Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27.1.-9.2.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3,4-Ethylenedioxythiophene
- Comercial name. EDOT
- Substance type: pure organic substance
- Physical state: liquid near colourless to pale yellow
- Analytical purity: > 99%
- Lot/batch No.: 091201
- Expiration date of the lot/batch: 21.1.2011
- Storage condition of test material: Keep in dark at room temperature. Stored in amber glass bottles under the argon atmosphere. Minimalize time for handling with substance without protective argon atmosphere as possible. Maximal time without protective atmosphere is 30 min.
- pH: approximately 4 (by contact of application form with universal indicator pH strip moistened with water, strip producer Lach-Ner, s.r.o. (Neratovice)

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118
- Age at study initiation: 8 to 10 weeks (at start of dosing)
- Weight at study initiation: 19.4 to 22.2 g (at start of dosing)
- Housing: group-wise five in macrolon cages with sterilized softwood shavings.
- Diet (e.g. ad libitum): Pelleted standard diet for experimental animals ad libitum. Microbiological control and content of nutrients was performed according SOP No. 75
- Water (e.g. ad libitum): Drinking tap water ad libitum. Water quality corresponded to Ministerial Decree No. 252/2004 Czech Coll. of Law
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3oC, permanently monitored
- Humidity (%): 30 – 70 %, permanently monitored
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle: 6am-6pm/6pm-6am


STUDY TIME SCHEDULE
Animal arrival/ start of acclimatization: 21. 01.2010
Pilot experiment: 27.- 30. 01. 2010
Main study:
First day of administration: 03.02.2010
End of treatment period: 05.02.2010
Application of radionuclide and necropsy: 08.02.2010

Study design: in vivo (LLNA)

Vehicle:
other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
Concentration:
30% (v/v) 300 µL/mL
3% (v/v) 30 µL/mL
0.3% (v/v) 3 µL/mL
No. of animals per dose:
5 animals
Details on study design:
RANGE FINDING TESTS:
- The highest test-substance concentration of 30% was administered to three animals to assess possible systemic toxicity. During the pilot experiment, clinical symptoms of systemic toxicity were observed (decreased sensory reactivity on acoustic stimuli, increased sensory reactivity on acoustic stimuli). No macroscopic changes (after necropsy) were found in all three animals.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Animals were subjected to a clinical examination (health check) shortly after arrival. Study animals were randomly allocated to the dose groups manually and assigned animal numbers.

EVALUATION OF RESULTS
Stimulation index
The SI is obtained by dividing the pooled radioactive incorporation for each treatment group by the incorporation of the pooled vehicle control group; this yields a mean SI. The response towards the test substance is considered positive, if the stimulation index (SI) is ≥ 3, and the response increases in dose-related manner (dose-response relationship). The response is considered negative, if the stimulation index (SI) is < 3 without the dose–response relationship. The response is considered ambiguous if the stimulation index is < 3, but the response increases in dose-related manner (dose–response relationship), and eventually statistical significance is observed.

Ear weight – irritation effect
If after treatment with the test substance a statistically significant increase of ear weight together with clear concentration dependence of the effect is recorded, the inflammatory effect is considered as irritation induced by the test substance.


TREATMENT PREPARATION AND ADMINISTRATION:
Dosage volume: 25 μL / ear / animal
Preparation for administration: All emulsions were prepared by suspending an appropriate amount of 3,4-Ethylenedioxythiophene in the vehicle to obtain a concentration of 30%, 3% or 0.3% (v/v). The emulsions were prepared before the start of application by mixing on magnetic stirrer and were still being mixed during application.
Application: The volume of the dose was constant for all groups of animals - 25 L of the appropriate dilution to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipette to avoid losses caused by draining from the ear.
Positive control substance(s):
other: DNCB (dinitrochlorbenzene)
Statistics:
For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. At first the global comparison of all three values of the concentration groups with vehicle control is performed by applying the non-parametric Kruskal-Wallis test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) was applied to all two-group comparisons.

Results and discussion

Positive control results:
The positive control substance DNCB produced a positive LLNA response at an exposure level expected to give an increase in the Stimulation Index SI of ≥ 3 over the negative control group, which was consistent with the expected mode of action of a contact allergen. The positive control also elicited a reaction pattern with a statistically significant increase in ear weight. These results demonstrate that the method performed under the conditions of our laboratory had sufficient sensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The SI for the test groups treated with the test substance was decreased in dose-related manner. At the highest dose level, the SI was 1.60, at the middle dose level the SI was 1.66, and at the lowest dose level, the mean SI was 2.87. The stimulation indexes of all test groups were below the threshold.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See table No.6

Any other information on results incl. tables

Table 6.  Summary table

Group

Radioisotope incorporation
in lymph nodes

Ear weight

Mean DPM

SI

Mean (mg)

NC

237.26

1.00

22.26

PC

4018.42

16.94+

33.02

30%

378.59

1.60

21.54

3%

394.58

1.66

22.66

0.3%

680.99

2.87

22.04

Notes:

Bold figures with cross = values ≥ 3  

NC – Negative control group

PC – Positive control group

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the given test conditions, the test substance, 3,4-Ethylenedioxythiophene, elicited negative response in LLNA test.
Executive summary:

The test substance, 3,4-Ethylenedioxythiophene, was tested for the assessment of skin sensitisation potential with the murine local lymph node assay. This study is a part of the test substance health hazard evaluation.

The Local Lymph Node Assay (LLNA) with radionuclides was used. The testing was conducted according to the EU Method B.42, Skin sensitization: Local Lymph Node Assay, Council Regulation (EC) No. 440/2008, published in O.J. L142, 2008.

 In this study the contact allergenic potential of 3,4-Ethylenedioxythiophene was evaluated after topical application to female BALB/c mice. Five mice per group were exposed by test and control substances on the dorsum of both ears once a day during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated by using radioactive labelling. The ratio of the proliferation in treated groups to that in vehicle controls, termed the Stimulation Index (SI), was determined. Statistical evaluation of ear weight was performed for elimination of potentially false positive findings with certain skin irritants.

Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and 3,4-Ethylenedioxythiophene: 30%, 3%, 0.3% (v/v) in the solvent mixture, DAE 433.

The animals exposed to the test substance at all dose levels showed no pathological skin reactions. Symptoms of systemic toxicity were observed at the highest dose level throughout the experiment (decreased response on stimuli, apathy, piloerection, reduction of body weights by 7.51% from Day 1 to Day 6,) and at the middle dose level (piloerection, decreased response on stimuli). The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and cell proliferation, the Stimulation Index reaching 16.94, which was consistent with its expected mode of action as a contact allergen.   

The comparison of the Stimulation Indexes between the treated groups and the control group revealed that the test substance 3,4-Ethylenedioxythiophene did not cause a significant increase in radioisotope incorporation into the DNA of proliferating lymphocytes.

In conclusion, at the given experimental conditions the result of LLNA study with test substance 3,4-Ethylenedioxythiophene was negative.