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Administrative data

Description of key information

In an LLNA assay, no sensitization was found. The results of the required 3 in vitro studies were equivocal, mandating the need for the LLNA

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Animal testing in the form of an LLNA was undertaked after the trio (DPRA, KeratinoSens, hClat) were undertaken and inconclusive results were obtained such that the in vivo study was required for classification and labelling.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:

Identification: 1,1'-[ethane-1,2-diylbis(thio)]bisbenzene

CAS Number: 622-20-8

Batch: 7061415

Purity: 100%

Physical state I Appearance: white solid

Expiry Date: 13 February 2019

Storage Conditions: room temperature in the dark
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

The animals were housed in suspended solid floor polypropylene cages furnished with softwood wood flakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to
70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 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.
Vehicle:
other: Butanone
Concentration:
20%, 10% or 5%
No. of animals per dose:
4
Details on study design:

Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in butanone. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 f.lL 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 four 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 0.25 mL (250 f.lL) of phosphate buffered saline (PBS) containing 3H-methyl thymidine eHTdR: 80 f.lCi/mL, specific activity 2.0 CilmmoL, ARC UK Ltd) giving a total of 20 f.lCi to each mouse.

Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity 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).

Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells 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 cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid ( TCA).
Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 21 00 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL ofTCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by P-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness 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. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer".
Positive control results:
Positive controls met the proper criteria for a valid study.
Parameter:
SI
Value:
1.33
Test group / Remarks:
25 mg/kg
Parameter:
SI
Value:
1.92
Test group / Remarks:
10 mg/kg
Parameter:
SI
Value:
1.74
Test group / Remarks:
5 mg/kg

Concentration (% w/w) in butanone Stimulation Index  Result
5 1.74 Negative
10 1.92 Negative
25 1.33 Negative
Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitizer under the conditions of the test.
Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary  screening test in which no clinical signs of toxicity were noted at a maximum attainable concentration  of 25% w/w, this concentration  was selected as the highest dose

investigated  in the main test of the Local Lymph Node Assay.  Three groups, each of four animals, were treated with 50 J.LL (25 J.LL per ear) of the test item as a solution in butanone at

concentrations  of 25%, 10% or 5% w/w.  A further group of four animals was treated with butanone alone.

Results

The Stimulation  Index expressed as the mean radioactive incorporation  for each treatment group divided by the mean radioactive incorporation  of the vehicle control group are as

follows:

5 mg/kg, Si = 1.74

10 mg/kg, Si = 1.92

25 mg/kg, Si = 1.33

Conclusion

The test item was considered to be a non-sensitizer under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification

Assessment of the Skin Sensitisation Potential of 1,1’-(ethane-1,2-diylbis(thio)bisbenzene was conducted in two key studies; according to OECD Test Guideline 442D (the KeratinoSens TM test method) and OECD TG 442C Direct Peptide Reactivity Assay (DPRA). After 48h exposure of cells with 12 concentrations (0.195- 400 ug/ml) of 1,1’-(ethane-1,2-diylbis(thio) bisbenzene , Luciferase measurements and MTT viability testing gave positive results in KeratinoSens method and 1,1’-(ethane-1,2-diylbis(thio)bisbenzene was classified as sensitizer. However, 1,1’-(ethane-1,2-diylbis(thio)bisbenzene gave overall result of 0% depletion mean values which places 1,1’-(ethane-1,2-diylbis(thio)bisbenzene in the reactivity class of “low” and therefore DPRA study predicted 1,1’-(ethane-1,2-diylbis(thio)bisbenzene as a non-skin sensitizer.

A subsequent LLNA study was run that gave no evidence of sensitization, and thus no classification is required