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Administrative data

Description of key information

The test substance was found to be non-sensitizing in an LLNA study conducted according to OECD guideline 429.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-04-27 to 200-05-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conforming study under GLP without deviations
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)
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., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 18.1 - 22.9 g
- Housing:single caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), 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), 25-58% (acclimation phase)
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Vehicle:
dimethylformamide
Concentration:
5, 10, and 25%
No. of animals per dose:
4
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 can be technically used was a 25 % (w/v) solution in dimethylformamide (DMF). Vortexing and warming to 37°C was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. sonicating). Hence DMF was elected as vehicle.

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 reported 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 10 and 25% 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, both animals (treated with 10 or 25%) showed erythema of the ear skin (Score 1).

Thus, the test item in the main study was assayed at 5, 10, and 25%. 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 5, 10, and 25% (w/v) in DMF. The application volume, 25 µL, was spread over the entire dorsal surface (  8 mm) of each ear once daily for 3 consecutive days. Two further groups each of 4 mice were treated with the vehicle (dimethylformamide 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

3H-methyl thymidine (3HTdR) was purchased from Hartmann Analytic, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL). Five days after the first topical application, all mice were administered with 250 µL of 81.7 µCi/mL 3HTdR (corresponds to 20.4 µCi 3HTdR per mouse) by intravenous injection via a tail vein.


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: 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).
Ear weights: In the pre-test: after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local irritation/ systemic toxicity): In the pre-test 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.
Positive control results:
The concurrent positive control yielded a S.I. of 6.25, demonstrating the validity of the study.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see depicted table below
Parameter:
SI
Value:
0.94
Test group / Remarks:
at concentrations of 5% test item concentration in dimethylformamide
Parameter:
SI
Value:
0.86
Test group / Remarks:
at concentrations of 10% test item concentration in dimethylformamide
Parameter:
SI
Value:
1.11
Test group / Remarks:
at concentrations of 25% test item concentration in dimethylformamide

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 388.3 was determined whereas DPM/node values of 366.7, 334.8, and 429.3 were determined for the test item groups with test item concentrations of 5, 10, and 25% (w/v), respectively. The Stimulation Indices calculated for these groups were 0.94 (at a test item concentration of 5%), 0.86 (at a test item concentration of 10%) and 1.11 (at a test item concentration of 25%). 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 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, recordedprior to the first application and prior to treatment with3HTdR, was within the range commonly recorded for animals of this strain and age.

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

In order to study a possible contact allergenic potential of the test item, three groups each of four female mice were treated daily with the test item at concentrations of 5, 10, and 25% (w/v) in DMF by topical application to the dorsum of each ear (left and right) for 3 consecutive days. Two negative control groups each of 4 mice were treated with the vehicle (dimethylformamide) 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. Five days after the first topical application the mice were injected with radio-labelled thymidine (3H-methyl thymidine) intravenously into a tail vein. Approximately 5 hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in a scintillation counter.

All treated animals survived the scheduled study period and no signs of toxicity were observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 0.94, 0.86, and 1.11 were determined with the test item at concentrations of 5, 10, and 25% (w/v) in DMF. 

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

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The results from a mouse local lymph node assay indicate the substance is not a skin sensitizer. As a result the substance does not meet the criteria for classification according to Directive 2001/59/EC, Annex VI, 3.2.7.2.

 

The results from a mouse local lymph node assay indicate the substance is not a skin sensitizer. As a result the substance does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, Part 3, 3.4.2.2.