaline phosphatase activity (ALP), Alanine amino-transferase activity (ALT), Aspartate amino-transferase (AST), Gamma-glutamyl transpeptidase activity (GGT), Glucose concentration (GLUC), Total bilirubin concentration (BILT), Total cholesterol concentration (CHOL), Total triglyceride concentration (TRIG), Creatinine concentration (CREA), Urea concentration, Total protein concentration (TP), Electrophoretic protein fractions (ALB, A-1, A-2, BETA, GAMMA), Albumin/globulin ratio (A/G), Sodium (Na) and Potassium (K), Chloride (Cl), Calcium concentration (Ca) and Inorganic phosphorus (Phos).
2) Reversibility study animals: Alanine amino-transferase activity (ALT), Glucose concentration (GLUC), Urea concentration, Total protein concentration (TP), Electrophoretic protein fractions (ALB, A-1, A-2, BETA, GAMMA), Albumin/globulin ratio (A/G) and Inorganic phosphorus (Phos).

URINALYSIS: Yes
- Time schedule for collection of urine: On day 30 of treatment and day 16 of reversibility overnight, urine samples were collected from all animals.
- Metabolism cages used for collection of urine: Yes. At approximately 17.00 hours each animal was placed in an individual metabolism cage without food or water, and urine was collected until approximately 09.00 hours the following day.
- Animals fasted: Yes, overnight. Animals were also deprived of water from approximately 12.30 hours
- Parameters checked: Appearance (APP), Volume (VOL), pH, Specific gravity (SG), Protein (PROT), Glucose (GLUC), Ketones (KET), Bilurubin (BIL), Blood and Microscopy [sediment from centrifugation was examined for epithelial cells (EP), polymorphonuclear leucocytes (P), erythrocytes (RBC), crystals (CRY), spermatozoa and precursors (S) or other abnormalities (A)].

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Euthanasia: All main study and reversibility phase animals were sacrified by carbon dioxide inhalation.
- Necropsy: All animals were subjected to a detailed necropsy. The necropsy procedure included a review of the history of each animal and a detailed examination of the external features and orifices, the neck and associated tissues and the cranial, thoracic, abdominal and pelvic cavities and their viscera. The requisite organs were weighted and external and cut surfaces of the organs and tissues were examined as appropriate. Abnormalities and interactions were noted and the required tissue samples preserved in fixative.
The retained tissues were checked against the protocol and the necropsy report reviewed.

ORGAN WEIGHTS: Yes
The following organs, taken from each animal, were dissected free of adjacent fat and other contiguous tissue and the weights recorded: Adrenals, Brain, Epididymides; Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles, Spleen, Testes.
The weight of each organ was expressed as a percentage of the bodyweight recorded immedialtely before necropsy.

HISTOPATHOLOGY: Yes
- Samples of the following tissues were preserved in 4% neutral buffered formaldehyde, except the testes which were initially placed in Bouin's fluid: Adrenals, Duodenum, Heart, Kidneys, Liver, Spleen, Stomach and Testes.
Samples of any abnormal tissues were also retained for histopathological examination.
- Tissue samples from all animals were dehydrated, embeded in paraffin wax, sectioned at approximately 5 µM thickness and stained with haematoxylin and eosin. The tissues subjected to histopathological processing included the following regions:
Adrenal-cortex and medulla
Heart-auricular and ventricular regions
Kidneys-cortex, medulla and papilla regions
Liver-section fom each of the left and median lobes.
For bilateral organs, sections of both were examined. A single section was prepared for each of the remaining tissues required for microscopic examination.

- Microscopic examination was performed on the tissues specified above for all animals.
- The tissues reported at macroscopic examination as being abnormal were also examined by microscopy for all animals of both the main study and reversibility phase.

- Samples of the tissues listed below were not processed histologically, but were held in fixative against any future requirement for microscopic examination: Aorta-thoracix, Brain, Bronchi, Caecum, Colon, Epididymides, Eyes and optic nerves, Harderian glands, Ileum, Pancreas, Pituitary, Prostate, Rectum, Salivary glands-submandibular, Sciatic nerves, Seminal vesicles, Skeletal muscle-thigh Skin, Jejunum, Lacrymal glands, Lungs, Lymph nodes - mandibular and mesenteric, Mammary glands-caudal and cranial, Oesophagus, Ovaries, Spinal cord, Sternum, Thymus, Thyroid with parathyroids, Tongue, Trachea, Urinary bladder, Uterus with cervix and Vagina. Femoral bone marrow smears were taken from all animals, air-dried, fiwed in methanol and stained using a May-Grünwald-Giemsa procedure.

Other examinations:

No further examinations.

Statistics:

The significance of inter-group differences in hematology, blood chemistry and urinalysis (volume, specific gravity and pH only) was assessed by Student's t-test using a pooled error variance. Statistical significances for eosinophil, basophil, monocyte and large unstained cell counts are not reported as these data are not normally distributed. For organ weights and bodyweight changes, homogeneity of variance was tested using Bartlett's test. Whenever this was found to be statistically significant a Behrens-Fisher test was used to perform pairwise comparisons, otherwise a Dunnett's test was used.
Inter-group differences in macroscopic pathology and histopathology were assessed using Fisher's Exact test.
Unless stated, group mean values or incidences for the treated groups were not significantly different from those of the Controls (p>0.05).
Because of the small number of animals in each group, the results of these tests cannot be considered definitive and are used merely as a guide in the interpretation of the results.

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):

significantly lower than controls during the first week of treatment in males at the 1000 mg/kg/day dose.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

on food consumption

Food efficiency:

effects observed, treatment-related

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

on water consumption

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- No mortality was observed during the study.
- Salivation associated with the dosing procedure was noted in virtually all animals receiving 1000 mg/kg/day from the second half of the first week of treatment onwards. Isolated incidences were also seen in the other treated groups.
- Routine clinical signs observations were restricted to some red staining, mainly dorsal, in animals receiving 1000 mg/kg/ day. Some residual red staining was noted during the reversibility period.
- There were no other signs that were considered to be related to treatment with Cerium sulphide.

BODY WEIGHT AND WEIGHT GAIN
- The bodyweight gains of males receiving 1000 mg/kg/day were significantly lower in comparison with the Controls during the first week of treatment. Overall, gains were not, however, significantly affected.
- The bodyweight gains of females receiving 1000 mg/kg/day and of the other treated groups were unaffected by treatment.
- During the reversibility period, the bodyweight gains of previously treated animals were broadly similar to those of the controls.

FOOD CONSUMPTION
- There was no clear dose-related effect of treatment upon the overall food consumption pf treated animals . However, food consumption of the males and females receiving 1000 mg/kg/day was slightly high in comparison with their Controls during the third and fourth weeks of treatment.
- The food consumption of animals that had previouvsly received 1000 mg/kg/day during the reversibility period was broadly similar to that of the Controls.

FOOD CONVERSION EFFICIENCY
- The efficiency of food conversion of males, and to a lesser extent females receiving the 1000 mg/kg/day dose, was inferior to that of the Controls.
- The food conversion efficiency of animals receiving 15 or 150 mg/kg/day was essentially the same as the Controls during the treatment period and the reversibility phase.

WATER CONSUMPTION
- An apparent effect upon water consumption was noted in treated animals on Day 4 of Week 3 of treatment, and formal water consumption measurements were instigated for the remainder of the study. These indicated markedly high water consumption in animals receiving 1000 mg/kg/day, and higher water consumption in females receiving 150 mg/kg/day. Males receiving 150 mg/kg/day and animals receiving 15mg/kg/day had water consumption broadly similar to the Controls.
- During the reversibility period, the water consumption of the males that received 1000 mg/kg/day remained slightly higher than their Controls, while that of the females was similar to their Controls.

HAEMATOLOGY
- On day 30 of treatment, significantly high erythrocyte counts associated with low mean cell haemoglobin and mean cell volumes were recorded in males and females receiving 1000 mg/kg/day. The mean cell haemoglobin concentrations in females receiving 1000 mg/kg/day and the mean cell volumes of males receiving 15 or 150 mg/kg/day were also significantly lower than Controls.
- The platelet counts of males and females receiving 1000 mg/kg/day were higher than Controls and, though dose-relationship was not evident, the prothrombin times and the activated partial thromboplastin times of all treated male groups were lower than tjeir Controls. After two weeks of recovery from treatment, the only effects considered to be related to treatment with Cerium sulphide comprised slightly lower mean cell haemoglobin concentration in the females and slightly shorter prothrombin times in the males.
- Other inter-group differences that were statistically significant (p<0.05) lacked dose-relationship, were minor or were confined to one sex and were therfore considered to be of no toxicological significance.

CLINICAL CHEMISTRY
- Biochemical investigations on Day 30 of treatment showed higher Alanine Amino-Transferase activities in females receiving 150 mg/kg/day and in males and females receiving 1000 mg/kg/day. Aspartate Amino-Transferase activities were also significantly higher when compared to Controls in males receiving 15 or 1000 mg/kg/day.
- Other biochemical changes that were considered to be relates to treatment comprised: low glucose and high phosphorus concentrations in animals receiving 1000 mg/kg/day, and higher urea concentrations in males receiving 1000 mg/kg/day.
- There were no evident residual biochemical disturbances after two weeks of respite from treatment.
- Other inter-group differences that were statistically significant (p<0.05) were either minor, lacked dose-relationship, or were confined to one sex, and were therfore not attributed to treatment.

URINALYSIS
- Urinalysis on Day 30 of treatment showed significantly higher urinary volumes and lower urinary specific gravities from male and females receiving 1000 mg/kg/day. The pH of the urine produced by females receiving 150 or 1000 mg/kg/day was also slightly but significantly more acidic than that of the Controls.
- Animals receiving 1000 mg/kg/day had a lower incidence of crystals and males at this dosage also had lower urinary protein.
- No disturbances of the urine were evident after two weeks of the reversibility period.

NEUROBEHAVIOUR

ORGAN WEIGHTS
Organ weights were not disturbed by treatment.

GROSS PATHOLOGY
- Macropathological examination at necropsy revealed thickening of the walls of the stomach and/or duodenumin four males and three females that received 1000 mg/kg/day, and in one male that received 150 mg/kg/day.
- The vasculature of the serosal surfaces of the stomach was also prominent in three of the affected high dosage males
- After 2 weeks of respite from treatment, areas of change in the glandular mucosa of the stomach and thickening of the duodenal wall were still evident in one male that received 1000 mg/kg/day. Another male animal that was dosed with 1000 mg/kg/day also exhibited abdominal adhesions involving multiple abdominal organs.

HISTOPATHOLOGY: NON-NEOPLASTIC (see Table in "Any other information on results incl. tables")
- The microscopic changes that were considered to be related to treatment with Cerium sulphide were seen in the stomach and duodenum. After 4 weeks of treatment, greater than normal numbers of acute inflammatory cells were present in the submucosa of the glandular region of the stomach in the five males and three of the females that received 1000 mg/kg/day. Hyperplasia of the keratinised mucosa in the non-glandular region and submucosal oedema were also noted in some of these animals.
- Mucosal hyperplasia, characterised by increased thickness and height of the villi, was seen in the duodenum of the five males and three of the females which received 1000 mg/kg/day, and one male which received 150 mg/kg/day of Cerium sulphide.
- After two weeks of recovery from treatment, two males which previously received 1000 mg/kg/day still showed increased numbers of acute inflammatory cells in the submucosa of the glandular region. However, this finding was also present at a similar severity in one male from the Control group.
- In one of the males which received the dose of 1000 mg/kg/day, macroscopic changes were noted at necropsy in the stomach and duodenum at the end of the reversibility period. There were, however, no microscopic changes to account for the necropsy findings.
- Multiple abdominal adhesions in one male which received 1000 mg/kg/day could potentially have arisen from an inflammatory focus in one of the affected organs and, as such, may have been associated with treatment, although no such inflammatory focus was detected histologically.
- All other microscopic findings were considered to be incidental and of no toxicological relevance.

OTHER FINDINGS
- No further findings.

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of toxicologically significant changes

2

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Complete or almost complete reversible of local irritation changes in the stomach and duodenum, and microcytic anaemia observed at the dosage of 1000 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table: Treatment-related findings of the main study in the stomach and duodenum

	Group and Sex
	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	15	150	1000	0	15	150	1000
Stomach
Keratinised region : epithelial hyperplasia	0	0	0	4*	0	0	0	1
Glandular region : increased acute inflammatory cells in the submucosa	0	0	0	5**	0	0	0	3
Submucosal oedema	0	0	1	0	0	0	0	1
Duodenum
Mucosal hyperplasia	0	0	1	5**	0	0	0	3

M: male, F: Female, 5 animals examined per group

Significantly different from Control (Group 1): * p<0.05, **<0.01

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions of this study, it is concluded that the oral administration of Dicerium trisulphide at a dose of 1000 mg/kg/day is associated with local irritant changes in the stomach and duodenum and with microcytic anemia. All changes showed complete or almost complete reversibility by the end of the 2-week recovery period.
Although some similar, but slight, localized microscopic changes were seen in an isolated animal dosed with 150 mg/kg/day, this dose can be considered to represent a No Observed Adverse Effect Level (NOAEL) for local effects.
As no toxicologically significant changes were observed at the 15 mg/kg/day dose, this is considered to be the No Observed Effect Level (NOEL).

Executive summary:

This study was performed to assess the toxicity of Dicerium trisulphide to the rat following sub-acute oral repeated exposure, and to assess the potential reversibility of any changes observed. The study was designed in accordance with the requirements of the Ministry of Health and Welfare for Japan – Part II of the Chemical Substance Control Law (1986).

 

Methods

Groups of five male and five female CD rats received Dicerium trisulphide by oral gavage, at doses of 15, 150 or 1000 mg/kg/day for 30 days. A similarly constituted control group received the vehicle, maize oil, alone and served to generate contemporaneous control data. A further five male and five female animals were assigned to each of the high dosage and control groups and were retained for further two weeks after cessation of treatment for investigation of reversibility.

 

Results

- There were no unscheduled deaths.

- Salivation associated with dosing was noted in treated animals, particularly those receiving 1000 mg/kg/day.

- Bodyweight gains were low in males receiving 1000 mg/kg/day during the first week of treatment, but overall gains were similar to the Controls. The bodyweight gains of other treated animals were unaffected by treatment.

- The food consumption of animals receiving 1000 mg/kg/day was slightly high in comparison with their Controls during the latter half of the treatment period but inter-group differences in the overall values lacked dose- relationship. The food consumption of the rats that had previously received 1000 mg/kg/day was broadly similar to that of the Controls during the reversibility period.

- The overall efficiency of food conversion of animals, particularly males receiving 1000 mg/kg/day was inferior to that of the Controls. There was no clear influence on efficiency following cessation of treatment.

- Higher water consumption was noted in animals receiving 1000 mg/kg/day and, to a lesser extent, in females receiving 150 mg/kg/day. Following cessation of dosing, the water consumption of males that received 1000 mg/kg/day remained slightly higher than their Controls.

- Higher erythrocyte and platelet counts, lower mean cell hemoglobin and mean cell volumes were noted in males and females receiving 1000 mg/kg/day. The mean cell hemoglobin concentrations of females receiving 1000 mg/kg/day were also significantly lower than the Controls, as were the mean cell volumes of males receiving 15 or 150 mg/kg/day. After two weeks of reversibility, slightly lower mean cell hemoglobin concentrations were still evident in the females that received 1000 mg/kg/day.

- Higher Alanine Amino-Transferase activities were noted in animals receiving 1000 mg/kg/day and in females receiving 150 mg/kg/day. Aspartate Amino­Transferase activities were also higher in males receiving 15 or 1000 mg/kg/day. Lower glucose and higher phosphorus concentrations were noted in animals receiving 1000 mg/kg/day and higher urea concentrations in males receiving 1000 mg/kg/day. None of these changes were evident at the end of the reversibility period.

- Higher urinary volumes and lower specific gravities were noted in animals receiving 1000 mg/kg/day. The urine of females receiving 150 or 1000 mg/kg/day was slightly more acidic than that of the Controls. Animals receiving 1000 mg/kg/day had lower numbers of crystals in the urine, and males receiving 1000 mg/kg/day had generally lower urinary protein. No inter-group differences were evident after two weeks of the reversibility period.

- Organ weights were undisturbed by treatment.

- Thickening of the walls of the stomach and/or duodenum and prominent vasculature of the serosal surfaces of the stomach was noted in several animals that received 1000 mg/kg/day. Thickening was also apparent in one male that received 150 mg/kg/day. Changes in these organs were also recorded at the end of the two week reversibility period in one male that received 1000 mg/kg/day.

- Histopathology revealed greater than normal numbers of acute inflammatory cells in the submucosa of the glandular region of the stomach and duodenal mucosal hyperplasia in most of the animals that received 1000 mg/kg/day. Duodenal mucosal hyperplasia was also evident in one male receiving 150 mg/kg/day. Hyperplasia of the gastric keratinized mucosa and submucosal oedema were also noted in some animals that received 1000 mg/kg/day. After 2 weeks of recovery from treatment, there were no changes that could be unequivocally attributed to previous treatment with Cerium sulphide. Although increased numbers of acute inflammatory cells were evident in the submucosa of two males that received 1000 mg/kg/day, this finding was also evident at a similar severity in one Control male.

 

Conclusion

Based on the experimental conditions of this study, it is concluded that the oral administration of Dicerium trisulphide at a dose of 1000 mg/kg/day is associated with local irritant changes in the stomach and duodenum and with microcytic anemia. All changes showed complete or almost complete reversibility by the end of the 2-week recovery period.

Although some similar, but slight, localized microscopic changes were seen in an isolated animal dosed with 150 mg/kg/day, this dose can be considered to represent a No Observed Adverse Effect Level (NOAEL).

As no toxicologically significant changes were observed at the 15 mg/kg/day dose, this is considered to be the No Observed Effect Level (NOEL).