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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-10-12 to 2011-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(adopted 22 July 2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(May 30,2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Manston Road Margate, Kent CT9 4LT United Kingdom
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 19.0- 22.6 g
- Housing: Group, Makrolon Type II / III, with wire mesh top
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2
- Humidity (%): 31 - 65
- Photoperiod (hrs dark / hrs light): 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Pre test: 25, 50 %
Main test: 5,10, 25%
No. of animals per dose:
Pre test: 2 animals
Main test: 5 animals
Details on study design:
RANGE FINDING TESTS:
- Irritation: The animals treated with 25% of test item did not show any signs of local skin irritation or systemic toxicity during the course of the study. The animals treated with 50% of test item did not show any signs of local skin irritation or systemic toxicity within the first two days of experiment. On day three this animal was sacrificed due to severe signs of systemic toxicity (reduced spontaneous activity, ruffled fur, and hunchback posture). Thus, the test item in the main study was assayed at 5, 10, and 25% (w/w) test item concentration.
- Lymph node proliferation response: The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
Second, that 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.

TREATMENT PREPARATION AND ADMINISTRATION
Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% (w/w) in N,N-dimethylformamide. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (diameter 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone.

Administration of 3H-Methyl Thymidine
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline (PBS) containing 20.1 μCi of 3HTdR (equivalent to approximately 80.4 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). After an appropiate reconditioning process the level of 3HTdR incorporation was measured on a β-scintillation counter.

OBSERVATIONS
Mortality / Viability At least once daily from experimental start to necropsy.
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany).
Ear weights: In the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear.
Lymph node weights: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Lymph node cell count: The lymph node cell count were determined in aliquots taken from the prepared single cell suspensions of the lymph nodes using a cell counter.
Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group.
For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test test was used for identification of possible outliers (performed with Microsoft Excel 2003).
However, both biological and statistical significance were considered together

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitisation properties in CBA/CaCrl mice. The periodic positive control experiment was performed with α-hexyl cinnamaldehyde dissolved in acetone:olive oil (4:1 v/v) ) using CBA/CaCrl mice in August 2011. The periodic positive control experiment was performed at intervals of not longer than 6 months.
Stimulation index: 0%: 1.00; 5%: 1.35; 10%: 2.18; 25%: 8.08
The estimated concentration for a S.I. of 3 (EC3) resulted 12.1 % (w/v).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Mean Stimulation Indices relative to the mean of the control group Vehicle 0%: 1.00 Test item 5%: 19.24 10%: 21.51 25%: 9.71 The EC3 value could not be calculated, since all S.I.´s are above the threshold value of 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM value (5 measures) corrected for mean background value (1 mL 5% trichloroacetic acid) Vehicle 0%: 870.3 Test item 5%: 16748.9 10%: 16748.9 25%: 8446.9

Any other information on results incl. tables

OBSERVATIONS

Viability / Mortality: No deaths occurred during the study period.

Clinical Signs: No systemic findings were observed during the study period. On day 4 and 5, the animals treated with 25% test item concentration showed an erythema of the ear skin (Score 1). In the animals treated with 10% test item concentration, this was seen on day 4 only. In the low dose group, no signs of local skin irritation were observed.

Body Weights: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Lymph Node Weights and Cell Counts: The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant increase in lymph node weight and lymph node cell count was observed in all dose groups in comparison to the vehicle control group (p<0.05). For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count exceeded this threshold by far in all test item treated groups (Indices of 4.6, 5.0, and 2.93).

Ear Weights: The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights was observed in the low dose group in comparison to the vehicle control group. For BALB/c mice, a threshold for the ear weight index of 1.1 was reported for a positive response. According to this criterion, the ear weight index determined for the low dose group (1.2) indicates a positive response regarding ear skin irritation. However, the mentioned cutoff-value has been determined using a different strain of mice and can thus not be implicitly adopted.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information