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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:
2011-04-27 to 2011-05-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conforming study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: EC 440 / 2008, B.42 (2008)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Appearance: Brown powder
Storage: At room temperature, protected from light and moisture



In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V.
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 18.3 - 22.3 g
- Housing: group
- Diet: pelleted standard diet, ad libidum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65 % (main experiment), 28-65% (acclimation phase)
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
2.5, 5, and 10% (w/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:

Vehicle and Dose Selection:
The highest test item concentration, which can be technically used was a 50 % (w/v) suspension in dimethylsulfoxide (DMSO). At a concentration of 5% and below, the test item could be dissolved in the vehicle. Hence DMSO was selected as the vehicle. Grinding of the test item in a mortar and vortexing were used to formulate the test item.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and documented in the raw data and study report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% (w/v) once daily for 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. Signs of local irritation were documented and a score was used to grade any 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.
Ear irritation is considered to be excessive if reddening of the ear skin of a score value ≥3 is observed at any observation time and/or if an increase in ear thickness of ≥25% is recorded on day 3 or day 6.

On day 4 and 5, the animal treated with 50% test item concentration showed erythema of the ear skin (Score 1). The animal treated with 25% test item concentration showed erythema of the ear skin (Score 1) only on day 5. Furthermore, in both animals, the measured increase in ear thickness exceeded the threshold value of ≥25%.

Therefore, a second pre-test was performed using test item concentrations of 5 and 10% (without using the correction factor of 1.107). At these concentrations, none of the treated animals showed signs of excessive local skin irritation or systemic toxicity.

Thus, the test item in the main study was assayed at 2.5, 5, and 10% (w/v). The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation in the pre-experiments.

MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 2.5, 5, and 10% (w/v) in DMSO. The application volume, 25 µL was spread over the entire dorsal surface (~ 8 mm) of each ear once daily for three consecutive days. Two further groups each of 4 mice were treated with the vehicle DMSO for the test item or acetone:olive oil (4+1) for the positive control item) only. A further group of four mice was treated with the positive control item.

ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE

Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). Approximately five hours after treatment with 3HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetíc acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a scintillation counter.

INTERPRETATION OF RAW DATA

The proliferation response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiliser 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.

OBSERVATIONS

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
-Mortality / Viability: At least once daily from experimental start to necropsy.
-Body weights: Prior to the first application and prior to sacrifice.
-Ear thickness: In the pre-tests: prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
-Ear weights: In the pre-tests: after sacrifice; biopsy punches were taken from each ear.
-Clinical signs (local / systemic): In the pre-tests and in the main experiment, clinical signs were recorded at least once daily. Especially the treatment sites were carefully examined.
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 and for the DPM values (group mean DPM ± SD)
A statistical analysis was conducted for assessment of the dose-response relationship and also to assess whether the difference was statistically significant between test item groups and negative control group. Statistical significance was at the five per cent level (p < 0.05). The EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:
The concurrent positive control yielded a S.I. of 6.25, demonstrating the validity of the study.

Experiment performed in May 2011 using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 1.30, 2.82, and 9.03, respectively.
The EC3 value calculated was 10.4 % (w/v). The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
2.9
Test group / Remarks:
2.5% (w/v) DMSO (4 animals)
Remarks on result:
other: see Remark
Remarks:
In this study Stimulation Indices of 2.90, 5.27, and 8.33 were determined with the test item at concentrations of 2.5, 5, and 10% (w/v) in DMSO. A clear dose response was observed and an EC3 value of 2.6% (w/v) was derived. The concurrent positive control yielded a S.I. of 6.25, demonstrating the validity of the study.
Parameter:
SI
Value:
5.27
Test group / Remarks:
5% (w/v) DMSO (4 animals)
Parameter:
SI
Value:
8.33
Test group / Remarks:
10% (w/v) DMSO (4 animals)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see depicted table below

Any other information on results incl. tables

CALCULATION OF RESULTS

Since the lymph nodes of the animals of a dose group were pooled, the number of radioactive disintegrations per minute per lymph node (DPM/node) was determined by dividing the measured DPM-value by the number of lymph nodes pooled (8 lymph nodes). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. For the control group (vehicle group for the test item), a DPM/node value of 596.9 was determined whereas DPM/node values of 1731.6, 3147.1, and 4970.1 were determined for the test item groups with test item concentrations of 2.5, 5, and 10% (w/v), respectively. The Stimulation Indices calculated for these groups were 2.90 (at a test item concentration of 2.5%), 5.27 (at a test item concentration of 5%) and 8.33 (at a test item concentration of 10%). A clear dose response was observed. The EC3 value calculated was 2.6 %. The concurrent positive control yielded a S.I. of 6.25, demonstrating the validity of the study.

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symtoms 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.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was found to be a skin sensitiser under the test conditions of this study.