Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 January 2012 to 18 January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Physical state: White to pale yellow powder with a slightly characteristic odour.
- Storage condition of test material: Room temperature (15-25°C), in the dark.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/J Rj mice
- Age at study initiation: 9 - 10 weeks old.
- Weight at study initiation: 19.0 – 20.2 g (The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight)
- Housing: Standard housing conditions in groups. Type II. polypropylene/ polycarbonate cages were used.
- Diet: Complete 'breeding and maintenance' diet for rats and mice was provided ad libitum.
- Water: Tap water from the municipal supply, ad libitum.
- Acclimation period: 20 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15-20 air exchange/hour.
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
10, 7.5, 5, 3.75 and 2.5 (w/v) %.
No. of animals per dose:
4 female mice per dose.
Details on study design:
MAIN STUDY

TREATMENT PREPARATION AND ADMINISTRATION:
- Formulation: The test material was formulated with propylene glycol to obtain appropriate concentrations. The dose levels chosen were based upon the maximum concentration obtained in the vehicle (solvent) and the sponsor's request. The test material was weighed and formulations prepared daily on a weight:volume basis ((w/v)%).
- Administration: During the assay, 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). There was no treatment on Days 4, 5 and 6.
- Injection of Tritiated Thymidine (3HTdR): On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
- Removal and Preparation of Draining Auricular Lymph Nodes: Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. 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.
- Preparation of Single Cell Suspension of Lymph Node Cells: 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. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
- Determination of Incorporated 3HTdR: After the final wash, supernatants 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.
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % TCA.

CONTROLS
- Negative control: Propylene glycol was used as a negative control.
- Positive control: The positive control group animals were treated with 25 (w/v) % α-Hexyl-cinnamaldehyde solution (dissolved in the same solvent as the test material) concurrent to the treatment groups.

OBERVATIONS
- Clinical Observations: During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
- Measurement of Body Weight: Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

EVALUATION OF RESULTS
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 (w/v) % TCA solutions was used as the background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation. Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

- Interpretation of Results: A test material is regarded as a sensitizer if both of the following criteria are fulfilled:
> That exposure to at least one concentration of the test material resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
> The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression

- Acceptability of the test: The Local Lymph Node Assay is considered acceptable if it meets the following criteria:
> The DPN value of the negative (vehicle) control falls within the range of historical laboratory control data,
> The positive control item should produce a significant lymphoproliferative response increases,
> Each treated and control group should include at least 4 animals,
> If the test material does not cause serious systemic or local toxicity.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Stimulation index values ranged from 1.0 to 2.1, see Table 1 for results.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The results of the proliferation assay are summarized in Table 1 and Figure 1.

Any other information on results incl. tables

CLINICAL OBSERVATION

No mortality, systemic toxicity or local irritation was observed during the study.

 

BODY WEIGHT MEASUREMENT

No treatment related effects were observed on animal body weights.

LYMPH NODES

The appearance of the lymph nodes was normal in the negative control group and the test material treated groups. Larger than normal lymph nodes was observed in the positive control group.

POSITIVE CONTROL

α-Hexylcinnamaldehyde (25 (w/v) % dissolved in PG) was used as a positive control to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 22.7) was noted for the positive control chemical and this result confirmed the validity of the assay.

INTERPRETATION OF OBSERVATIONS

Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test material to cause lymphoproliferation in the Local Lymph Node Assay. The lack of any positive result under these exaggerated test conditions is considered to be good evidence that the test material is not a sensitizer.

Table 1: LLNA Assay Results

Test Group [(w/v) % in PG]

Measured DPM/group

Group DPM

No. of Nodes

DPN

Stimulation Index Values

10

939

899.5

8

112.4

2.1

7.5

830

790.5

8

98.8

1.9

5

786

746.5

8

93.3

1.8

3.75

657

617.5

8

77.2

1.5

2.5

489

449.5

8

56.2

1.1

Background (5 (w/v) % TCA)

39.5

 

-

 

 

Negative Control PG

462

422.5

8

52.8

1.0

Positive Control 25 % HCA in PG

9610

9570.5

8

1196.3

22.7

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, under the conditions of the present assay the test material formulated in propylene glycol, was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay.
Executive summary:

The skin sensitization potential of the test material was determined in a Local Lymph Node Assay. The study was performed under GLP conditions and in line with standardised guidelines OECD 429 and EU Method B.42.

Four female CBA/J Rj mice per group were dosed at test concentrations of 10, 7.5, 5, 3.75 and 2.5 (w/v) %, formulated in propylene glycol. Test solutions 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 (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality, systemic toxicity or local irritation was observed during the study.

No treatment related effects were observed on animal body weights in any other treated groups. Stimulation index values of the test material were 2.1, 1.9, 1.8, 1.5 and 1.1 at treatment concentrations of 10, 7.5, 5, 3.75 and 2.5 (w/v) %, respectively.

Under the conditions of the assay, the test material was shown to have no sensitization potential and was concluded to be a non-sensitizer.