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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, unpublished report available, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test Item: Sulphur Technical
Common name (active ingredient): Sulfur
Chemical name (IUPAC): Sulfur
CAS No. : 7704-34-9
Batch No. : SML/RD/T/S-191
Manufactured and Supplied by: Sulphur Mills Limited; 604/605, Plot No. 349, Business Point; 6th Floor, Western Express Highway; Andheri (E), Mumbai - 400 069, INDIA; Tel.: 91-22-5691 0011; Fax.: 91-22-56910308
Date of manufacture: January 2005
Date of expiry: December 2006
Purity stated in the report: 99.6% w/w
Physical appearance: Yellow coloured solid powder
Storage conditions: Ambient (+18 to +36°C)

Test animals

Species:
rat
Strain:
other: Hsd Cpb: WU
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Species: Hsd Cpb: WU rats conventionally bred (In-house random bred)
Source: Toxicology Department; Rallis Research Centre; Rallis India Limited; Bangalore - 560 058, INDIA
No. of rats/group: 20 (10 males + 10 females)
Age at the start of treatment : 6 – 7 weeks
Mean body weights (g): Males 191 g, Females 138 g
Acclimatization: After veterinary examination, for ascertaining good health and suitability for the study, the rats were acclimatized for five days before
start of the treatment. Females used in the study were nulliparous and non-pregnant.
Housing: Two rats per sex were housed in sterilized suspended polypropylene rat cages (size: L 410 x W 282 x H 150 mm) with stainless steel mesh bottom and stainless steel top grill having facilities for holding pellet food and drinking water in glass bottles with stainless steel sipper tubes.
Food: ad libitum: Ssniff rats/mice pellet food-maintenance meal-low in germs manufactured by Ssniff Spezialdiäten GmbH., Ferdinand-Gabriel-
Weg 16, D-59494 Söest, GERMANY was provided to animals.
Water: ad libitum: Deep borewell water passed through activated charcoal filter and exposed to UV lamp in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai - 400 001, INDIA in collaboration with Electrolux, SWEDEN was kept in glass bottles with stainless steel sipper tubes.

ENVIRONMENTAL CONDITIONS:
Air conditioned with adequate filtered fresh air supply (12 - 15 air changes/hour)
Temperature: 21 - 24°C
Relative humidity: 30 - 70%
Photo period: 12 hour light and 12 hour dark cycle.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxymethyl cellulose containing 0.1% Tween-80
Details on oral exposure:
The test item suspensions were prepared on a daily basis. Quantities of 0.75 g (G2), 3.00 g (G3) and 15.0 g (G4/G4R) of test item were weighed and suspended in 0.5% carboxymethyl cellulose containing 0.1% Tween-80 and the volume was made up to 75 ml for G2 and G3 and 150 ml for G4/G4R with 0.5% carboxymethyl cellulose containing 0.1% Tween-80 and stirred for 3 minutes using magnetic stirrer to get test item concentrations of 10 mg (G2), 40 mg (G3) and 100 mg (G4/G4R)/ml, respectively. Carboxymethyl cellulose was chosen as the vehicle since the test item was not soluble in water.
For control and vehicle control recovery groups (G1/G1R), vehicle was stirred for 3 minutes using a magnetic stirrer and was administered at an equivolume dose of 10 ml/kg bw.
The weight of test item, volume of the test item preparation and volume of administration were varied depending on the body weights of the rats recorded during different intervals of the treatment period.
The dose volume was calculated for individual animals on the first day of the treatment and was adjusted according to the body weights recorded during different intervals of the treatment period.
Homogeneity of the test item suspensions during gavaging was maintained by constant stirring using a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sulphur content in the sample was estimated by principally adopting the CIPAC method 18/1/M/1.
Principle: The sulphur is converted by refluxing with sodium sulphite to sodium thiosulphate. The thiosulphate was then titrated with standard iodine solution.
Gavage preparations on treatment day 1 and at the beginning of months 2 and 3 were analysed for test item concentrations. One composite sample was collected from each group from the samples which were prepared for administration.

Homogeneity and Stability (H/S): Stability and homogeneity tests were carried out before start of the treatment i.e, on day 2 of acclimatization period at 10 and 100 mg/ml concentrations. The stability of the test item in the gavage sample was tested for ‘0’ and ‘4’ hours using the procedure given for
estimation of sulphur content in the sample. The mean of homogeneity test results were used as the ‘0’ hour results for stability test. For stability and homogeneity test, quantities of 0.5 and 5.0 g of the test item were initially mixed with 10 ml of 0.5% CMC containing 0.1% Tween-80 and then the volume was made up to 50 ml. The was stirred for 3 minutes using a magnetic stirrer to get test item concentrations of 10 and 100 mg/ml. For homogeneity, samples were taken from top, middle and bottom layers and analysed for the test item concentration using the procedure given for estimation of sulphur content in sample.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 400, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Three dose levels of 100, 400 and 1000 mg/kg bw/day were selected in consultation with the sponsor.

The test item was administered by oral gavage at doses of 100, 400 and 1000 mg/kg bw/day to low (G2), mid (G3) and high dose (G4/G4R) groups of rats, respectively. The concurrent control and recovery group (G1/G1R) recieved 0.5% carboxymethyl cellulose containing 0.1% Tween 80 without the test item. The treatment was discontinued for the high dose recovery group (G4R) at the end of 90th day of treatment. The rats of both G1R and G4R groups were maintained on normal food for a further 28-days periods.

Examinations

Observations and examinations performed and frequency:
a. Veterinary examination: Veterinary examination was carried out prior to the initiation of treatment and weekly thereafter (± 1 day) during treatment and recovery periods.
b. Clinical examination: Clinical examination was carried out prior to initiation of treatment and once weekly (± 1 day) during treatment and recovery periods.
c. Ophthalmological examination: Ophthalmological examination of all animals was done with an ophthalmoscope, before the start of the treatment and at the end of the treatment and recovery periods, prior to sacrifice. Mydriasis was induced before examination using a solution of 1% Tropicamide.
d. General clinical signs and pre-terminal deaths: General clinical signs were observed once daily. All animals were observed for morbidity and pre-terminal deaths twice daily.
e. Neurological Examination: The following neurological examinations were conducted at the end of the treatment period (13th week of the treatment period).
- Home cage observations: Observations on rats were made in their home cages and while opening the cages. The rats were observed for
* Absence or presence of convulsions
* Absence or presence of tremors
* Palpebral (eyelid) closure was observed
- Handling observations: Rats were removed from the cage and then observed for the following reactions.
* Ease of removal from the cage
* Ease of handling animal in hand: the following observations were made, while handling the animal.
* Lacrimation: Rats were observed for whether lacrimation was there or not. If lacrimation was present then it was observed for its severity i.e.,
whether it is slight or severe.
* Chromodacryorrhea (red tears): Rats were observed for presence or absence of red tears.
* Salivation: Rats were observed for whether salivation was there or not. If salivation was present, then it was observed for its severity i.e.,
whether it is slight or severe (active salivation drooling).
* Piloerection: Hair coat was observed whether piloerection was there or not. If piloerection was present, then it was observed for its severity
whether it is slight or severe.
* Palpebral (eyelid) closure was observed
* Respiratory character: Rats were observed for the following changes in respiratory character
* Eye prominence
* Muscle tone: Musculature of the limbs was palpated between the thumb and forefinger
- Open field observations: To carry out open field observations, the animal was placed in open field arena (Dimension 850 x 587 x 200 mm) and
evaluated during a 2 minutes observation period for:
* Mobility: Scoring was done within 30 seconds of placing the animal in open field arena.
* Backing: Recording was done for the number of times animal takes backward steps during the 2 minute observation period in the open field
arena.
* Grooming: Recording was done for the number of times the animal grooms itself during the 2 minute observation period in the open field
arena. Grooming included wiping/rubbing face and head with forepaws, scratching head or body with hind paws and biting the fur.
* Gait: Gait of the animal was observed
* Convulsions: Rats were observed for presence or absence of convulsions.
* Tremors: Rats were observed for presence or absence of tremors.
* Arousal
- Sensory observations: The following sensory observation tests were performed in the open field arena.
* Startle response (Auditory response): A finger click sound was produced directly above the rat’s head and the rats were observed
* Touch response (Tactile response): The rump was touched with a pencil tip and reaction by the rat was observed
* Pupil response (visual response): Using a pen torch, light was shone into the one eye of the rat and the other eye was shielded from the light.
The response of the pupil i.e., constriction of the pupil present or absent was recorded. The same procedure was followed for the other eye also
* Response to nociceptive stimuli: The tail was gently pressed with a forceps and response from the rat was recorded
* Righting reflex
- Neuromuscular observations: The following neuromuscular observation tests were performed:
* Grip strength: The grip strength of fore limbs and hind limbs was done using Digital force Measurement Instrument (Chatillon grip strength
apparatus). Each animal was allowed to grip the T-shaped bar with the paws of the fore limbs and hind limbs and each animal was pulled
backwards gently until the grip was broken and the displayed readings were recorded. Three readings each for fore limbs and hind limbs were
recorded.
* Motor activity: Motor activity was done using photoactometer. Each animal was placed inside the activity cage of the instrument
(photoactometer chamber) for 15 minutes and at the end of 15 minutes session, the displayed score was recorded.
Hind limbs foot splay: The heel pads of the hind feet of each rat were painted with ink and the rat was dropped down onto a sheet of white
blotting paper from a height of 30 cms above the table. The distance in centimetres between the centres of the backs of the heel prints was
measured. Three readings were recorded for each rat.
- Physiological observations:
* Body temperature: Thermometer was inserted into the rectum and the displayed temperature in degree Fahrenheit (°F) was recorded after
the beep sound indicating completion of equilibration.
f. Body Weights: Individual body weights were recorded before the start of treatment and weekly thereafter except for week 13 wherein the animals were weighed on day 6 of that week.
g. Food Intake: Determinations were performed at weekly intervals.
h. Clinical Laboratory Investigations
* Blood smear: Blood smears were made two to three days prior to sacrifice from all rats
* Blood collection: At the end of the treatment period for main groups and at the end of recovery period for recovery groups, all surviving rats
were fasted overnight (water allowed) and blood was collected from abdominal aorta under ether anaesthesia.
* Haematology: Blood was analysed for the following haematological parameters: Haemoglobin (Hb), Red Blood Corpuscles (RBC), White Blood
Corpuscles (WBC), Haematocrit (Hct), Platelets (Plat). The following calculated RBC associated indices were recorded from the haematology
analyser: Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Haemoglobin Concentration (MCHC).
Prothrombin time analysis was carried out after blood collection.
* Clinical chemistry: Plasma was analysed for the following parameters: Fasting Glucose (Glu) mmol/l, Blood Urea Nitrogen (BUN) mmol/l, Urea
(mmol/l), Total Plasma Protein (Tot.Pro.) g/l, Aspartate Amino transferase (AST) U/l, Alanine Amino transferase (ALT) U/l, Alkaline phosphatase
(Alp) U/l, Gamma Glutamyl Transpeptidase (GGT) U/l, Creatinine (Creat) μmol/l, Albumin (Alb) g/l, Total Cholesterol (Chol) mmol/l, Sodium (Na)
mEq/l, Potassium (K) mEq/l
Sacrifice and pathology:
Gross necropsy: All rats in the study were subjected to gross necropsy and the findings were recorded. The rats sacrificed at term were fasted overnight (water allowed), anaesthetized using ether as per random numbers generated for the study, weighed and blood was collected. After blood collection, the animals were subjected to detailed necropsy by the Pathologist.
Tissue collection: Tissue samples from the following organs were preserved in 10% buffered neutral formalin and archived:
1. Liver
2. Kidneys
3. Lungs
4. Spleen
5. Heart
6. Aorta
7. Thymus
8. Stomach
9. Duodenum
10. Pancreas
11. Jejunum
12. Ileum (with Peyer’s patches)
13. Cecum
14. Colon
15. Rectum
16. Mesenteric lymph nodes
17. Trachea
18. Esophagus
19. Thyroids with Parathyroids
20. Adrenals
21. Urinary bladder
22. Ovaries
23. Uterus
24. Testes
25. Epididymides
26. Prostate
27. Seminal vesicles and coagulating glands
28. Female mammary gland
29. Brain including medulla/pons cerebellum and cerebrum
30. Pituitary
31. Spinal cord at 3 levels - cervical, mid thoracic and lumbar.
32. Sciatic nerves
33. Sternum with bone marrow
34. Bone marrow smear
35. Axillary lymph node
36. Eyes
37. Skin
38. Salivary glands
39. All gross lesions
Organ weights: The following organs were weighed from all animals: liver, adrenals, kidneys, ovaries, testes, epididymides, uterus, thymus, spleen, brain and heart. The organ weight ratios was determined as percentage of body weights.
Histopathology: Histopathological evaluation was carried out on all the tissues collected from control and high dose group animals and gross lesions from low, mid and recovery groups. The tissues were processed for routine paraffin embedding and 5 micron sections were stained with Harris Haematoxylin Eosin stain. Unused tissues were archived.
Statistics:
Using specific computer programmes, functional observation battery, body weights, net body weight gains, food consumption and laboratory investigation (haematology and clinical chemistry) data were compared by Bartlett's test for homogeneity of intra group variances. When the variances prove to be heterogeneous, the data were transformed using appropriate transformation. The data with homogeneous intra group variances were subjected to one-way analysis of variance (ANOVA).
Organ weight and organ weight ratio data were analysed by Student ‘t’ test.
Following ANOVA, when ‘F' value was significant, Dunnett's pairwise comparison of means of treated groups with control group mean was done individually. In the case of recovery groups (reversal study), data of treatment period and recovery period (no treatment period) was tested using the methods stated above. When a significant difference in values over the control group was observed in a minimum of two treatment groups with linear increase or decrease, the dose correlation coefficient was estimated and subjected to ‘t’ test.
All analyses and comparisons were evaluated at the 5% (P<0.05) level.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
- No toxic/clinical signs were observed at any of the tested doses either during treatment or recovery periods.
- No pre-terminal deaths were observed at any of the tested doses in both sexes.
- No treatment related changes in weekly mean body weights and cumulative net body weight gains in either sex during treatment and recovery periods.
- Food intake showed no inter group difference in either sex during treatment and recovery periods.
- No treatment-related changes in haematological parameters were observed at any of the tested doses in both sexes.
- No treatment-related changes in biochemical parameters were observed at any of the tested doses in both sexes.
- There were no treatment related changes in the terminal fasting body weights, organ weights and organ weight ratios.
- There were no treatment related gross and microscopic findings.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 other: mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No treatment related adverse effects were observed.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the lack of treatment-related effects, the subchronic toxicity NOAEL was determined to be 1000 mg/kg bw/day.
Executive summary:

Sulfur was tested in a repeated dose (90 days) oral toxicity study in Wistar rats (OECD guideline 408 and GLP compliant). It was administered by oral gavage at doses of 100, 400 and 1000 mg/kg bw/day to low, mid and high dose (including recovery group) groups of rats, respectively. The concurrent control and control recovery group received 0.5% carboxymethyl cellulose containing 0.1% Tween 80 without the test item. Treatment was discontinued for the high dose recovery group at the end of 90th day of treatment. The rats of both the control recovery and high dose recovery group were maintained on normal food for a further 28 days. All the groups consisted of 10 male and 10 female rats.All rats were observed for clinical signs, physical abnormalities, changes in body weights, food consumption and pre-terminal deaths. Haematological and clinical chemistry investigations were performed at sacrifice. The rats were subjected to detailed necropsy at terminal sacrifice and the organs were weighed. Histopathological evaluation was carried out on an extensive range of tissues collected from control and high dose group animals as well as on any gross lesions from lower dose and recovery groups. No changes of toxicological significance were noted up to the highest tested dose of 1000 mg/kg bw/day. This dose level is considered to be the NOAEL for sulfur in Wistar rats.