Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.02.2011-01.11.2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
EU Method B42
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): XP-7866
- Analytical purity: 99.5%
- Purity test date: May 16, 2011
- Lot/batch No.: #10188-1
- Expiration date of the lot/batch: Feb. 28, 2015

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon , UK
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing:
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 deg. C
- Humidity (%): 30 to 70%
- Photoperiod: 12hrs dark /12 hrs light):

IN-LIFE DATES: From:August 9, 2011 To: August 23, 2011

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
suspension
Concentration:
10, 25 and 50% w/w in DMF
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
One mouse was treated daily with 25 micro-l of the test item at a concentration of 50% w/w in DMF to the dorsal surface of each ear for 3 consecutive days. The mouse was obbserved twice dauly on days 1, 2, and 3 and once daily on days 4, 5, and 6. ocal irritation was scored daily. Any clinical signs of toxicity were also recorded. Body weight was recorded on day 1 pre-dosing and day 6. Ear thickness was measured using an Oditest micrometer pre-dose on day 1, post dose on day 3 and 6. A mesn ear thickness increase of equal or greater than 25% was considered to represent excessive irritation.
- Choice of vehicle DMF was the only vehicle tested that was suitable for dosing and gave a homogenous suspension after vortexing
- Irritation: No skin irritation was observed in one animal after adminstration of a 50% test substance suspension in DMF. Observation period: daily day 1 to day 6.
- Ear thickness: predose: 0.235 mm, day 3: 0.243 mm, day 6 0.260 mm

MAIN STUDY
Groups of 4 randomly assigned female mice were treated with 25 micro-l of the test item at concentrations of 0 (vehicle contorl), 10, 25 or 50% w/w in DMF to the dorsal surface of each ear for 3 consecutive days. 5 days after the first topical application (day 6) all mice were injected via the tail vein 250 micro-l 3H-methyl thymidine (3HTdR: 80 microCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd.) in phosphate buffered saline. (20 micro-Ci per mouse.

Observations:
Clinical observations: twice daily on days 1, 2, and 3, once daily on days 4,5, and 6. Any clincal signs of ill health or toxicity were recorded.
Body weight: recorded for each animal on day 1 pre-dosing and day 6 (prior to termination)
Terminal procedures: 5 h following the 3HTdR administration all mice were killed by carbon dioxide asphyxation. The draining auricular lymph nodes were excised and pooled for each group. For each group 1 ml of PBS (phosphate buffered saline) was added to the pooled lymph nodes. A single cell suspension of the pooled lymph nodes was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze and cetriguged at 1400 rpm (appr. 190xg) for 10 min. After resuspension in 10 ml PBS and re-pelleting the radioactive material was precipitated by resuspension in 3 ml of 5% trichloroacetic acid (TCA)
Determination of 3HTdR incorporation: After appr. 18 h of incubation at 4 deg. C the precipitate was centrifuged at 2100 rpm (appr. 450 xg) for 10 min, resuspended in 1 ml of TCA and transferred to 10 ml of scinitllation fluid (optiphase Trisafe). The 3HTdR incorportaion was measured by ß-scintillation counting. The scintillation vials with the fluid were left in the sample charger of the scintillator for appr. 20 min in order to reduce the influence of luminescence. After appr. 20 min the vials were shaken vigorously and the numbe rof disintegrations per min was measured using a Beckman LS6500 scintillatiion system (Beckman Instruments Inc., ullerton, CA, USA).
- Criteria used to consider a positive response:
The proliferation response of the lymph node cells was expressed as the number of radioactive disintegrations per min per lymph node and as the ratio of 3HTdR incorporation into lymph node cells of test nodules relative to that recorded for control nodes (stimualtion index).
A threefold or greater increase in 3HTdR incorporation compared to control values is regarded a positive response (skin sensitizer).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
no statisitcal evaluation performed

Results and discussion

Positive control results:
15% v/v of alpha-Hexylcinnamaldehyde in DMF resulted in a stimulation index of 4.77 and thus gave the expected positive response.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see table below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see table below.

Any other information on results incl. tables

Disintegration per minute (dpm) and stimulation index for the 4 test group and the vehicle control:

dpm/node dpm/8 as 8 lymph nodes were pooled per group.

 

 Concentration (% w/w in DMF)  dpm  dpm/node  Stimulation index  Result
 Vehicle  7581.72  947.72  na  na
 10  6774.54  846.82  0.89  negative
 25  11200.94  1400.12  1.48  negative
 50  6852.84  856.61  0.9  negative

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item did not produce any indications of skin sensitization in the local lymph node assay in mice at concentrations of up to 50% w/w in DMF.
Executive summary:

The skin sensitization of the test item was investigated in a local lymph node assya in CBA/Ca strain female mice according to OECG Tg 429 and EU method B42 and under GLP. The concentrations applied were determined in a prelimianry study and no signs of toxicity or irritation were observed at a concentration of 50% w/w of the test article in dimethyl formamide (DMF). In the main study groups of 4 mice were applied 25 micro-l of a 0 (vehicle control), 10, 25 or 50% suspension of the test article on the dorsal parts of both ears for 2 consecutive days. At day 5 3H-Thymidine was injected into the tail vein of each animal (20 micro-Ci/mouse). The stimulationm index was determined from the preparation of single cell suspensions of the auricular lymph nodes of each mouse pooled per dose group by liquid scintillation counting. The stimulation index was expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group. The criterium for a positve result was a 3 fold increase compared to controls. The test item did not show a positive response in this assay. The stimulation idices were 0.89 (10% test item in DMF), 1.48 (25% test item in DMF) and 0.9 (50% test item in DMF)