Life Power® L1

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

other: fine dust

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J Rj

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/J Rj mice
Source: ELEVAGE JANVIER; France
Hygienic level during the study: Standard housing conditions
Sex: female, nulliparous, non pregnant
Age of animals at starting: 8 weeks old
Body weight range at starting: 19.0 – 20.5 grams
Acclimatization time: 6 days

Husbandry
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene/ polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: 15-20 air exchange/hour
Feeding: ssniff SM R/M-Z+H "Autoclavable complete diet for rats and mice – breeding and maintenance"
Water: tap water.

Study design: in vivo (non-LLNA)

No. of animals per dose:

.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

A Preliminary Irritation/Toxicity Test was performed using two doses, at test item concentrations of 100 and 50% (w/v), respectively.
Test item concentrations of 100, 50 and 25 % w/v were used in the main study.

No. of animals per dose:

4 animals / treatment group

Details on study design:

Each mouse was topically dosed on the dorsal surface of each ear with 25 μl of the appropriate formulation applied using a pipette. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3).
On Day 6, each mouse was intravenously injected via the tail vein with 250 μl of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR. Once injected, the mice were left for 5 hours (± 30 minutes).
Five hours after intravenous injection the mice were euthanized. The draining auricular lymph nodes were excised. Once removed, the nodes of mice from each test group were pooled and collected in separate Petri dishes containing a small amount (1-2 ml) of PBS to keep the nodes wet before processing.
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe.
After washing, supernatant were removed leaving a small volume (<0.5 ml) of supernatant above each pellet. Each pellet was gently agitated before suspending the LNCs in 3 ml of 5% TCA (trichloroacetic acid) for precipitation of macromolecules. After incubation with 5% TCA at 2-8 °C overnight (approximately 18 hours) precipitate was recovered by centrifugation at 190 x g for 10 minutes, supernatants were removed and pellets were suspended in 1 ml of 5% TCA and dispersed using ultrasonic water bath. Each precipitate was transferred to a suitable sized scintillation vial with 10 ml of scintillation liquid and thoroughly mixed. The vials were loaded to a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Negative control, DMF: SI = 1.0 Test substance, 100 %: SI = 1.1 Test substance, 50 %: SI = 1.3 Test substance, 25 %: SI = 1.1 Positive control, HCA: SI = 12.4

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See the attachment.

Any other information on results incl. tables

No mortality or sign of systemic toxicity or local irritation were observed during the study. Test item precipitate was observed in all test item treated groups.

No treatment related effects were observed on animal body weights.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The substance was shown to have no sensitization potential in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to determine the skin sensitization potential of the substance following dermal exposure. The test item was dissolved N,N-Dimethylformamide (DMF). The test item formed an applicable formulation in DMF at 100 and 50 % (w/v). The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using two doses (test item concentrations of 100 and 50% (w/v)).

In the main assay, twenty female mice were allocated to five groups of four animals each:

- three groups received the appropriate formulation of the substance at concentrations of 100, 50 and 25% (w/v),

- the negative control group received DMF,

- the positive control group received 25 % α-Hexylcinnamaldehyde in DMF.

The solutions of the test item were applied on the dorsal surface of ears of experimental animals (25 μl/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine and the values obtained were used to calculate stimulation indices (SI).

No mortality or sign of systemic toxicity or local irritation were observed during the study. No treatment related effects were observed on animal body weights in any treated groups. No relevant clinical signs were observed.

Stimulation index values of the test item were 1.1, 1.3 and 1.1 at treatment concentrations of 100, 50 and 25% (w/v), respectively.

A significant lymphoproliferative response (SI = 12.4) was noted for the positive control chemical and this result confirmed the validity of the assay.

In conclusion, under the conditions of the present assay the substance, tested in a suitable vehicle, was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay.