Registration Dossier

Administrative data

Description of key information

LLNA (OECD 429, Harlan 2012): sensitizing

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a GLP compliant study according to OECD guideline 429 the possible skin sensitisation potential of (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid was studied (Harlan CCR, 2012). Three groups each of five female mice were treated once daily with the test item at concentrations of 2.5, 5, and 10% (w/w) in methyl ethyl ketone by topical application to the dorsum of each ear for three consecutive days. The test item could be dissolved in the vehicle. The appropriateness of the used concentrations was previously assessed by two pre-experiments. A control group of five mice was treated with the vehicle (methyl ethyl ketone) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes were excised, pooled per animal and immediately weighed. Furthermore, after excision of the lymph nodes, both ears of the mice were punched at the apical area using a biopsy punch and were immediately weighed pooled per animal using an analytical balance. Afterwards, single cell suspensions of lymph node cells were prepared from lymph nodes pooled per animal. An aliquot of each cell suspension was used for determination of lymph node cell count. Subsequently the suspensions were washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a β-scintillation counter. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On application day 2 and 3, the animals treated with test item concentrations of 5 and 10% showed an erythema of the ear skin (Score 1). On day 4, only the animals of the high dose group showed an erythema of the ear skin (Score 1). A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p<0.05). However, this was considered to be not biologically relevant, as the observed increase did not exceed the threshold value of 25% for excessive local skin irritation mentioned in OECD guideline 429. Furthermore, for BALB/c mice, a cutoff-value of 1.1 was reported for a positive response of the ear weight index regarding ear skin irritation. None of the indices determined for the test item treated groups exceeded this threshold. A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 3.42, 8.41, and 14.20 were determined with the test item at concentrations of 2.5, 5, and 10% (w/w) in methyl ethyl ketone, respectively. An EC3 value could not be calculated because all obtained Stimulation Indices exceeded the threshold value of 3 for a positive response. An outlier was identified in the vehicle control group (DPM values determined for animal number 1). However, as exclusion of the outlier only slightly changed the mean value of the vehicle group and had no influence on the overall test result, the value in question was not excluded from calculation. A statistically significant and biologically relevant increase in DPM value, lymph node weight and –cell count was obtained in all test item treated groups in comparison to the vehicle control group (p<0.05) and a clear dose response was observed. Furthermore, the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was exceeded in all dose groups (indices of 2.08, 3.55, and 4.94). The test item (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid was a skin sensitiserunder the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the findings of the available studies, the test substance has to be classified R43 (Directive 67/548/EEC) and Skin sensitisation Cat. 1 (CLP). However based on the available data, no subcategorization into 1A/1B is possible.