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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 guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (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
Details on test material:
- CAS-No.: 1312296-85-7
- Appearance: Beige solid
- Purity: 100.4 g/100 g, determined by 1H-NMR spectroscopy
- Expiration Date: February 22, 2012
- Homogeneity: The test substance appeared to be homogeneous
- Storage: At room temperature, under N2

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: pre-test: 10-11 weeks (beginning of treatment); main study: 10-11 weeks (beginning of treatment)
- Weight at study initiation: 21.4 ± 1.4 g (mean ± SD)
- Housing: group housing (5 animals per group) in Makrolon Type III cages with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet: Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
- Water: Tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Relative Humidity (%): 33 - 65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. - 6.00 a.m. / 6.00 a.m. - 6.00 p.m.)

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10, 25, 50% (w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 50% (w/w) solution of the test item. Test item solutions at different concentrations were prepared using dimethylformamide as vehicle. Vortexing was used to formulate the test item.
- Irritation: To determine the highest non-irritant test concentration that does not induce signs of systemic toxicity at the same time, a pre-test was performed in two animals. Two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% (w/w) once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible reddening of the ear skin. Furthermore, 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). Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm²) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if reddening of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6. The measured ear weight was also considered in this evaluation. On day 3 and day 4, both treated animals showed an erythema of the ear skin (Score 1). Other signs of irritation or signs of systemic toxicity were not observed. Thus, the test item in the main study was assayed at 10, 25, and 50 % (w/w).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: 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:
The test item was placed into an appropriate container on a tared balance and dimethylformamide was added to achieve the required test item concentration. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied. Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 10, 25, and 50% (w/w) in dimethylformamide. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ~ 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.
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 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 periodic positive control experiment was performed with α-hexyl cinnamaldehyde dissolved in acetone:olive oil (4:1 v/v) (compound listed in OECD 429 Guideline) using CBA/CaCrl mice in August 2011. An EC3 value of 12.1% (w/v) was calculated.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Stimulation Indices (S.I.) of 1.36, 1.42, and 1.10 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in dimethylformamide, respectively. An EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: A statistically significant or biologically relevant increase in DPM values was not observed in any test item treated group in comparison to the vehicle control group.

Any other information on results incl. tables

Calculation of Stimulation Indices per Dose Group

Test item concentration

Group Calculation

Mean DPM
   per animala)

SDb)

S.I.

Vehicle
(dimethylformamide)

742.3

323.4

1.00

10 % test item

1007.1

315.2

1.36

25 % test item

1056.7

322.5

1.42

50 % test item

817.9

216.7

1.10

a)     Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group

b)     SD = Standard Deviation

The EC3 value could not be calculated, since all S.I.'s are below the threshold value of 3.

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

 

Clinical Signs: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

 

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 and biologically relevant increase in lymph node weight or lymph node cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group. 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 did not exceed this threshold.

 

Ear Weights: The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in ear weights was not observed in any test item treated 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. This threshold was not exceeded in any dose group. Furthermore, according to OECD guideline 429, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation. This threshold was not exceeded in any test item treated group.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item was not a skin sensitiser under the test conditions of this study.
Executive summary:

The study was performed according to OECD guideline 429 in compliance with GLP.

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in CBA/CaOlaHsd mice. Test item solution at 10, 25, and 50% (w/w) was prepared in the vehicle dimethylformamide. The highest concentration tested was the highest concentration that could be technically used.

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, the cutoff-value for a positive response regarding the ear weight index of 1.1 reported for BALB/c mice was not exceeded in any dose group.

Stimulation Indices (S.I.) of 1.36, 1.42, and 1.10 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in dimethylformamide, respectively. A statistically significant or biologically relevant increase in DPM value, lymph node weight and cell count was not observed in any treated group in comparison to the vehicle control group. Furthermore, the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was not exceeded in any dose group.

Conclusion: The test item was not a skin sensitiser under the test conditions of this study.

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