Morpholinium, 4-[2-[2-[(2-hydroxyphenyl)methylene]hydrazinyl]-2-oxoethyl]-4-methyl-, chloride (1:1)

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

LLNA

The skin sensitizing potential of Tinocat ES 96000 was assessed using the radioactive Murine Local Lymph Node Assay according to OECD 429 guideline and GLP (BASF, 2011). The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears.

Groups of 5 female CBA/J mice each were treated with 5%, 10% and 25% w/w preparations of the test substance in 1% aqueous Pluronic® or with the vehicle alone. A 50% preparation was the maximum technically applicable concentration. The pre-test revealed that the 50% concentration is unsuitable for appropriate application on the ears and therefore a 25% preparation was selected as the high concentration.

The study used 3 test groups and 1 control group. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation, applied to the dorsal surfaces of both ears for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone.

Three days after the last application the mice were injected into the tail vein with 20 μCi of 3H-thymidine in 250 μL of sterile saline. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

Besides a slight reduction of the mean body weight of test group 4, no signs of systemic toxicity were noticed during the observation period.

When applied as 25% preparation in 1% aqueous Pluronic®, the test substance induced a statistically significant and biologically relevant (increase above the cut off Stimulation Index of 3) increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes.

The 5% concentration caused a statistically significant, but not biologically relevant increase of 3H-thymidine incorporation.

The increase in the auricular lymph node cell counts was statistically significant and at the border of biological relevance (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) at 25%. The increase at 5% was statistically significant, but not biologically relevant.

Additionally, concentration dependent and statistically significant increases were noted in lymph node weights at all concentrations, as well.

There was no relevant increase in ear weights at all concentrations, demonstrating the absence of ear skin irritation. However, the increase at the 5% concentration was statistically significant. Residues of the test substance were observed on the ear skin of the animals treated with the 25% test-substance preparation on day 2, only.

Thus, it is concluded that Tinocat ES 96000 exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was ca. 25% for 3H-thymidine incorporation. Due to the difference in the concentration-effect relationships of 3H-thymidine incorporation and cell count, no congruent estimated concentrations were obtained. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was

calculated by linear regression from the results of the 10% and 25% concentrations to be 22%, while the SI of 1.5 for cell count was >25%.

In vitro:

In a GLP-compliant Direct Peptide Reactivity Assay (DPRA) the reactivity of the test item, Tinocat ES 96000, towards synthetic cysteine (C)- or lysine (K)-containing peptides is evaluated (BASF SE, 2011). The test substance is incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm. The peptides are custom material containing phenylalanine to facilitate UV-detection and either cysteine or lysine as the reactive center. The test substance was solved at a 100 mM concentration in highly de-ionized water. One (K-peptide) or two test runs (C-peptide) were performed. Per test run three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). The peptide depletion of test-substance incubated samples was compared to the peptide depletion of the NC samples and expressed as relative peptide depletion. For the test substance the mean peptide depletion as average of C- and K-peptide depletion is calculated and used for evaluation of the chemical reactivity. The negative peptide depletion obtained with the C-containing peptide indicates that a peak is interfering with the C-peptide peak, thereby increasing the peak area to a value above the value of the respective vehicle control. The test-substance control showed that this peak is not caused by the unchanged test substance. Therefore the interference could be caused by a reaction product of the test substance with the peptide. Visual observation after the 24-hour incubation time revealed precipitates in the samples of the test substance with the K-containing peptide. Thus the samples were filtered prior to HPLC analysis. The mean of both peptide depletions could not be calculated due to the artifacts obtained with the C-peptide. However, the K-peptide depletion was determined to be 12.5%. Thus based on the observed results the prediction model proposed in Gerberick et. al (2007) can not be applied but there was some chemical reactivity of Tinocat ES 96000 in the DPRA under the test conditions chosen.

A GLP compliant myeloid U937 skin sensitization test, a dendritic cell activation test, was performed to predict the skin sensitizing potential (BASF SE, 2011). The test is performed using the human pro-monocytic cell line U937 as surrogate for dendritic cells. The test substance was tested in a concentration range of 3.9 to 61.8 µg/mL based on a preliminary cytotoxicity study.Three independent experiments were performed.As readout, the change in the expression of the cell membrane marker CD 86 measured by flow cytometry after 48 hours of test substance exposure is determined. A test substance is predicted to activate dendritic cells when CD 86 cell surface expression exceeds the threshold in relation to vehicle control in atleast three independent experiments.Cell viability was decreased just below 70% at 30.9 µgin the first experiments 1 while an induction of the expression of CD 86 was observed. Induction of CD 86 expression was also observed in the second experiment, while cell viablilty was just above the set threshold of ≥70%. Therefore a third experiment with a refined concentration spacing was performed and an induction of the expression of CD 86 was observed at several sufficiently non-cytotoxic (cell viability≥70%) concentration.The expression levels of the controls were within the range of the historical negative and positive control data.In summary, after 48 hours of exposure to test substance CD 86expression was induced in U937 cells at concentrations affording at least 70% viability.From this it has to be concluded that test substance does inducedendritic cell activation.

A GLP-compliant LuSens assay was performed using a luciferase reporter cell line (LuSens cells) to assess the keratinocyte activating potential of the test substance (BASF SE, 2012). The cell line LuSens was treated with 6 test substance concentrations, between 5.61 – 13.96 µg/mL, for 48 hours in at least two independent experiments with each 3 replicates. Cells were lysed and luciferase induction was evaluated by measuring luminescence signal after substrate addition. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. The CV75 value was derived from the concentration response curve. The CV75 is the estimated concentration that affords 75% cell viability and was determined to be 9.69 µg/mL for the test substance under the chosen exposure conditions on LuSens cells. The luciferase activity level of the controls were within the range of the historical negative and positive control data. After 48 hours of exposure to the test substance luciferase activity in LuSens cells was not induced at concentration between 5.61 and 13.96 µg/mL affording at least 70% viability. From this it has to be concluded that test substance has no keratinocyte activating potential.

Due to the complexity of the skin sensitization process a single in vitro assay is not sufficient to adequately assess this toxicological endpoint (BASF SE, 2013). Therefore, a combination of several in vitro methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD, has been conducted to assess the skin sensitizing potential of the test substance: protein reactivity (DPRA), activation of keratinocytes (LuSens) and activation of dendritic cells (MUSST) has been used. The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al., 2012. Based on the performance standards of the OECD test guideline no. 429 (LLNA, OECD 2010) the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95% compared to results in humans (for comparison: for the same data set the LLNA yielded an overall accuracy of 86%). A skin sensitizing (quantitative) potency assessment using the reported results was not validated at the time of writing this report. In the test battery evaluation a weight of evidence approach is used: Any two of the three tests determine the overall results, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer. Based on the results summarized in the above described individual test systems and applying the evaluation criteria the sensitizing potential of the test substance cannot be predicted.

Migrated from Short description of key information:
LLNA (BASF SE, 2011): sensitizing

Justification for classification or non-classification

Based on data from the LLNA, that indicate a sensitising potential, classification is warranted in accordance with EU Directive 67/548 (R43) and Eu Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 (Skin Sens. Cat. 1B).