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Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- February from the 12th to the 20th, 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- Adopted 22th July 2010
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
Test material
Constituent 1
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- CBA
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: TOXI-COOP ZRT, Budapest.
- Females: female, nulliparous, non-pregnant.
- Age at study initiation: young adult mice. 10-11 weeks old at start of the preliminary test; 10-12 weeks old at start of the main test.
- Weight at study initiation: 18.2-22.5 g. The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing: 4 animals/cage. Cage type II, polypropylene / polycarbonate. Mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.
- Diet: animals received ssniff® SM R/M-Z+H complete diet for rats and mice, ad libitum.
- Water: tap water from municipal supply, as for human consumption, from a bottle, ad libitum.
- Acclimation period: 7 days.
- Animal health: only healthy animals were used.
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 – 70 %
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Study design: in vivo (LLNA)
- Vehicle:
- dimethylformamide
- Concentration:
- 10 %, 5 %, 2.5 % and 1 % (w/v) as formulations in N,N-Dimethylformamide (DMF)
- No. of animals per dose:
- 28 animals, 4 animals/treatment group
- Details on study design:
- PRE-SCREEN TESTS:
- Compound solubility: the test item was adequately soluble in DMF with a maximum concentration of 10 % (w/v). The other vehicles (i.e. Acetone: Olive oil 4:1 (v/v) mixture and purified water) were not suitable at the same concentration (10 %, w/v) and above. Based on this DMF was used for the test item formulations in the main test.
- N. of animals per dose: 6 animals (2 animals/treatment groups).
- Concentration: the test item was formulated in the selected vehicle (DMF) and tested at concentrations of 10 %, 5 % and 2.5 % (w/v).
- Frequence of application: once daily for 3 consecutive days.
- Irritation: no signs of significant irritation (indicated by an increased ear thickness of ≥ 25 % on any day of measurement) were observed in the treatment groups. Although erythema could not be reliably observed due to intensive orange colour of the test item an erythema score < 3 is considered.
- Systemic toxicity: no mortality or any signs of systemic toxicity were observed during the preliminary test. No significant, treatment related effect on body weights was observed.
- Erythema scores:
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4
MAIN STUDY
TEST PROCEDURES
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 (DMF or AOO as negative control groups) using a pipette, on the dorsal surface of each ear. After the treatments animals were 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 animals showed symptoms of systemic toxicity or excessive skin irritation, and no technically failed treatment was observed during the test: all animals treated were processed. Therefore no any treatment group was excluded from the evaluation.
Injection of 3HTdR
On Day 6, each mouse was intravenously injected via the tail vein with 250 μl of sterile PBS (1 x PBS, diluted from 10x concentrate with purified water) containing 20 μCi of 3H-methyl-thymidine using a hypodermic needle with 1 mlsterile 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 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 the mice from each test group were pooled and collected separately in a Petri dish 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 lymph node cells (LNCs), pooled according to groups, 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). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 ml supernatant above each pellet. The pellets were gently agitated before making up to 10 ml with PBS and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
Determination of Incorporated 3HTdR
After the final wash, each supernatant was removed leaving a small volume (< 0.5 ml) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 ml of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for 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, than 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 expresses the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 ml aliquots of 5 % TCA.
OBSERVATIONS
Clinical Observations
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test. 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 the Results
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value. 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 pooled lymph nodes).
The stimulation index (SI = the DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Based on the results EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated. Dose-response relationship was evaluated by linear regression. All calculations were made by Microsoft Excel Software.
Interpretation of the Results
The test item is considered as a skin sensitizer, if: exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.
ASSAY ACCEPTANCE CRITERIA
- Lymph nodes from all 4 animals in each dose groups were pooled for a valid experiment.
- Skin sensitizing effect was observed at the applied concentration of the positive control (the stimulation index was greater than 3). - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
Results and discussion
- Positive control results:
- No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
Significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 7.0). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.
In vivo (LLNA)
Resultsopen allclose all
- Parameter:
- SI
- Value:
- 1.1
- Test group / Remarks:
- at the concentration of 10 %
- Parameter:
- SI
- Value:
- 0.6
- Test group / Remarks:
- at the concentration of 5 %
- Parameter:
- SI
- Value:
- 0.5
- Test group / Remarks:
- at the concentration of 2.5 %
- Parameter:
- SI
- Value:
- 0.9
- Test group / Remarks:
- at the concentration of 1 %
- Cellular proliferation data / Observations:
- Body Weight Measurement: no significant, treatment related effect on body weights was observed during the test.
Clinical Observations and Signs of Irritation: no mortality was observed during the study. No significant, treatment related effect on body weights or any other signs of systemic toxicity were observed in any treatment group during the test. No signs of significant irritation or any other significant local effect were observed at the treatment site (ears) in any treatment group. Although erythema could not be reliably observed due to intensive orange colour of the test item an erythema score < 3 is considered in all dose groups.
Proliferation Assay: visually larger lymph nodes than the vehicle control observed in the positive control group only. Visual appearance of the lymph nodes was normal in the negative control groups (both DMF and AOO) and in the test item treated groups.
No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMF) was noted for test item at the tested concentrations. The stimulation index values were 1.1, 0.6, 0.5 and 0.9 at concentrations of 10 %, 5 %, 2.5 % and 1 %, respectively. No dose-related response was observed (r = 0.56, p = 0.44, evaluated by linear regression using SI values).
ASSAY ACCEPTANCE CRITERIA
The assay acceptance criteria was fulfilled, hence the test was valid.
Any other information on results incl. tables
DPM, DPN and Stimulation Index Values for all Groups in the Main Test
Test Group | Measured DPM/group | Group* DPM | DPN (DPM/node) | Stimulation Index |
Vehicle control for the positive control: AOO | 8621 | 8599.5 | 1074.9 | 1.0 |
Positive control: 25 % HCA in AOO | 60045 | 60023.5 | 7502.9 | 7.0 |
Test item 10 % in DMF | 6112 | 6090.5 | 761.3 | 1.1 |
Test item 5 % in DMF | 3621 | 3599.5 | 449.9 | 0.6 |
Test item 2.5 % in DMF | 2714 | 2692.5 | 336.6 | 0.5 |
Test item 1 % in DMF | 5171 | 5149.5 | 643.7 | 0.9 |
Vehicle control for the test item: DMF | 5720 | 5698.5 | 712.3 | 1.0 |
HCA = α-Hexylcinnamaldehyde; AOO = Acetone: Olive oil 4:1 (v/v) mixture; DMF= N,N-Dimethylformamide; *Group DPM = measured DPMgroup- average DPMbackground Average DPMbackground = 21.5
Applicant's summary and conclusion
- Interpretation of results:
- other: not classified, according to the CLP Regulation (EC) No 1272/2008
- Conclusions:
- not sensitising
- Executive summary:
The maximum achievable test item concentration was 10 % (w/v) in N,N-Dimethylformamide (DMF). Based on the preliminary test results the test item was examined in the LLNA as 10 %, 5%, 2.5 % or 1 % (w/v) formulations in DMF according to the OECD guidelines 429. In the main test 28 female CBA/Ca mice were allocated. No mortality, no significant treatment related effect on body weights, any other signs of systemic toxicity or any sign of significant irritation or any other local effect were observed in any treatment group during the test. Visually larger lymph nodes than the control were observed in the positive control group only. Visual appearance of the lymph nodes was normal in the test item treated groups and in the negative control groups (both DMF and AOO). No significantly increased lymphoproliferation (indicated by an SI > 3) compared to the vehicle control was noted for test item at the tested concentrations. The stimulation index values were 1.1, 0.6, 0.5 and 0.9 at concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. No dose-related response was observed (p = 0.44, r = 0.56).
The positive control item (25 % HCA in AOO) induced the appropriate (SI > 3) stimulation over the control (SI value was 7.0), thus confirming the validity of the assay.
Conclusion
According to evaluation criteria of the relevant guidelines the lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum achievable concentration (based on solubility) of 10 % (w/v) and the lack of a dose-related response are considered evidence that test item has no skin sensitization potential.
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Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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