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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard methods and GLP and is considered adequate, reliable and relevant for classification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, The Netherlands
- Age at treatment: 9-11 weeks
- Weight at study initiation: 20.6-23.2 g
- Housing: Individually in solid floor standard polysulfone cages (Size: approximately L 360 x B 205 x H 140 mm), with stainless steel top grill ; steam sterilized corn cob was used as bedding and changed along with the cage twice a week. Cages were placed on 5 tier racks.
- Diet (e.g. ad libitum): Ad libitum standard pelleted food ( Ssniff mice pellet food-maintenance ,manufactured by Ssniff Spezialdiäten GmbH., Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany)
- Water (e.g. 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) ad libitum in polycarbonate bottle with stainless steel sipper tubes
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20 to 22°C
- Humidity (%): 56-66%
- Air changes (per hr): 13.2
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: February 6, 2012 To: February 27, 2012

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Remarks:
DMSO
Concentration:
Irritation Screening Test: 5, 10, 15, 30 and 50% w/v Tin disulfide (CAS 1315-01-1) in dimethyl sulfoxide (DMSO)
Main LLNA Study: 5, 10, 15, 30 and 50% w/v Tin disulfide (CAS 1315-01-1) in dimethyl sulfoxide (DMSO)
No. of animals per dose:
Irritation Screening Test: 1
Main LLNA Study: 5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Based on the miscibility/solubility results, rheology of the suspensions and our previous experience, dimethyl sulfoxide was selected as the vehicle. Tin disulfide was not miscible/soluble at 90 to 60% w/v dilutions. Homogeneous suspension at ≤ 50% w/v dilutions.
-Both ears of six female mice (one mouse/concentration) were topically treated once daily for three consecutive days with one of the concentrations of the test item. The test item was administered using an adjustable micropipette with disposable tips. All mice received 25 µL (12 and 13 µL to each ear) of one concentratien of the test item, spread over the dorsal surface of each ear in a manner to prevent test item loss. Similarly the vehicle was applied to the ears of one anima!. Both ears were observed prior to application of the test item, and erythema was evaluated on Days 2, 3 and 6. All mice were weighed on Days l and 6. Ear thickness was measured using Digimatic micrometer (Mitutoyo, Japan) on Day I (pre-dose), Days 3 and 6. Additionally, on Day 6, ear thickness was determined by ear punch weight determination, after animals were euthanized. Erythema scores, ·ear thickness and body weight data following the test item applications were compared to the response of the animals treated with vehicle alone. Irritation to mouse ears is only relevant in the context of the LLNA and should not be interpreted as an indicator for an irritation potential in humans.
- Irritation: There were no clinical signs, erythema, no significant increase in the ear thickness and ear punch weights and no effect on body weight.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Animals were randomly distributed to different groups by body weight stratification method. The mice procured for the study were weighed and grouped into body weight ranges. These body weight stratified animals were distributed to all the study groups. Mice with extreme body weights were removed from the study.
- Criteria used to consider a positive response: The test item Tin disulfide (CAS 1315-01-1) produces a Stimulation Index (SI) < 3, it is considered “Negative” for sensitization, and therefore an EC3 was not determined.

TREATMENT PREPARATION AND ADMINISTRATION:
- The application of the test item (25 µL/ear) was made on the dorsurn of both ears as described above. Five female mice/group received the vehicle (DMSO, or the positive control substance (30% a-hexylcinnamaldehyde), or 5, 10, 15, 30 and 50% w/v of Tin disulfide (CAS 1315-01-1), once daily for three consecutive days. Ears were inspected prior to each application of the test item solution, and erythema was evaluated on days 2, 3 and 6. All mice were weighed on days 1 and 6. On day 6, a volume of 250 µL (20 µCi) of 3H-TdR in PBS was administered to each mouse via the lateral tail vein using 1.0 mL disposable syringe fitted with 26 G x 1/2 inch needle.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
On day 6, uptake of 3H-methyl thymidine into the auricular lymph nodes draining the site of test item application was measured five hours post administration. Proper conduct of the LLNA was confirmed via a positive response using 30% alfa-hexylcinnamaldehyde, a contact sensitizer. A mean dpm (desintegrations per minute) value ± SD (standard deviation) was calculated for each group and the stimulation index (SI) was calculated using the absolute dpm value for each mouse as the numerator, and the mean dpm value from the vehicle-treated mice as the denominator.

1.The % increase in ear thickness was calculated for each ear using the following equation:
% Ear swelling = (B – A) x 100/A
Where, A = ear thickness measurement on Day 1(µm)
B = ear thickness measurement on Day 3 or 6 (µm)

2.The SI was calculated for each mouse using the following equation:
SI = Disintegrations per minute (dpm) of individual mouse / Average dpm of the vehicle control mice

Means and SD were generated for body weight data (absolute and gain) and LLNA response (dpm and SI values). The body weight and dpm data were analysed by one-way analysis of variance. When the differences were indicated by the ANOVA, a comparison of treated vs. control groups was done using a Dunnett’s t-test (P<0.05).

Statistically significant differences (P<0.05), indicated by the aforementioned tests, are designated by the superscripts throughout the report as stated below:
+/- : Significantly higher (+)/ lower (-) than the vehicle control group

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.42
Test group / Remarks:
5% w/v Tin disulfide (CAS 1315-01-1)
Parameter:
SI
Value:
0.41
Test group / Remarks:
10% w/v Tin disulfide (CAS 1315-01-1)
Parameter:
SI
Value:
1.98
Test group / Remarks:
15% w/v Tin disulfide (CAS 1315-01-1)
Parameter:
SI
Value:
2.35
Test group / Remarks:
30% w/v Tin disulfide (CAS 1315-01-1)
Parameter:
SI
Value:
2.87
Test group / Remarks:
50% w/v Tin disulfide (CAS 1315-01-1)

Any other information on results incl. tables

Table 2. Summary of Disintegrations Per Minute (DPM) for3H-Methyl Thymidine Incorporation in Auricular Lymph Nodes and Stimulation Index (SI)

Values: Mean ± SD

 

Refer Appendix 4

Group and

Dose concentration

No. of mice

DPM/ Mouse

 

SI

 

G1

 

1278.20

1.00

Vehicle : DMSO

5

287.33

0.23

 

 

+

 

G2

 

7792.60

6.10

30% v/v HCA in DMSO

5

2284.66

1.79

 

G3

5% w/v Tin disulfide (CAS 1315-01-1)in DMSO

 

5

-

530.60

0.42

157.50

0.12

 

G4

10% w/v Tin disulfide  (CAS 1315-01-1)in DMSO

 

5

-

527.40

0.41

137.28

0.11

 

 

G5

15% w/v Tin disulfide (CAS 1315-01-1)in DMSO

 

5

+

2529.80

1.98

1135.32

0.89

 

 

 

G6

30% w/v Tin disulfide (CAS 1315-01-1)in DMSO

 

5

+

 

3008.40

2.36

656.62

0.51

 

 

G7

50% w/v Tin disulfide  (CAS 1315-01-1)in DMSO

 

5

+

3668.20

2.87

724.27

0.56

 

 

+ /- : Significantly higher / lower than the vehicle control group

DMSO: Dimethyl sulfoxide 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
A topical application with 5, 10, 15, 30 and 50% w/v Tin disulfide (CAS 1315-01-1) elicited a stimulation index (SI) of 0.42, 0.41, 1.98, 2.35 and 2.87, respectively. The test item Tin disulfide (CAS 1315-01-1) did not demonstrate dermal sensitization potential in the mouse LLNA, as the lymph nodes draining the area of topical application did not elicit a proliferative response greater than the 3X threshold.
Executive summary:

This Local Lymph Node Assay (LLNA) was conducted to evaluate the potential of Tin disulfideto cause contact sensitization by measuring lymphocyte proliferative response from auricular lymph nodes following topical application of the test item to the female CBA/Ca mouse ear. 

Screening Study: Daily topical application for three days of 5, 10, 15, 30 and 50% w/v of Tin disulfide in dimethyl sulfoxide (DMSO) were given to one animal at each dose level. There were no clinical signs, no erythema, no increase in the ear thickness and ear punch weights and no effect on body weight. Results from this study were used to determine the dosing concentration for the main study.
LLNA main study: Five female CBA/Ca mice/group received the vehicle (DMSO) or 30% α-hexylcinnamaldehyde (HCA: positive control in DMSO) or 5, 10, 15, 30 and 50% w
/v of Tin disulfide in DMSO on days 1 to 3. On day 6, a volume of 250 µL (20 µCi) of 3H-TdR in PBS was administered to each mouse via the lateral tail vein, and uptake into the auricular lymph nodes draining the site of test item application was measured five hours post administration.
Proper conduct of the LLNA was confirmed via a positive response using 30% α-hexylcinnamaldehyde, a contact sensitizer, which elicited proliferation with a Stimulation Index (SI) value of 6.10, in comparison to vehicle-treated mice.The test itemTin disulfide (CAS 1315-01-1) did not elicit erythema, no clinical signs and no effect on body weight gain at the tested concentrations. The test itemTin disulfideat dose concentrations of 5, 10, 15, 30 and 50% w/v elicited proliferative response with SI of 0.42, 0.41, 1.98, 2.35 and 2.87, respectively in comparison with the vehicle-treated mice. Tin disulfide did not demonstrate dermal sensitization potential in the mouse LLNA, as the lymph nodes draining the area of topical application did not elicit a proliferative response greater than the 3X threshold.