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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study available as unpublished report, no restrictions, fully adequate for assessment.

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)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
Toxi-Coop ZRT., Arácsi út 97., 8230 Balatonfüred, Hungary
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Petol PM 410-4N
- Physical state: yellow-brown (apparently yellow), viscous, clear liquid
- Storage condition of test material: Store in tightly closed container, in a dry and well ventilated area, between 20-30°C.

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. H-1103, Budapest, Cserkesz u. 90.
- Age at study initiation: Young adult mice, 10 weeks old (at start of Experiment 1), 9 weeks old (at start of Experiment 2)
- Weight at study initiation: 17.7-22.6 g in Experiment 1, 16.9-20.3 g in Experiment 2
- Housing: Grouped in small groups during acclimatization period. During the test in groups of 4 per Type II. Polypropylene / polycarbonate cage with deep wood sawdust bedding (Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG, D-73494, Rosenberg, Germany, Holzmühle 1)
- Diet: Ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494, Soest, Germany, ad libitum
- Water: tap water from municipal supply, ad libitum
- Acclimation period: 7 days (in both Experiments)

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3
- Humidity: 30 – 70
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Experiment 1: concentrations of 75 %, 50 %, 25 %, 10 % and 5 %
Experiment 2: concentrations of 2.5 %, 1 %, 0.5 % and 0.25 %
No. of animals per dose:
28 animals in Experiment 1
24 animals in Experiment 2
(4 animals/treatment group in both Experiments)
Details on study design:
RANGE FINDING TESTS:
The undiluted test item was not applicable on the ears of animals due to high viscosity. Two groups of 2 CBA/Ca mice were treated with the 75 % or the 50 % formulation once daily for 3 consecutive days. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to the first treatment (Day 1) and prior to termination (Day 6). 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.

MAIN STUDY
- In vivo Treatment: AOO was used as negative (vehicle) control for both the test item and the PC substance in both Experiments. Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance or the vehicle 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. This procedure was followed in both Experiments.
- Proliferation Assay: On Day 6 each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (1 x PBS, diluted from 10 x concentrate with purified water) containing 20 μCi of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes). 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. 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. 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) trichloracetic 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 MAIN TEST:
- Clinical Observations: Based on the preliminary test results beside erythema scoring, measurement of ear thickness was also used for additional monitoring of local irritation in the main test (in both Experiments). Ear thickness measurements was performed using digital micrometer on Day 1 (pre-dose), on Day 3 (approximately 48 hours after the first dose) and on Day 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 in both Experiments.

EVALUATION:
DPM (disintegration per minute) was measured for each treatment group in both Experiments. 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. EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated.

INTERPRETATION OF RESULTS:
The test item is considered as a skin sensitizer, if the following criteria are fulfilled:
- That 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.
- 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Significance of the dose-response was evaluated linear regression

Results and discussion

Positive control results:
The positive control group animals were treated with 25 % (w/v) HCA solution (dissolved in AOO) concurrent to the test item groups in both Experiments. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. Significant lymphoproliferative response (SI ≥ 3) was noted for HCA in both Experiments: the observed SI values were 9.1 and 11.1 in Experiment 1 and Experiment 2, respectively.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks:
EXPERIMENT 1
Value:
20.4
Test group / Remarks:
75% dose-group
Parameter:
SI
Remarks:
EXPERIMENT 1
Value:
18.9
Test group / Remarks:
50% dose-group
Parameter:
SI
Remarks:
EXPERIMENT 1
Value:
21.7
Test group / Remarks:
25% dose-group
Parameter:
SI
Remarks:
EXPERIMENT 1
Value:
18.6
Test group / Remarks:
10% dose-group
Parameter:
SI
Remarks:
EXPERIMENT 1
Value:
15
Test group / Remarks:
5% dose-group
Parameter:
SI
Remarks:
EXPERIMENT 2
Value:
15.8
Test group / Remarks:
2.5% dose-group
Parameter:
SI
Remarks:
EXPERIMENT 2
Value:
5.5
Test group / Remarks:
1.0% dose-group
Parameter:
SI
Remarks:
EXPERIMENT 2
Value:
2.6
Test group / Remarks:
0.5% dose-group
Parameter:
SI
Remarks:
EXPERIMENT 2
Value:
3.1
Test group / Remarks:
0.25% dose-group
Cellular proliferation data / Observations:
disintegrations per minute (DPM)
EXPERIMENT 1: 75% dose group: 29450.5 50% dose-group: 27353.1 25% dose-group: 31342.4 10% dose-group: 26838.4 5% dose-group: 21745.3 0% dose-group: 1446.6
EXPERIMENT 2: 2.5% dose-group: 14312.8 1.0% dose-group: 4991.8 0.5% dose-group: 2337.4 0.25% dose-group: 2772.3 0% dose-group: 903.2

Any other information on results incl. tables

Dose-range finding study:

No mortality was observed during the test. Loss of the body weights was observed in both treatment groups (the maximum value was 14 % in the 75 % dose group) but no other signs of systemic toxicity were observed. No signs of significant 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 although increased ear thickness values were observed in both treatment groups (the maximum value was 23.8 % in the 50 % dose group). The observed effects were considered equivocal. Based on the preliminary test results the 75 % concentration was used in the main test as the maximum test concentration in order to test highest concentration possible. On the other hand the effects observed in the preliminary test could not be disregarded hence, four additional, lower concentrations (50 %, 25 %, 10 % and 5 %) were also examined in the main test to ensure that no unexpected adverse effect interferes the evaluation of the test.

Body Weight Measurement:

Significant loss of body weights (> 5%) was observed in Experiment 1 at test item concentrations of 75%, 50% and 25%: the mean values decreased by 9%, 9% or 7%, respectively. No significant, treatment related effect on body weights was observed in Experiment 1 in the control groups and in the 10% and 5% dose groups. Similarly, no significant, treatment related effect on body weights was observed in Experiment 2.

Clinical Observations and Signs of Irritation:

No mortality was observed during the test (in any of the Experiments). No symptoms of systemic toxicity other than loss of body weights were observed in any treatment group in Experiment 1. As a local effect loss of hair around the treatment site (ears) was observed in Experiment 1 at the 25%, 10% and 5% dose groups. The symptom was observed on Days 3, 4, 5 (2/4 animals) and on Day 6 (4/4 animals) in the 25% dose group, on Day 6 in the 10% dose group (4/4 animals) and on Day 6 in the 5% dose group (4/4 animals). As a sign of a possible irritation effect of the test item increased ear thickness values were observed in all test item treated groups. The increase was significant (≥ 25%) in the 25% and 10% dose groups: the maximum values measured on Day 6 were 33.3% or 42.9%, respectively. Although erythema (scored as 1 or 2) was observed in all test item treated groups, it was not significant: no erythma scored as ≥ 3 was observed during Experiment 1. Based on the loss of body weights, loss of hair, increased ear thickness, and erythema it was considered that these effects may indicate irritation and/or toxic effect of the test item at higher concentrations which may contributed to increase of the lymphoproliferation. The lack of a significant dose-response relationship at the tested concentration range also can confirm this supposition. In Experiment 2 no any symptom of systemic toxicity, no visible signs of irritation (indicated by an increased ear thickness of ≥ 25% or erythema score ≥ 3) or any other local effect were observed in any treatment group. Therefore, the proliferation values obtained are considered to reflect the real potential of the test item to cause sensitization in the Local Lymph Node Assay.

Proliferation Assay:

In Experiment 1 visually larger than the control lymph nodes were observed in the positive control group and in all test item treated groups. Appearance of the lymph nodes was normal in the negative control group (AOO). In Experiment 2 visually larger than the control lymph nodes were observed in the positive control, the 2.5 % and the 1 % test item treated groups. Appearance of the lymph nodes was normal in the negative control group (AOO) and in the 0.5 % or 0.25 % dose groups. In Experiment 1 significant (SI ≥ 3) lymphoproliferative response was noted for Petol PM 410-4N at all tested concentrations. Significance of the dose-response was evaluated by linear regression using the SI values. No statistical significance was observed (p = 0.36; r value = 0.53). In Experiment 2 significant (SI ≥ 3) lymphoproliferative response was noted for Petol PM 410-4N at test concentrations of 2.5 %, 1 % and 0.25 %, however the lymphoproliferative response observed at concentration of 0.5 % was also on the borderline. Statistically significant dose-related response was observed (p = 0.01; r value = 0.99).

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance is, in a GLP compliant OECD 429 guideline study, considered to be a skin sensitser.
Executive summary:

In a GLP compliant skin sensitisation study, performed according to OECD Guideline 429the skin sensitisation potential of Petol PM 410-4N was investigated in a mouse Local Lymph Node Assay (LLNA). The main test consisted of two independent experiments. In experiment 1, five groups of 4 female CBA mice each were treated with 75, 50, 25, 10, and 5% of the test substance and in experiment 2, four groups of 4 females each were treated with 2.5, 1, 0.5, and 0.25% of the test substance. In both experiments the test substance was applied by means of open application of 25μL to each ear for three consecutive days. Three days after the last treatment, all animals received an intravenous injection of 3H-thymidine. Five hours later, the 3H-thymidine incorporation in the draining auricular lymph nodes was determined. The results were compared with those of a negative control group which was treated with the vehicle (acetone/olive oil, 4:1 v/v). In experiment 1 significant loss of body weights was observed at test item concentrations of 75 %, 50 % and 25 %: the mean values decreased by 9 %, 9 % or 7 %, respectively. As a local effect loss of hair around the treatment site was observed at the 25 %, 10 % and 5 % dose groups. The ear thickness increase was 33.3 % or 42.9 % at day 6 in the 25 % and 10 % dose groups, respectively. The stimulation indices ranged between 15.0 and 21.7. Based on the local and systemic effects observed it was considered that the observed effects may indicate irritation and/or toxic effect of the test item at higher concentrations which could contribute to increase of the lymphoproliferation. This suggestion was supported by the lack of a significant dose-response relationship at the tested concentration range. Therefore, a second experiment was performed. In experiment 2 no systemic and local effects were observed. The stimulation indices in experiment 2 were 15.8, 5.5, 2.6, and 3.1 for the 2.5, 1.0, 0.5, 0.25% dose-group, respectively. Based on these results, the test substance is considered to be a skin sensitizer.