Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19th April 2011 to 4th May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A GLP study performed in compliance with appropriate guidelines.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: off-white solid
- Storage condition of test material: room temperature in the dark

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: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: individually in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): 2014C Teklad Global Rodent Diet ad libitum
- Water (e.g. ad libitum): mains tap water ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25ºC
- Humidity (%): 70-30%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Study design: in vivo (LLNA)

Vehicle:
other: emulsion in ethanol/distilled water (7:3)
Concentration:
10%, 25% and 50% test substance concentration.
No. of animals per dose:
Four
Details on study design:
RANGE FINDING TESTS:
One individual mouse was treated with three consecutive daily applications (days 1-3) of 25 µL/ear of the test substance on the dorsal ear. The test substance was administered using an automatic micropipette and spread over the dorsal surface of the ear. The animal was observed twice daily on days 1-3 and once daily on days 4-6. Local irritation and clinical signs of toxicity were recorded and bodyweights taken on days 0 and 6. The thickness of the ear was measured using an Oditest micrometer on days 1 (pre-dose), 3 and 6. A mean ear thickness increase equal or greater to 25% over the course of the study was considered to indicate excessive irritation and limited biological relevance to sensitisation.

MAIN STUDY
Individual animals received daily applications of 25 µL/ear on the dorsal surface for three consecutive days (days 1-3). The test substance was administered using an automatic micropipette and spread over the dorsal surface of the ear. A further group of four animals received only vehicle in the same manner. Five days after the first topical application (day 6), all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each animal.

OBSERVATIONS
All animals were observed twice daily on days 1-3 and once daily on days 4-6 and any signs of ill health or toxicity recorded. The bodyweight of each animal was recorded on days 1 (pre-dosing) and day 6.

TERMINAL PROCEDURES
Five hours following the administration of 3HTdR all animals were killed by carbon dioxide asphyxiation. Draining auricular lymph nodes were excised and pooled for each test substance concentration. For each group 1 mL of PBS was added to the pooled lymph nodes.

A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze and rinsed through the gauze with PBS. The pooled lymph node cells were centrifuged at 1400 rpm for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. radioactive material was precipitated out by resuspending the pellet in 3 mL of 5% trichloroacetic acid (TCA).

After approximately 18 hours incubation at 4 ºC, the precipitates were recovered by centrifugation at 2100 rpm for ten minutes, resuspended in 1 mL of TCA and measured by scintillation counting. Prior to counting the vials were left in darkness for 20 minutes to reduce the risk of luminescence.
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 (dpm/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the controls.

The test substance is considered to be a sensitiser if at least one concentration of the test substance results in a three-fold or greater increase in 3HTdR incorporation compared to controls.

Results and discussion

In vivo (LLNA)

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

Any other information on results incl. tables

Range-finding test

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. White residual test substance on the ears was noted after dosing on days 1 to 3. Based on this information, the dose levels selected for the main test were 10%, 25% and 50% w/w in ethanol/distilled water (7:3).

Clinical observations and mortality data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the study. White residual test substance was noted on the ears after dosing on days 1 and 2 in all animals treated at the 50% w/w concentration and after dosing on day 3 in all animals treated at the 25% and 50% w/w concentrations.

Bodyweight

Bodyweight gains of the animals between days 1 and 6 were comparable to those observed in the control group over the same period.

Table 1: Indiviual bodyweights and bodyweight gains

 Concentration (% w/w) Animal number Bodyweight (g) Bodyweight change (g)
     Day 1  Day 6  
 Vehicle 1-1 18 18 0
  1-2 18 18 0
  1-3 19 19 0
  1-4 19 19 0
 10 2-1 18 20 2
  2-2 18 18 0
  2-3 19 19 0
  2-4 18 18 0
 25 3-1 20 21 1
  3-2 17 18 1
  3-3 17 17 0
  3-4 19 19 0
 50 4-1 16 17 1
  4-2 17 17 0
  4-3 18 17 -1
  4-4 18 18 0

Disintegrations per minute and Stimulation Index

Table 2: Disintegrations per minute, Disintegrations per minute/node and Stimulation Index

 Concentration (% w/w) dpm dpm/Node* Stimualtion Index Result
 Vehicle 6341.61 792.70 N/A N/A
 10 10733.41 1341.68 1.69 Negative
 25 5841.62 730.20 0.92 Negative
 50 5933.06 741.63 0.94 Negative

* dpm/Node is calculcated by dividing dpm by 8 (total number of lymph nodes)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the study the Stimulation Index scores of 3HTdR incorporation into lymph node cells was less than three for all tests concentrations. The test substance was therefore considered to be a non-sensitiser.
Executive summary:

In a GLP compliant skin sensitisation study conducted in line with OECD Guideline 429 and EU Method B.42, the skin sensitisation of the test substance was investigated. The Stimulation Index scores for 3HTdR incorporation into lymph node cells were less than three at all test concentrations and the substance was therefore considered to be a non-sensitiser.