Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-23-08 to 2010-09-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed in accordance with OECD principles of GLP with no deviations. Furthermore, the study fulfilled the validity criteria described in the OECD guideline.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
PC2414
IUPAC Name:
PC2414
Details on test material:
- Name of test material (as cited in study report): PC2414
- Lot/batch No.: Y5UB376
- Expiration date of the lot/batch: 2013-03-01

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Nulliparous and non-pregnant females of mouse substrain CBA/CaOlaHsd
- Age at study initiation: Approximately 8-12 weeks at start of treatment
- Weight at study initiation: Between 15-23g
- Housing: Individually housed, in suspended polypropylene cages fitted with stainless mesh lids and furnished with softwood woodflakes
(Datesand Ltd., Chesshire, Uk)
- Diet (e.g. ad libitum): rodent 2018C Teklad Global Certified Diet (Harlan Laboratories UK Ltd. Oxon, UK)
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): Twelve hours of continuous artificail light in each twenty-four hour period

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
10, 25 and 50 % (w/w) in DMSO
No. of animals per dose:
0% (w/w): 5 animals
10% (w/w): 5 animals
25% (w/w): 5 animals
50% (w/w): 5 animals
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: 50% (w/w)
- Irritation: no clinical sings of toxicity was noted. White coloured residual material on the ears was noted on Days 2 to 5. Based on this information the dose levels selected for the main test were 10%, 25% and 50% (w/w) in dimethyl sulphoxide.
- Lymph node proliferation response: not performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA study using individual approach
- Criteria used to consider a positive response: A stimulation index (SI) of ≥ 3, together with consideration of dose-response and where appropriate a statistical significance.

TREATMENT PREPARATION AND ADMINISTRATION: Groups of five mice was treated with 10%, 25% and 50% (w/w) of PC2414 in DMSO. 25 µl was applied to the entire dorsal surface of each ear of each mouse by daily application for three consecutive days (Days 1, 2 and 3). Five days following the first topical application (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR:80 µCi/ml) giving a total of 20 µCi to each mouse. Five hours later, the draining Auricular lymph nodes from each individual mouse was excised and processed into PBS (maximum of 2 nodes per animal). A single cell suspension of lymph node cells (LNC) was prepared from each mouse by gentle mechanical disaggregation of the nodes through a 200-mesh stainless steel gauze. The LNC was rinsed and pelleted two times at 1400 rpm (approximately 190g) for 10 minute. Cells were precipitated with 5% trichloroacetic acid at 40C for 18 hours followed by measurement of 3HTdR incorporation using β-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Individual and group mean dpm values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of significant result from the ANOVA, pairwise comparisons were perfromed between control and treated groups. For homogenous datasets Dunnett´s Multiple Comparison test was used and for non-homogenous datasets Dunnett´s T3 Multiple Comparison Method was used.

Results and discussion

Positive control results:
An SI value of 3.25 was obtained using 15% (v/v) α-Hexylcinnamaldehyde in DMSO. The result was from the latest positive control study (09 June 2010 to 15 June 2010) performed by the testing laboratory used (Harlan Laboratories). A group of five animals was treated with 50 ul (25 ul per ear) of
α-Hexylcinnamaldehyde (tech. 85%) as a solution in DMSO at aconcentration of 15% (v/v). A further control group of five animals was treated with DMSO alone.
The positive result for α-Hexylcinnamaldehyde in DMSO was although in the low part of range within the range of positive control data used by the testing laboratory, thereby validating the study performed with PC2414.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 0% (w/w): SI=NA 10% (w/w): SI: 3.54 25% (w/w): SI: 3.32 50% (w/w): SI: 3.50 Positive control group: SI=3.25
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 0% (w/w): dpm=1475.24 ( ± 567.28) 10% (w/w): dpm=5215.94 ( ± 971.58) 25% (w/w): dpm=4903.32 ( ± 1657.17) 50% (w/w): dpm=5169.47 ( ± 2934.68)

Any other information on results incl. tables

Table 1. Disintegrations per Minute and Stimulation Indices.

Concentration
(% w/w) in
dimethyl sulphoxide

Animal Number

dpm/
Animal
a

Mean dpm/Animal
(Standard Deviation)

Stimulation Indexb

Result

Vehicle

1-1

2225.47

1475.24
(±567.28)

na

N/A

1-2

1390.68

1-3

1785.04

1-4

717.82

1-5

1257.17

10

2-1

4960.24

5215.94**
(±971.58)

3.54

Positive

2-2

3701.35

2-3

6302.02

2-4

5595.89

2-5

5520.19

25

3-1

4469.66

4903.32*
(±1657.17)

3.32

Positive

3-2

7725.67

3-3

3416.92

3-4

4124.55

3-5

4779.82

50

4-1

3223.67

5169.47
(±2934.68)

3.50

Positive

4-2

3596.35

4-3

6772.32

4-4

9575.95

4-5

2679.04

dpm = Disintegrations per minute

a = Total number of lymph nodes per animal is 2

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

** = Significantly different from control group p<0.01

* = Significantly different from control group p<0.05

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
PC2414 was found to be a skin sensitiser in the LLNA study using concentrations of 10, 25 and 50%. Based on the SI values obtained in this study, the result is evaluated to be a “borderline result” because no dose response was established and significant results only obtained using 10 and 25% PC2414. Further, the SI values were all only very slightly above 3.

Overall, because of the result being a “borderline result”, it is evaluated that the positive result obtained in this study is a "false positive" and that PC2414 is not a skin sensitizer.

Executive summary:

The dermal sensitization of PC2414 to mice (CBA/CaOlaHsd) was determined using the local lymph node assay in accordance with OECD Guideline for Testing of Chemicals 429 and EEC method B42. Groups of 5 female mice were dosed at: 0% (control), 10%, 25% and 50% solutions of PC2414 in DMSO. Stimulation indexes (SIs) were 3.54, 3.32 and 3.50 for the 10%, 25% and 50% dose groups, respectively. The EC3 value could not be calculated, due to flat dose response. Based on the SI values, PC2414 was found to be a skin sensitizer in this assay.

Positive and vehicle controls were within the range to ensure the validity of the test. With the positive control a S.I. of 3.25 was obtained at a concentration of 15%. This result was from the latest positive control study (09 June 2010 to 15 June 2010) performed by the testing laboratory used (Harlan Laboratories). The result was in the low part of range but still within the range of positive control data.

Even though PC2414 was found to be a skin sensitizer based on the SI values, it is though noted that no dose response was established and significant results only obtained using 10 and 25% PC2414. In the vast majority of assays conventional dose responses are recorded with sensitizing chemicals such that increasing concentrations of the allergen provoke increasingly more vigorous proliferative responses. In some instances, as in this study, the dose response profile was relatively flat suggesting either that saturation kinetics for absorption have been achieved or that maximal immune stimulation has been induced. 

Based on the SI values obtained in this study, the result is evaluated to be a “borderline result” because no dose response was established and significant results only obtained using 10 and 25% PC2414. Therefore, it is evaluated that the results obtained could be categorized as a "false positive", especially because an initial qualitative prediction of the possible toxicity of PC2414 based on the chemical structure was performed using the DEREK for Windows system (LHASA Limited). A number of toxicological endpoints, including skin sensitization, were investigated by this predictive software and no alerts were trigged by the structure of PC2414. Further, data form testing showed that PC2414 was not corrosive and not irritating to skin and eye (see section 7.3.1 and 7.3.2).

Overall, because of the result being a “borderline result”, it is evaluated that the positive result obtained in this study is a "false positive" and that PC2414 is not a skin sensitizer.