Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
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other: GLP compliant, guideline study, unpublished report available, no restrictions, fully adequate for assessment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:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Common Name (active ingredient): sulfur
Name to be used in the report: Sulphur Technical
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.: 91-22-5691 0308
Date of manufacture: January 2005
Date of expiry: December 2006
Purity to be stated in the report: 99.6 % w/w
Density: 2.07g/mL (rhombic)
Physical appearance: Yellow coloured solid powder
Storage conditions: Ambient (+18 to +36°C)
Analysed purity (as per certificate of analysis): 98.6% w/w

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Source: Rallis Research Centre; Toxicology department; Bangalore –560 058; India
No. of mice per group: 10 (5 males + 5 females)
No. of groups: Three (One test item concentration, one positive control and one vehicle control).
Age at the start of acclimatisation: 12 weeks
Body weight range at the start of treatment: Male : 28.19 – 35.58 g; Female : 20.92 – 27.21 g
Acclimatisation: Five days under experimental conditions after veterinary examination.
Grouping: Mice were assigned to 3 study groups by body weight stratification during acclimatisation.
Identification: At the start of acclimatisation, mice were identified by serial numbers and crystal violet body marking. After grouping, they were identified by turmeric body marking, cage cards and permanent accession numbers.
Accommodation: The mice were housed individually during acclimatization and treatment in standard polypropylene mice cages (size: Length 290 x
Width 220 x Height 140 mm) with stainless steel top grill having free access to feed and drinking water in glass bottles with stainless steel sipper tubes.
Bedding: Clean paddy husk was provided and changed thrice a week.
Feed: Pelletted Ssniff rats/mice diet-maintenance meal, low in germs (manufactured by Ssniff Spezialdiäten GmbH., Ferdinand-Gabriel-Weg 16, D-59494 Soest, GERMANY) was provided ad libitum.
Water: Bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard water filter cum purifier (manufactured by Eureka Forbes Ltd., Mumbai 400 001, INDIA) was provided ad libitum in glass bottles.

ENVIRONMENTAL CONDITIONS
Air conditioned with 12 to 15 air changes per hour, temperature 21 to 24°C, relative humidity 30 to 70% and artificial lighting with 12 hours light / dark cycle.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
0.5% aqueous carboxymethyl cellulose (CMC) with Tween 80 (1 ml/l)
Details on exposure:
Route of Administration: oral, as gavage. This route is most commonly used because it maximizes the effect of the dose on the target tissue.

The test item and the positive control were administered orally as gavage twice with a 24 hour interval to the treatment group and to the positive control group respectively at an equivolume of 10 ml/kg body weight. The mice in the vehicle and the positive control groups were handled in an identical manner to those in the treatment group, however, they were administered the vehicle [0.5% aqueous carboxymethyl cellulose (CMC) with Tween 80
(1 ml/l)] and cyclophosphamide (monohydrate), respectively. The sampling of the femur bone marrow cells was done 23 to 24 hours after the second treatment.
Duration of treatment / exposure:
2 days
Frequency of treatment:
the test item was administered twice at 24 hour interval
Post exposure period:
23-24 hour after the second treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (monohydrate)

Examinations

Tissues and cell types examined:
femur bone marrow cells
Details of tissue and slide preparation:
Terminal Sacrifice
All mice sacrificed at term were subjected to gross pathological observations. The mice were killed by cervical dislocation and the femora from both sides were removed after clearing the musculature. Femur heads were trimmed to expose the marrow canals, the bone marrow was flushed with 0.9% sodium chloride and collected in a centrifuge tube.

Bone Marrow Smear Preparation
The cell suspensions were centrifuged at 2500 rpm for 10 minutes and supernatants discarded. Approximately 10 μl of the cell suspension was spread evenly on a glass slide and air dried. The slide was etched with the study number, mouse accession number, slide number and sex immediately thereafter. The smears were fixed in methanol for 30 minutes. Four to five slides were prepared for each animal.

Staining
Slides were stained by a combination of May-Gruenwald and Giemsa stain in succession. The slides were blow dried, immersed in xylene and cover slips mounted with DPX. The slides were then coded after which blind evaluation was carried out.

Microscopic Evaluation of Erythrocytes
A minimum of 2000 polychromatic erythrocytes (PCE) was scored from each animal for the incidence of PCE with micronuclei. The proportion of immature erythrocytes among total RBC (number of PCE divided by number of total RBC) was determined for each animal by counting 386 to 821 erythrocytes per animal. From these observations, the following were derived for each animal:
• Total RBC/erythrocytes scored
• No. of PCE differentiated
• No. and percentage of PCE with micronuclei
• Mean and SD of PCE with micronuclei
• Ratio of PCE : total RBC

Clinical Signs and Mortality
Twice a day (except on weekends, holidays and on the days of sacrifice when mice were observed once).

Body Weight
The body weights were recorded on day 1 (initial), day 2 and day 3 (sacrifice).
Evaluation criteria:
A statistically significant increase in the number of micronucleated polychromatic erythrocytes indicates a positive result and a test item, which does not meet the above criterion, is considered non-mutagenic.
Statistics:
Intragroup comparison: The body weight on day 2 and at sacrifice was compared with the initial body weight and the body weight at sacrifice was also compared with the body weight on day 2 by the paired T-test.
Intergroup comparison: The net body weight change was analysed by Bartlett’s test for homogeneity of intragroup variances followed by ANOVA and Dunnett's test.
The data of the treatment group and the positive control was compared with the vehicle control by Bartlett's test followed by ANOVA and Dunnett's test for the percentage of PCE with micronuclei and the proportion of PCE among total RBC.
The statistical analyses was carried out using in-house developed and validated software.
All analyses and comparisons are evaluated at 5% (P<0.05) level.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The stability of the test item in gavage sample was tested for ‘0’ and ‘3’ hours for 200 mg/ml concentration. The test item was found to be stable after 3 hours at the dose level of 200 mg/ml when admixed with the test medium and stored at ambient condition. The homogeneity of the test item was carried out at 200 mg/ml concentration and the analysed test item concentration conformed to the nominal concentration in all the layers (top, middle and bottom) within the acceptable limits of ± 10%.
The analysed test item concentration conformed to the nominal concentration (200 mg/ml) with in the acceptable limits of ± 10% level.

There were no clinical signs, mortality and necropsy findings in the vehicle control, positive control and the treatment (limit dose) group except for an incidence of liver and kidney congestion in a male mouse in the positive control group.

The intra group comparison of the body weights in the treatment group showed a statistically significant increase in the sacrifice body weights
1. of males and combined sex as compared to their initial body weights.
2. as compared to the body weights at 24 hour in combined sex
The inter group comparison of net body weight change in the treatment group showed a statistically significant increase in males and combined sex
as compared to the vehicle control group. These results clearly indicate that the body weights of mice were unaffected by the treatment.
In the positive control group, the intragroup comparison of body weights showed a statistically significant decrease in
• sacrifice body weights as compared to the initial body weights in males,
• body weight at 24 hours as compared to initial body weight in males, females and combined sex.
The net body weight change in the positive control group did not differ from the vehicle control group.

MICRONUCLEUS ASSAY IN ERYTHROCYTES
Vehicle Control Group: The percentage of micronucleated PCE and the PCE : total RBC ratio in the concurrent vehicle control group in males, females and combined sex was within the historical data range.
Treatment Group: The percentage of micronucleated PCE in males was comparable to the vehicle control group. There was no incidence of micronucleated PCE in females in the limit dose group. Although there was a statistically significant reduction in the PCE : total RBC ratio in the limit dose group in females and combined sex as compared to the vehicle control group, the decrease was marginal and the values were within the historical data range and thus was not of biological significance.
Positive Control Group: Cyclophosphamide (monohydrate) significantly increased the percentage of micronucleated PCE and significantly altered (reduced) the PCE : total RBC ratio in males, females and combined sex. In the positive control group, the percentage of micronucleated PCE and the
PCE : total RBC was within the historical data range in males, females and combined sex. This result indicated the effectiveness of the methodology adopted and that the test system used was sensitive to a known mutagen.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Sulfur was not clastogenic in the micronucleus test in Swiss albino mice at the tested limit dose of 2000 mg/kg bw.
Executive summary:

Sulfur was tested for its potential to induce cytogenetic damage in bone marrow cells of Swiss albino mice by the micronucleus test (OECD guideline 474 and GLP compliant). The test item was administered twice with at 24 hour interval by oral gavage at the limit dose of 2000 mg/kg bw at the dosage volume of 10 ml/kg. A concurrent vehicle control group (0.5% aqueous carboxymethyl cellulose (CMC) with Tween 80 (1 ml/L)) and a concurrent positive control group (cyclophosphamide) were also included. The mice were sacrificed 23 to 24 hour after the second treatment. From each animal, a minimum of 2000 polychromatic erythrocytes (PCE) were scored for the incidence of micronucleated PCE. Sulfur, at the tested limit dose of 2000 mg/kg bw, did not affect the body weights of mice and there were no clinical signs, mortality and necropsy findings except for an incidence of liver and kidney congestion in a male mouse in the positive control group. The percentage of micronucleated PCE in the treatment group was comparable to the concurrent vehicle control group. Cyclophosphamide significantly increased the percentage of micronucleated PCE and significantly altered (reduced) the PCE:total RBC ratio in males, females and combined sex indicating the effectiveness of the methodology adopted and the sensitivity of the test system.

The test item sulfur was not clastogenic in this micronucleus test in Swiss albino mice at the tested limit dose of 2000 mg/kg bw.