Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-08 to 2017-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc di(benzimidazol-2-yl) disulphide
EC Number:
221-203-2
EC Name:
Zinc di(benzimidazol-2-yl) disulphide
Cas Number:
3030-80-6
Molecular formula:
C7H6N2S.1/2Zn
IUPAC Name:
2-{[(1H-1,3-benzodiazol-2-ylsulfanyl)zincio]sulfanyl}-1H-1,3-benzodiazole
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: OUCHI SHINKO CHEMICAL INDUSTRIAL CO., LTD.; 608011
- Expiration date of the lot/batch: 2018-08-31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep to the storeroom with suitable ventilation. Avoid fire, direct sunlight and moisture. Store at room temperature.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90., Hungary
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 18.1 – 21.6 g
- Housing: groupwise
- Diet: ad libitum, ssniff® Rat/Souris-Elevage E complete diet (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water: ad libitum, tap water
- Acclimation period: 14 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
5, 10, 25 %
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
Two groups of 2 CBA/Ca mice were treated with the appropriate formulations once daily for 3 consecutive days. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site during the preliminary test. Body weights were recorded prior to the first treatment (on Day 1) and prior to termination (on Day 6). Both ears of each mouse were observed for erythema and scored according to criteria depicted in Table 2. Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity were observed. No sign of significant irritation (indicated by an erythema score ≥ 3 and/or an increase of ≥ 25 % of ear thickness observed on any day of measurement) was observed. Loss of hair (located at the top of the head) was observed in the 25 % (w/v) dose group for 1 animal of the 2 as a possible local effect. The symptom was considered not significant since it was observed for 1 animal and on Day 6, only.

MAIN STUDY
INDUCTION
- Frequency of application: once daily for a total of 3 days (days 1, 2 and 3)
- Site of application: The dorsum surface of both ears
- Route of application: epidermal
- Volume applied: 25 µL/ear
- Concentrations: 5 %, 10 % and 25 % in vehicle

Injection of 3H-methyl thymidine (Amersham Pharmacia Biotech, NOTOX Substance 105624)
- Day of injection: 3 days after last treatment (day 6)
- Site of injection: tail vein
- Vehicle: PBS
- Volume injected: 0.25 mL
- Specific radioactivity: 20 µCi/injection

SACRIFICE
- Time schedule: 5 hours after injection of 3H-methyl thymidine
- Method: injection of phenobarbital

TISSUE PROCESSING AND MEASUREMENTS
- Lymph node processed: auricular lymph nodes
- Pooling of lymph nodes: yes
- Measurement of radioactivity: Tri-Carb 3100TR Liquid Scintillation Analyzer

OTHER
The test animals were checked once daily for mortality/viability and at least once a day for toxicity signs. Bodyweight of the test animals was taken on day 1, prior to treatment and on day 6. On day 3 (3 - 4 hours after treatment), the skin reactions were assessed. If possible, skin reactions were graded following the Draize numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The positive control group animals were treated with 25 % HCA solution (formulated in AOO) concurrent to the test item treated groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. The positive control substance induced the appropriate stimulation compared to the relevant control (AOO). The calculated SI value was 21.1.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
< 5
Key result
Parameter:
SI
Value:
9.8
Test group / Remarks:
25 %
Key result
Parameter:
SI
Value:
10.1
Test group / Remarks:
10 %
Key result
Parameter:
SI
Value:
4.8
Test group / Remarks:
5 %
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Visually larger lymph nodes compared to the relevant vehicle controls (AOO or DMSO) were observed in the positive control group and in all test item treated groups. Appearance of the lymph nodes was normal in the negative control groups (AOO or DMSO).
Significant lymphoproliferative response (SI ≥ 3) was observed for the test item at all test concentrations. The observed stimulation index values were 9.8, 10.1 and 4.8 at test item concentrations of 25 %, 10 % and 5 % (w/v), respectively. The response was considered dose-related although no strong linear correlation was observed.

EC3 CALCULATION
EC3 is normally calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve. In this test no SI value below 3 was observed (hence no calculation of the EC3 value was possible), but based on the recent test results an EC3 value of < 5 % (w/v) is considered for the test item.

CLINICAL OBSERVATIONS
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group.

BODY WEIGHTS
No significant, treatment related effect on the body weights was observed in any treatment group. Loss of body weight > 5 % was observed in the DMSO control group only (1 of the 4 animals, 6 % decrease) but the mean body weight did not decrease hence the effect was considered not significant.

Any other information on results incl. tables

Interpretation of observations

A formulation evaluation and a Dose Range Finding test (DRF) were performed to find an appropriate vehicle and the maximum applicable concentration according to the relevant guidelines. Solubility of the test item in vehicles preferred in the LLNA was evaluated using concentration series recommended by the guidelines. Although the test item was soluble in N,N-Dimethylformamide (DMF), in Dimethyl sulfoxide (DMSO) and in Absolute ethanol: water 7:3 (v/v) mixture (EtOH) only partial solubility was observed even at 0.025 % (w/v) concentration.

Based on this the test item was examined in the LLNA as suspension formulation with the aim of testing as high concentrations as possible above the solubility limit. The maximum feasible concentration resulting in an adequately stable and homogeneous formulation (suspension) was 25 % (w/v). Formulations were prepared with DMSO according to the Sponsor’s request. According to results of the DRF, where no adverse effect was observed up to this maximum concentration, the test item was examined in the main test at concentrations of 25 %, 10 % or 5 % (w/v).

Since the test was valid and no confounding effects of irritation or systemic toxicity were observed during the main test, the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay.

Significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMSO) was noted for Zinc di(benzimidazol-2-yl) disulphide at all applied test concentrations. The observed stimulation index values were 9.8, 10.1 and 4.8 at test item concentrations of 25 %, 10 % and 5 % (w/v), respectively. Dose-related response was considered although no strong linear correlation was observed.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Under the conditions of the above assay, Zinc di(benzimidazol-2-yl) disulphide tested at the maximum feasible concentration of 25 % (w/v) and also at concentrations of 10 % or 5% (w/v) as suspension formulations in a suitable vehicle (Dimethyl sulfoxide, DMSO) was shown to have skin sensitization potential in the Local Lymph Node Assay.
Executive summary:

The skin sensitization potential of Zinc di(benzimidazol-2-yl) disulphide was determined following dermal exposure in the Local Lymph Node Assay according to OECD guideline 429. The pooled treatment group approach was used in this test.

The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and a Dose Range Finding test (DRF). According to results of the DRF, where no adverse effect was observed up to this maximum concentration, the test item was examined in the main test at concentrations of 25 %, 10 % or 5 % (w/v).

Appropriate positive control (α-Hexylcinnamaldehyde, HCA), furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed. The positive control item [25 % (w/v) HCA in Acetone:Olive oil 4:1 (v/v) mixture, AOO] induced significant stimulation over the relevant control (SI = 21.1) thus confirming the validity of the assay.

No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group.

Significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMSO) was noted for Zinc di(benzimidazol-2-yl) disulphide at all applied test concentrations. The observed stimulation index values were 9.8, 10.1 and 4.8 at test item concentrations of 25 %, 10 % and 5 % (w/v), respectively. Dose-related response was considered although no strong linear correlation was observed.

According to evaluation criteria of the relevant guidelines, the significantly increased lymphoproliferation (indicated by an SI ≥ 3) at the maximum attainable concentration of 25 % (w/v) as well as at lower concentrations are considered as evidence that Zinc di(benzimidazol-2-yl) disulphide is a skin sensitizer.

Chemicals can be classified according to their relative skin-sensitization potency using the EC3 value (dose calculated to induce a stimulation index of 3) which is normally calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve according to published method [7]. In this test no SI value below 3 was observed (hence no calculation of the EC3 value was possible), but based on the recent test results an EC3 value of < 5 % (w/v) is considered for the test item.

Under the conditions of the present assay, Zinc di(benzimidazol-2-yl) disulphide tested at the maximum feasible concentration of 25 % (w/v) and also at concentrations of 10 % or 5 % (w/v) as suspension formulations in a suitable vehicle (Dimethyl sulfoxide) was shown to have skin sensitization potential in the Local Lymph Node Assay.