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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
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other: GLP compliant, guideline study, unpublished report available, no restrictions, fully adequate for assessment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
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sulfur
EC Number:
231-722-6
EC Name:
Sulfur
Cas Number:
7704-34-9
Molecular formula:
S
IUPAC Name:
sulfur
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, Business Point, 6th Floor, Plot No. 349, 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
Physical appearance: Yellow coloured solid powder
Storage conditions: Ambient (+18 to +36°C)
Purity to be stated in the report: 99.6 % w/w
Density: 2.07g/mL (rhombic)
Analysed purity (as per certificate of analysis): 98.6% w/w

Test animals

Species:
rat
Strain:
other: HsdCpb: WU rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Species: HsdCpb: WU rats conventionally bred (In-house random bred)
Source: Toxicology Department; Rallis Research Centre; Bangalore - 560 058, INDIA
No. of rats/group: 10 (5 males + 5 females). Animals with healthy skins were used. Females were nulliparous and non-pregnant.
Date of birth: 24.01.2005 to 27.01.2005
Age at start of treatment: 12 – 13 weeks
Mean body weights (g): Males 279g, females 217g
Acclimatization : 5 days under experimental conditions after veterinary examination.
Housing: The rats were individually housed in sterilized suspended polypropylene rat cages (size: L 410 x B 280 x H 140 mm) with stainless steel top grill having facilities for pelletted food and drinking water in glass bottles. Cages were changed once a week.
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 bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier (manufactured by Eureka Forbes Ltd., Mumbai - 400 001, INDIA in collaboration with Electrolux of SWEDEN) was provided to animals 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

Type of coverage:
occlusive
Vehicle:
other: de-ionised water
Details on exposure:
Based on the individual body weight, the test item at doses of 100 (G2), 400 (G3) and 1000 (G4 and G4R) mg/kg were separately weighed on aluminium foil and moistened with sufficient quantity of de-ionised water and made into a paste. This paste was completely transferred on to the cotton gauze (Males: 9 x 6 cm2; Females: 8 x 6 cm2 - 6 ply) and the cotton gauze along with the test item was applied on the clipped area of the skin of main (G2, G3 and G4) and recovery (G4R) group rats. The cotton gauze was held in contact with the skin with a non-irritating crepe bandage, Dyna crepe (Johnson and Johnson Limited, Mumbai - 400 036, INDIA). The test item contact with the skin was for at least 6 hours during all treatment days (during this period drinking water was withheld). After removal of the dressing, the treated skin area was washed with tap water and Johnson’s baby moisturising soap (Johnson – Johnson, Mumbai – 400 036).
The animals of the vehicle control (G1) and vehicle control recovery (G1R) groups were handled similar to the treatment groups and sufficient amount of the de-ionised water (vehicle) without the test item was instilled on to the cotton gauze which was applied over the prepared area of the skin.
The dose was calculated based on the individual animal body weight on the first day of treatment and subsequently the dose was adjusted according to the weekly body weights thereafter.

Approximately 24 hours before first test item application, the hair on the dorsolateral thoracic region of the rats was clipped (approximately: 6.5 x 11 cm) with an electric clipper (Aesculap® - Germany). Repeat clipping was done twice a week. During clipping care was taken to avoid causing skin abrasions.

The test item was applied to the maximum possible area.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
5 days/week, 6 hours/day
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 400 and 1000 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Doses for the study were selected based on the results of the prestudy (Study No.: 4190/05/1) in which doses of 100, 400 and 1000 mg/kg body weight were tested. The test item at the doses of 100 (G2), 400 (G3) and 1000 (G4) mg/kg body weight was made as a paste with de-ionised water and loaded on a cotton gauze and applied to 4 (2M + 2F) rats/dose for 2 weeks (10 applications). The vehicle control group (G1) was treated similarly with de-ionised water, but without the test item.
The results of pre-study were as follows: There were no toxic signs, pre-terminal deaths and local skin reactions. There was no effect of the test item on body weight, food consumption and there were no abnormalities detected at necropsy. Based on the above results of the pre-study, the doses of 100 (G2), 400 (G3) and 1000 (G4/G4R) mg/kg body weight were selected for the main study.

Recovery period: The vehicle control recovery (G1R) and high dose recovery (G4R) groups were treated similar to the vehicle control (G1) and high dose (G4) groups for 28 days (5 days a week for four weeks), thereafter no treatment was given for 14 days (recovery period).

Examinations

Observations and examinations performed and frequency:
a. Veterinary examination: Veterinary examination was done prior to the initiation of treatment and weekly thereafter during treatment and recovery periods.
b. Clinical examination: Clinical examination was done prior to initiation of treatment and once weekly during treatment and recovery periods.
c. Ophthalmological examination: Ophthalmological examination of all animals was done with an ophthalmoscope before the start of treatment, at the end of treatment period for all groups and at the end of recovery period for recovery groups. Mydriasis was induced before examination by using 1% Tropicamide solution.
d. General clinical signs and pre-terminal deaths: General clinical signs and pre-terminal deaths were observed twice daily on treatment days and once daily on other days. The treated skin areas were examined once daily on all treatment days prior to the next application and daily during recovery period. The skin reactions were assessed by the Draize Method.
e. Body weights: Individual body weights were recorded shortly before the test item was first applied and at weekly intervals thereafter.
f. Food intake: The following method was adopted for the measurement of weekly food consumption. Day 1a: Food input 250 g Food output on day 8
a: Day ‘1’ denotes food input at the start of each week.
The weekly cagewise food consumption was calculated by dividing the total food consumed in 7 days by the number of animals per cage to determine the food intake/rat/week. The visual estimation of the food spillage was recorded during each food output and litter paper change session and added to the food output data for the calculation of weekly food consumption. The weekly consumption/rat was divided by the number of days (7) to obtain food consumption (g)/rat/day. This was repeated throughout the treatment and recovery periods.
g. Blood smear: Blood smears were made one day before sacrifice from all the animals of the main groups and recovery groups by the tail clipping method.
h. Blood collection: At the end of the treatment period and the recovery period, all the animals were fasted overnight (water allowed) and blood was collected from the abdominal aorta under ether anaesthesia.
i. Haematology: The following haematological parameters were determined Haemoglobin (Hb), Red Blood Corpuscles (RBC), White Blood Corpuscles (WBC), Haematocrit (Hct) and Platelets (Plat)
j. Clinical Chemistry: Plasma was separated and analysed for the following parameters: Fasting Glucose (Glu) mmol/l, Blood Urea Nitrogen (BUN) mmol/l, Total 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, Total Bilirubin (Tot. Bil) μmol/l, Creatinine (Creat) μmol/l, Albumin (Alb) g/l, Inorganic Phosphorus (Pi) mmol/l, Calcium (Ca) mmol/l, Chloride (Cl) mEq/l, Sodium (Na) mEq/l, Potassium (K) mEq/l
Sacrifice and pathology:
a. Gross necropsy: All rats in the study were subjected to gross necropsy and the findings recorded. The animals sacrificed at term were fasted overnight (water allowed), anaesthetised as per random numbers generated for the study, weighed, exsanguinated and subjected to detailed necropsy by a pathologist.
b. Organ weights: The following organs were weighed: liver, adrenals, kidneys and testes. The relative organ weights as a percentage of body weight were also determined.
c. Tissue collection: The following organs and tissues were collected from all rats and preserved in 10% buffered neutral formalin:
- Treated skin
- Untreated skin
- Liver
- Kidneys
d. Histopathology: Histopathological examination was performed on all preserved organs and tissues of the high dose group and the control group including gross lesions. All gross lesions and treated skin in the lower dose groups (both males and females) and recovery groups were examined. Unused tissues were archived.
Statistics:
Using specific computer programmes, body weight, net body weight gain, 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 proved 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. Following ANOVA, when ‘F' value was significant, Dunnett's pairwise comparison of means of treated groups with control group mean was done individually.
Organ weight and organ weight ratio data were analysed by Student ‘t’ test. 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 co-efficient was estimated and subjected to ‘t’ test. 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.
All analyses and comparisons were evaluated at 5% (P<0.05) level.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
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):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
- No toxic/clinical signs were observed at any of the tested doses either during treatment and recovery periods.
- No pre-terminal deaths were observed at any of the tested doses in both sexes.
- No local skin reactions and changes in skin/fur 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 in males and females.
- There were no treatment related gross changes.
- Histopathology: In both males and females, treated skin was examined in the lower dose groups and recovery groups as higher incidence of hyperkeratosis was observed at high dose. The significant increase in the incidence of hyperkeratosis in high dose males and females was considered treatment related. This change was minimal in severity and considered reversible as in the recovery groups the incidence was nil in high dose recovery males and a single incidence was observed in high dose recovery females.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
400 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Microscopically the treated skin shows higher incidences of hyperkeratosis in the high dose males and females which was considered treatment related.
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No adverse systemic effects were observed.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the lack of systemic toxicity effects observed in this study, the NOAEL for systemic effects was determined to be 1000 mg/kg bw/day. The NOAEL for local dermal effects was 400 mg/kg bw/day.
Executive summary:

Sulfur was tested for its dermal toxicity potential in a 28 day study (OECD guideline 410 and GLP compliant) using Wistar rats. The test item was applied at doses of 100, 400 and 1000 mg/kg bw/day. It was applied as a paste (moistened with de-ionised water) with the dose adjusted based on individual body weight. The paste was transferred on to a cotton gauze pad and applied to a prepared area of skin for 5 days a week for 4 consecutive weeks. A cotton gauze pad moistened with a similar amount of deionised water served as the concurrent vehicle control treatment. The study design included control and high dose recovery groups group, which were maintained for 2 weeks (14 days) after treatment ended. All the rats were observed for clinical signs, local skin reactions, changes in skin/fur, changes in body weight and food consumption. Haematological and clinical chemistry investigations were performed at termination. The rats were subjected to a detailed necropsy at sacrifice and the organs were weighed. Histopathological evaluation was carried out on all the tissues collected from control and high dose group animals and treated skin from all groups, along with any gross lesions present in the lower dose groups and recovery groups. No systemic effects were observed at any dose level tested. Regarding local effects, a higher incidence of hyperkeratosis (apparent after microscopic examination of treated skin and present in the high dose males and females only) was considered treatment related. This change was considered reversible as in the recovery groups the incidence was nil in high dose recovery males and a single occurrence was present in high dose recovery females.

In view of the results observed, the NOAEL for systemic effects was 1000 mg/kg bw/day. The NOAEL for local dermal effects was 400 mg/kg bw/day.