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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February - March 2014
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
according to guideline
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Details on test animals and environmental conditions:
On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatization period of nine days the animals were selected at random and a cage card was prepared with details of test item, project number, dose level, sex, study dates and the initials of the Study Director. At the start of the study, the animals were in the weight range of 15 to 23 g, and were approximately eight to twelve weeks old.

The temperature and relative humidity remained within 19 to 25 °C and 30 to 70%, respectively, during the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Based on results from the preliminary study the following doses were selected for the main study: 0, 10, 25, and 50%, w/w in dimethyl formamide.
No. of animals per dose:
Details on study design:
Following a preliminary screening test in which no clinical signs of systemic toxicity or excessive local skin irritation nor irritation indicated by an equal or greater than 25% increase in mean ear thickness were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test.

Groups of five mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in dimethyl formamide. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of five mice received the vehicle alone in the same manner.

Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd), giving a total of 20 µCi to each mouse.

Clinical Observations: All animals were observed twice daily (pre- and post-dose) on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any clinical signs of toxicity, skin irritation, or signs of ill health during the test were recorded.

Body Weights:
The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.

Preparation of Single Cell Suspension:
A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was re suspended in 10 mL of PBS and re pelleted. The pellet was re suspended in 3 mL of 5% Trichloroacetic acid (TCA) and incubated for approximately 18 hours at approximately 4 °C to precipitate DNA.

Determination of 3HTdR Incorporation: The TCA precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, followed by resuspension in 1 mL of TCA. Each resuspended pellet of each individual animal was transferred to a ‘’Poly QTM’’ vial containing 10 mL of scintillation fluid (Optiphase 'Trisafe'). The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously in the dark. 3HTdR incorporation was measured as the number of radioactive disintegrations per minute by beta scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Data was processed to give group mean values for disintegrations per minute (dpm) per animal (dpm/animal) and standard deviations were appropriate. Individual and group mean dpm values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). A significant effect was identified by the ANOVA; therefore, pairwise comparisons were performed between control and treated groups. Dunnett's Multiple Comparison test was also used due to the significant results achieved from ANOVA.

Results and discussion

Positive control results:
Postiive control study (October 18 - 24, 2013), with a Hexylcinnamaldehyde (85%) yielded a positive result and confirmed to be a sensitizer under the conditions of the positive control test when compared to the control DMF.

In vivo (LLNA)

Resultsopen allclose all
Test group / Remarks:
Group 10%
Test group / Remarks:
Group 25%
Test group / Remarks:
Group 50%
Remarks on result:
not determinable
because no dose response observed
Cellular proliferation data / Observations:
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Pale yellow colored residual test item on the ears was noted on all the test mice: Day 1 (post dose) and Days 2 to 4 at a concentration of 50% w/w in dimethyl formamide; Days 1, 2 and 3 (post dose) at a concentration of 25% w/w in dimethyl formamide; and Days 2 and 3 (post dose) at a concentration of 10% w/w in dimethyl formamide.

Any other information on results incl. tables

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Pale yellow colored residual test item on the ears was noted on all the test mice: Day 1 (post dose) and Days 2 to 4 at a concentration of 50% w/w in dimethyl formamide; Days 1, 2 and 3 (post dose) at a concentration of 25% w/w in dimethyl formamide; and Days 2 and 3 (post dose) at a concentration of 10% w/w in dimethyl formamide.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione is considered to be a sensitizer under the conditions of the test.
Executive summary:

In a LLNA (OECD 429), conducted according to GLP, 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione was tested at 10%, 25% and 50% concentration in dimethyl formamide, and resulted in a positive response in mice, with a stimulation index of 7.71, 10.03 and 6.11, respectively. 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione is therefore considered a skin sensitiser. No EC3 could be determined in the absence of dose-response.