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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Toxi-Coop ZRT.
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Test material form:
solid: crystalline
Details on test material:
- Name of test material: Thymidine

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Young adult mice, 10-11 weeks old (at start of the preliminary test)
- Weight at study initiation: 17.9 – 22.8 g, with weight variation not exceeding ± 20 % of the mean weight
- Housing: Grouped caging (5 animals/cage) in type II polypropylene / polycarbonate cages with laboratory bedding
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
- From: 19 Sept. 2012
- To: 25 Sept. 2012

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
Based on the preliminary test results the test item was examined in the main test as formulations in Dimethyl sulfoxide (DMSO). The formulations in this vehicle were real solutions. The test item was weighed and formulations prepared daily on a weight: volume basis at concentrations of 25 %, 10 %, and 5 % (w/v) in a final volume of 1 mL using calibrated volumetric vials and mechanical agitation. Formulations were freshly prepared just before the treatments.
No. of animals per dose:
Preliminary test: 2 animals per treatment group
Main test: 2 animals per treatment group
Details on study design:
DOSE RANGE FINDING TEST
- The maximum dose selection was performed according to the relevant guidelines. The preliminary irritation/toxicity screen was conducted in a similar experimental manner to the exposure phase of the main test except there was no assessment of lymph node proliferation and fewer animals were used.
- The test item was dissolved in the selected vehicle (DMSO) and tested at concentrations of 25 %, 10 % and 5 % (w/v). The formulations (apparently solutions) were adequately applicable on the ears of animals.
- Three groups of 2 CBA/Ca mice were treated with the appropriate formulations once daily for 3 consecutive days. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site during the preliminary test. Body weights were recorded prior to the first treatment (Day 1) and prior to termination (Day 6, where applicable). Both ears of each mouse were observed for erythema and scored using criteria depicted in Table 2. Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
- No mortality was observed during the test. No significant, treatment related effect on the body weights was observed in any treatment groups. No signs of systemic toxicity or local irritation (indicated by an erythema score ≥ 3 and/or an increase of ear thickness ≥ 25 %) were observed at the treatment site (ears) at the tested concentrations.
- Based on the preliminary test results the maximum attainable concentration (based on solubility) of 25% was used in the main test. The test item was tested also at two additional, lower concentrations (10 % and 5 %) according to the relevant guidelines.

MAIN TEST DESIGN
- The test item was administered at three different concentrations according to the results of the dose range finding test. The positive control substance (HCA) was tested at one concentration.

IN VIVO TREATMENT
- Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance (positive control group) or the vehicles (negative control groups) using a pipette, on the dorsal surface of each ear. After the treatments animals returned to their cages. 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.

PROLIFERATION ASSAY
- No symptoms of systemic toxicity, excessive skin irritation or technically failed treatment were observed during the test. Lymph nodes from all five animals per dose groups were processed for evaluation.

INJECTION OF 3H-METHYL-THYMIDINE (3HTdR)
- On Day 6, the animals were intravenously injected via the tail vein with 250 μL of sterile PBS (diluted from 10x concentrate with purified water) containing 20 μCi# of 3HTdR using a hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
# Remark: Braking down of [methyl-3H]-Thymidine in aqueous solution (about 3 % per month) was taken into account when 3HTdR solution was prepared.

REMOVAL AND PREPARATION OF DRAINING AURICULAR LYMPH NODES
- Five hours (± 30 minutes) after intravenous injection the mice were humanely sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps. Once removed, the nodes of individual mice were collected separately in a Petri dish containing a small amount (1-2 mL) of PBS (diluted from 10x concentrate with purified water) to keep the nodes wet before processing.

PREPARATION OF SINGLE CELL SUSPENSION OF LYMPH NODE CELLS
- A single cell suspension (SCS) of lymph node cells (LNCs) of each individual animal was prepared and collected in disposable tubes by a gentle mechanical disaggregation of the lymph nodes through 200 μm-mesh stainless steel gauze using the plunger of a disposable syringe. The cell strainer (stainless steel gauze) was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4°C. After centrifugation, the majority of the supernatant was aspirated off, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated and the LNCs were resuspended in 10 mL of PBS. This washing procedure was repeated twice. This procedure was repeated for lymph nodes of each individual animal.

DETERMINATION OF INCORPORATED 3HTDR
- After the final washing step, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before resuspending the LNCs in 3 mL of 5 % (w/v) trichloracetic acid (TCA, dissolved in purified water) to allow for the precipitation of the macromolecules. After incubation with 5 % TCA at 2-8 °C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4°C and decanting the supernatants and the pellets were re-suspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and stored at room temperature protected from light until measurement.
- For the measurement of incorporated radioactivity the vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % TCA.
- Instrument used for the measurement: Packard Tri-Carb 2300 TR Liquid Scintillation Analyzer (Serial Number: 406 117)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis was performed by SPSS/PC+ (4.0.1) software package. The measured DPM values corrected with the mean background (TCA) value were used for statistical analysis of the proliferation data.
The heterogeneity of variance between the groups treated with the test item and the relevant vehicle controls was checked by Bartlett's test. Since significant heterogeneity was detected, the normal distribution of data was examined by Kolmogorow-Smirnow test followed by the non-parametric method of Kruskal-Wallis One-Way analysis of variance. As a results of this analysis the inter-group comparisons was performed using Mann-Whitney U-test to assess the significance of inter-group differences.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Visually larger lymph nodes than the relevant control (AOO) was observed in the positive control group. Visual appearance of the lymph nodes was normal in both negative control groups and in the test item treated groups. No significant lymphoproliferation (SI ≥ 3) compared to the relevant control (DMSO) was noted for the test item at the tested concentrations. The observed stimulation index values were 1.2, 1.4 and 1.7 at test item concentrations of 25 %, 10 % and 5 %, respectively. No dose-related response was observed. - The positive control group animals were treated with 25 % (w/v) HCA solution (dissolved in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. The positive control substance induced the appropriate, statistically significant stimulation compared to the control (SI = 14.3; p < 0.01). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed sensitivity and validity of the assay.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Individual DPM values (corrected with the mean background (TCA) value) were statistically evaluated in all test item treated groups and in the control groups (both positive and negative control). Statistically significant increase of the proliferation values compared to the relevant negative control was observed in the positive control group (p < 0.01, Mann-Whitney U-test versus AOO control) and in the 10 % test item treated group (p < 0.05, Mann-Whitney U-test versus DMSO control). No statistical significance was observed for the other test item treated groups (Mann-Whitney U-test versus DMSO control).

Any other information on results incl. tables

BODY WEIGHT MEASUREMENT

- No significant, treatment related effect on the body weights was observed in any treatment group.

OBSERVATIONS

- No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of significant irritation (indicated by an erythema score ≥ 3) or any other local effect was observed in any treatment groups.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and of the preliminary irritation/toxicity test. The maximum attainable test concentration based on solubility was 25 % (w/v) test item in Dimethyl sulfoxide (DMSO). Based on the preliminary test results the test item was examined in the main test as formulations (apparently solutions) in DMSO at concentrations of 25 %, 10 % and 5 %.
Since the test was valid and no sign of systemic toxicity or irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.
No significant lymphoproliferative response indicated by a Stimulation Index ≥ 3 of LZ3547 was observed at the applied concentrations. The proliferation values observed for the test item at concentrations of 25 % and 5% did not differ significantly from the relevant control values. Although statistical significance was observed at 10 % concentration it has no biological relevance since no dose-related response was observed.
According to evaluation criteria of the relevant guidelines the lack of a significantly increased proliferation (indicated by an SI ≥ 3) up to the maximum attainable concentration of 25 % and the lack of a dose-related response are considered evidence that the test item is not a sensitizer.
Executive summary:

A study was carried out according to EU Method B.42, OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay) and OPPTS 870.2600. The aim of this study was to determine the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. Selection of test item concentrations was based on the results of a formulation evaluation and a preliminary irritation/toxicity tests. In the main study concentrations of 25 %, 10 % and 5 % in DMSO were tested. Female CBA/Ca mice were allocated to 6 groups of five animals each: - three groups received the test item at three different concentrations of 25 %,10 % and 5 %, - the negative control group used as control for the groups treated with the test item formulations received the vehicle of the test item (DMSO) only, - the positive control group received α-Hexylcinnamaldehyde (HCA) at concentration of 25 %, - the negative control group used as control for the positive control group received the vehicle of the positive control substance (AOO). Each substance was applied on the external surface of each ear (25 μL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR) and sacrificed approximately 5 hours after the injection. Auricular lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI). The positive control item (25 % HCA in AOO) induced the appropriate, statistically significant stimulation compared to the control No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control was noted for the test item at the tested concentrations. The observed stimulation index values were 1.2, 1.4, and 1.7 at test item concentrations of 25 %, 10 % and 5 %, respectively. No dose-related response was observed. According to evaluation criteria of the relevant guidelines, the lack of a significantly increased proliferation (indicated by an SI ≥ 3) up to the maximum attainable concentration of 25 % and the lack of a dose-related response are considered evidence that the test item is not a sensitizer.