Reaction mass of N,N-ethanediylbis- Hexadecanamide, N-[2-[(1-oxohexadecyl)amino]ethyl]-Octadecanamide, Hexadecanoic acid, 12-hydroxystearic acid, reaction products with ethylenediamine, N,N-ethanediylbis-Octadecanamide and Octadecanoic acid, 12-hydroxystearic acid, reaction products with ethylenediamine

Endpoint summary

Description of key information

In a LLNA the test item suspended in ethanol:deionised water (7 +3) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 5, 10 and 20 %. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S. I.) of 1.2, 1.0 and 1.5 were determined with the test item at concentrations of 5, 10 and 20 % (w/v) in ethanol:deionised water (7 +3), respectively. The test item was not a skin sensitiser in this assay.

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:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Test Item Preparation:
The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle (ethanol:deionised water (7+3)) was quantitatively added. The test item was sonicated for at least 45 minutes until a homogeneous suspension was achieved. The test item concentrations were prepared individually. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer. The preparations were made freshly before each dosing occasion. To determine the highest non-irritant test concentration or the highest technically applicable concentration, a non-GLP pretest (non GLP-study 881506) was performed in two mice. The data showed that the highest test item concentration, which could be technically used was a 20 % suspension. Even warming and sonicating could not achieve a higher concentration. In other vehicles used, e.g. acetone : olive oil (4+1), DMF, DMSO or propylene glycol, higher concentrations could also not be achieved. At the concentration of 20 % the treated mouse did not show any signs of irritation. The test item in the main study was, therefore, assayed at 5, 10 and 20 % (w/v).

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Age: 6 - 12 weeks (beginning of acclimatization)
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.TEST ANIMALS
- Source: Harlan Netherlands B.V. Postbus 6174
- Females (if applicable) nulliparous and non-pregnant: yes
- Housing: Single caging in Makrolon Type I, with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness will be used for the study.
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3°C
- Humidity (%): 30-85%
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES:
From: 22.05. To: 01.08.2006

Vehicle:

other: ethanol:deionised water (7+3)

Concentration:

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5, 10 and 20% (w/v) in ethanol:deionised water (7+3). The application volume, 25 μl, was spread over the entire dorsal surface (diameter ca. 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

No. of animals per dose:

5 animals per group per dose

Details on study design:

Administration of 3H-Methyl Thymidine:
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 μl of 79.3 μCi/ml 3HTdR (corresponds to 19.825 μCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of Incorporated 3HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental (Trapanal, Altana, D-78467 Konstanz). The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Interpretation of Raw Data:
The proliferative 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 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.
The decision to select a STIMULATION INDEX (S.I.) of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.

Observations:
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability once daily (week day) from experimental start tonecropsy.
Body weights prior to the first application and prior to treatment with 3HTdR.
Clinical signs (local / systemic) at 1 – 2 hours after each application. Especially the treatment sites were observed carefully.

Statistical Analysis:
The mean values and standard deviations were calculated in the body weight tables.

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.

Key result

Parameter:

EC3

Test group / Remarks:

The EC3 Value could not be calculated, since all S.I.'s are below 3.

Remarks on result:

not determinable

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 weights 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.

Interpretation of results:

other: not a skin sensitiser

Conclusions:

The test item was not a skin sensitiser under the described conditions.

Executive summary:

In the study the test item suspended in ethanol:deionised water (7 +3) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 5, 10 and 20 %. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S. I.) of 1.2, 1.0 and 1.5 were determined with the test item at concentrations of 5, 10 and 20 % (w/v) in ethanol:deionised water (7 +3), respectively. The test item was not a skin sensitiser in this assay.

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

Licolub FA 6 did not show skin sensitizing properties in the Local Lymph Node Assay according to the current OECD guidelines. Allergic skin reactions or case reports of acute contact dermatitis to Licolub FA 6 have not been described in the literature. Therefore, the substance does not have to be classified as a skin sensitizer.