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Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from October 24, 2012 to November 06, 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

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

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
IUPACname: N ,N' -( methylenedi -p-phenylene )bis[hexahydro-2-oxo-1 H-azepine-1-carboxamide]
ECnumber: 258-981-8
Molecu/ar formula: C27H32N404
Molecu/ar weight: 476.57
Purity: 98.97%(w/w)
Appearance: White solid powder
BatchNo.: 9114761/024
Stability/Expiration: 11.7.2013
Storage during the study: The substance was stored at laboratory conditions.

In vivo test system

Test animals

Details on test animals and environmental conditions:
Experimental animals
Animals species and strain: Y oung adult female mice (nulliparous and non-preguant), strain BALB/CBYJICO (referred to as BALB/c in this document)
Source: Breeding farm VELAZ s.r.o., Kolec u Kladna, Czech Republic, RCHCZ21760118
Number of animals per group:
Pilot experiment- 3 females
Exposed groups - 15 females ( 5 animals in three groups)
Positive control group - 5 females
Negative control group -5 females
Reserve group - 2 females
(Reserve animals were intended for possible replacement of the animals in other groups during the acclimatisation period)
Total: 30 animals
Age: 8 to 10 weeks (at start of dosing)
Body weight range: 16.50- 19.66 g (at start of dosing)
Health examination: All animals were examined during the acclimatisation period
Acclimatisation: At least 5 days
Animal quarterslhusbandry
Animal rooms: Monitared conditions, microbiologically defined background, according to SOP No.40
Microclimatic conditions: Room temperature: 20.4 - 21.6 °C permanent1y monitared
Relative humidity: 44.9 - 55.0% permanently monitared
Light: 12 hours Iight/dark cycle: 6am-6pm/6pm-6am
Animal caging: Animals in groups: maximum six in macrolon cages (35x20x15 cm) with sterilized softwood shavings
Water: Drinking tap water ad libitum. Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry
Diet: Pelleted standard diet for experimental animals ad libitum (ST 1 BERGMAN, manufacturer: Ing. Miroslav Mrkvicka - V)rroba krmnych smesi, Mlyn Kocanda No. 19, 252 42 Jesenice u Prahy). Microbiological control and content of nutrients is performed according internal SOP No. 72.
Prophylactic arrangement: Cleaning and disinfection of animal room was regularly performed, as it is described in internal SOP No.10.
Animal identification
Cage identification: By cage number, study number and group specific colour.
Animal identification: By feit tip marking (from 1 to 5 per each group and group specific colour)
Random selection: According to internal SOP No.42

Study design: in vivo (LLNA)

other: DAE 433- mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
in vehicle (DAE 433): 50%, 5%, 0.5% (w/v).
No. of animals per dose:
30 females
Details on study design:
pilot experiment:
The test substance was administered to three animals to assess andlor discard a possible systernic toxicity or high irritation of skin. In pilot experiment one animal was used o per dose group.
The pilot experimentwas conducted under the conditions identical to the main study, except there was no assessment of 1ymph node proliferation. The appropriate dilution of the test substance (50%, 5%, 0.5% w/v) was applied to three animals in volume 25 fll to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slow1y by micropipette. The route of administration was the same as in the main study. Both ears of each mouse was observed for erythema and scored and subsequently thickness was measured using digital thickness gauge. Body weights were recorded before application and prior to termination (Day 6). According to the results of pilot experiment these doses were confirmed for the main experiment:
The test substance was administered in the form of suspension in DAE 433.
Concentration in formu1ation: 50% (w/v) ....................... 500 mg/mL
5% (w/v) ........................... 50 mg/mL
0.5% (w/v) .......................... 5 mg/mL
Main experiment
Concentration of test substance in DAE433 % (w/v)
Group 1Vehicle 0
Group 2Positive control substance 0.5
Group 3 Test substance 50
Group4 Test substance 5
Group 5 Test substance 0.5
Group 6 Reserve animals -
5 animals per dose
The volume of the application form was constant at all groups of animals - 25 fll of the appropriate dilution to the dorsum of each ear once a day moming for 3 consecutive days. The application was performed very slowly by micropipette. Losses caused by draining from the ear must be minimized. This route of administration is listed in the guideline and it is similar to expected exposure conditions at the workplace. The application form oftest substance was prepared immediately before administration.
Experimental schednle
Day 1:
Open application of 25 µl (in the morning, by pipette) of appropriate suspensions of the test substance, the vehicle alone or the positive control to the dorsum of each ear.
Days 2 and3:
The application procedure repeated as carried out on day 1.
Days4 and 5:
No treatrnent.
Day 6:
The weight of animals was recorded. Injection 250 µl of phosphate-buffered saline (PBS) containing 7.86 x105 Bq of 3H-methyl thymidine into all test and control micevia the tail vein. Five hours later, the animals were killed.
Criteria for the evaluation of results
Test validity criteria
The test is considered valid, if the positive control substance DNCB will produce positive LLNA response and if the stimulation index SI is :>- 3 over the negative control group.
Test results criteria
Cell proliferation
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 ifthe Stimulationindex 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 will be considered as irritation induced by the test substance.
Positive result in cell proliferation reveals that the test substance could be a contact allergen. When positive irritation effect in animals is demonstrated simultaneously, the possibility can not be ruled out, that the evaluation based on cell proliferation could be a false positive.
Positive control substance(s):
other: Dinitrochlorobenzene (DNCB), CASNumber: 97-00-7
For statistical calculations the software Statgraphic ® Centurian (version XV, USA) was used. Statistical evaluation of measured parameters was performed at first by applying the non-parametric Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group, as global test, and then the non-parametric two-group Mann-Whitney rank test (probability Ievel 0.05) for all two-group comparisons.

Results and discussion

Positive control results:
The positive control substance DNCB produced positive LLNA response at an exposure Ievel expected to give an increase in the Stimulation Index SI :,. 3 over the negative control group, which was in congruence with the expected mode of action of a contact allergen. The positive control also elicited a reaction pattern with statistically significant increase in ear weight.

In vivo (LLNA)

Resultsopen allclose all
Test group / Remarks:
0.5% test substance
Test group / Remarks:
5% test substance
Test group / Remarks:
50% test substance
Test group / Remarks:
positive control (DNCB, dinitrochlorobenzene)
Test group / Remarks:
negative control (vehicle, 40% dimethylacetamide, 30% acetone, 30% ethanol)
other: disintegrations per minutes (DPM), mean
SD = 13.39
Test group / Remarks:
0.5% test substance
other: disintegrations per minutes (DPM), mean
SD = 36.35
Test group / Remarks:
5% test substance
other: disintegrations per minutes (DPM), mean
SD = 54.80
Test group / Remarks:
50% test substance

Any other information on results incl. tables

Results of pilot experiment

Body weight: Individual body weight of all females before administration and before necropsy was relatively well balanced. During the pilot experiment no significant reduction of body weight was recorded.

Mortality, clinical observations and results of macroscopic necropsy: During the pilot experiment no test substance related effects were found in all animals. No clinical symptoms of systemic toxicity were found in all animals. The thickness of ears of all animals were similar during whole pilot experiment. During the pathological examination no pathological changes were found.

Results of main experiment

Body weight of animals: Individual body weight of females before administration and before necropsy was relatively well balanced (result of random selection of animals into groups). Body weight increment was calculated from values of day 6 before necropsy and day 1 before first application.

Mortality and clinical Observations: No animal died during the main experiment. No symptoms of toxicity and no erythema on application site were observed in animals from the negative control group and from groups administered by the test substance. In groups of animals administered with test substance, a statistically significant weight increase of ear targets was not found out.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Based on the results given in this LLNA study, the test article caused no sensitisation to mice's skin.
Executive summary:

This study was conducted to assess the sensitization potential of test substances at dose levels of 50%, 5%, and 0.5% in vehicle (DAE 433) with 30 female mice.The test substance did not show a tendency to increase ear weight - it means the test substance did not cause irritation of skin. The test substance showed a dose dependent increase in radionuclide incorporation but the Stimulation Indexes of all treated groups were < 3. Statistically significantly increased radionuclide incorporation was recorded at concentration of 50% and 5%. The highest concentration was the technically practicable maximum concentration. The increase in the cell proliferation cannot be attributed to irritation effects because no increase in the ear weight was seen in concurrence.

Under the experimental conditions, the test substance dosed up to the maximum concentration of 50% did not Iead to irritation and provided a stimulation index below the classification threshold in LLNA study.