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Description of key information

Key value for chemical safety assessment

Skin sensitisation

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

There are valid study data available to assess the sensitising potential Fatty acids, C18-unsatd., dimers, ethoxylated. No valid information for Fatty acids, C18-unsatd., dimers, ethoxylated on respiratory sensitisation is available.

In vitro sensitisation studies:

At first 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 Fatty acids, C18-unsatd., dimers, ethoxylated . The test battery evaluation uses the results of three individual assays testing reflecting three key events along the adverse outcome pathway (AOP) leading to skin sensitization: peptide reactivity (DPRA), keratinocyte activation (LuSens or KeratinoSens) and dendritic cell activation (MUSST or h-CLAT).

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 (Local Lymph Node Assay, 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%). Therefore this test battery is used as replacement for the animal test.

Protein reactivity (DPRA) test:

The reactivity of Fatty acids, C18-unsatd., dimers, ethoxylated) towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA) (BASFSE, 2013, 64V0072/12A114). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm. As no molecular weight of the test substance was available a gravimetric procedure was applied. The test substance was solved in isopropanol at a concentration of 3.76% (corresponding to a theoretical molecular weight of ca. 375 g/mol). Three samples of the test substance were incubated with each peptide in ratios of 1:5 (for C-peptide) or 1:24 (for K-peptide) concerning absolute mass. Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. Additionally, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were additionally analyzed by measuring UV absorbance at 258 nm and the area ratio 220 / 258 was calculated as a measure of peak purity. The following results were obtained in the DPRA: The test substance was soluble in isopropanol. However when mixed with the peptide stock solutions the samples became cloudy directly after preparation. After 24 hours precipitates were noticed in the samples with the C-peptide, additionally. Thus all samples were centrifuged prior to HPLC analysis. The samples containing the Cpeptide were additionally filtered as a separation could not be achieved by centrifugation. The mean C-peptide depletion, caused by the test substance was determined to be 21.8%. The mean K-peptide depletion, caused by the test substance was determined to be -1.3%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 10.9%. No co-elution of test substance and peptides was noticed.

Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that Fatty acids, C18-unsatd., dimers, ethoxylated shows a low chemical reactivity in the DPRA under the test conditions chosen.

Activation of keratinocytes (LuSens):

The keratinocyte activating potential of test substance Fatty acids, C18-unsatd., dimers, ethoxylated was evaluated in the LuSens assay (BASFSE, 2013, 66V0072/12A116) . For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and ARE-dependent luciferase activity was measured in a luminometer. The test substance was soluble in 1% DMSO at a concentration of 2000 μg/mL. In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test substance (0.5 μg/mL up to 2000 μg/mL) and cytotoxicity was determined thereafter by MTT assay. No decrease in cell viability below 70% was observed. In the main test, test substance was used at eight final concentrations. After 48 hour exposure luciferase activity was measured in a luminometer. The test substance was concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeded 1.5 fold induction with respect to the vehicle control and at concentrations that did not reduce a viability below 70%. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. The LuSens showed the following results: The test substance was soluble in DMSO. The dilutions of the test substance were solutions in DMSO and suspensions in culture medium at a concentration of 558 μg/mL onward. However, after 48 hours precipitates were noticed in the samples. In summary, after 48 hours of exposure to test substance Fatty acids, C18-unsatd., dimers, ethoxylated (NIO AD1) luciferase activity in LuSens cells was was not induced at concentration affording at least 70% viability. From this it has to be concluded that Fatty acids, C18-unsatd., dimers, ethoxylated does not have a keratinocyte activating potential.

Activation of dendritic cells (MUSST):

The test substance was not soluble at sufficiently high concentrations therefore the Test was stopped in accordance with GLP conditions.

 

Summery of results of in vitro sensitization battery:

 

 

Project Number

Test Method

Test Result

Test Evaluation

64V0072/12A114

Direct Peptide Reactivity Assay (DPRA)

10.4% mean peptide depletion (21.8% cysteine peptide depletion, -1.3%* lysine peptide depletion)

Positive

66V0072/12A116

Keratinocyte Activation assay – LuSens

In at least two independent experiments no ARE-dependent luciferase activity induction above 1.5-fold was observed.

Negative

65V0072/12A115

Dendritic Cell Line Activation Assay

Myeloid U937 Skin Sensitizing Test (MUSST)

The test substance was not soluble at sufficiently high concentrations

n/a

*Negative depletions were considered to be ”zero” for calculation of mean peptide depletion.

 

Based on the results summarized in the Table and applying the evaluation criteria for the in vitro sensitization battery the sensitizing potential of Fatty acids, C18-unsatd., dimers, ethoxylated cannot be predicted.

Hence further test are needed to address the skin sensitization of Fatty acids, C18-unsatd., dimers, ethoxylated

Guideline conform studies:

The dermal sensitizing potential of Fatty acids, C18-unsatd., dimers, ethoxylated

was investigated according to one of the methods recommended in the OECD Guideline No. 406. The aim of the study was to evaluate the possible allergenic activity of the test item after intradermal and topical administration in guinea pigs (GPMT). The study was started with a reduced number of animals (5 control and 10 test group animals) according to the recommendations given in the OECD guideline. After induction (intradermic injection at 5% in corn oil and topical application at 100%) of 10 Guinea Pigs of treated group with the test item Fatty acids, C18-unsatd., dimers, ethoxylated (NIO AD1) and a 10- day rest phase, the challenge phase, under occlusive dressing for 24 hours, consisted to a single topical application of the test item undiluted (100%) and of a negative control (liquid paraffin). The experimental protocol was established according to the OECD guideline No. 406 dated July 17th, 1992 and the test method B.6 of the council regulation No. 440/2008, US EPA OPPTS guideline 870.2600 (March 2003). No cutaneous reaction was recorded in animals from the treated group, 24 hours after the challenge phase, on the treated area with the test item at 100%. A slight to moderate erythema was recorded in 80% (8/10) and in 60% (6/10) of the animals from the treated group, 48, and 72 hours respectively after the challenge phase, on the treated area with the test item at 100%. No cutaneous reaction was recorded in animals from the negative control group, 24 hours after the challenge phase, on the treated area with the test item at 100%. A slight erythema was recorded in 80% (4/5) and in 60% (3/5) in animals from the negative control group, 48, and 72 hours respectively after the challenge phase, on the treated area with the test item at 100%. As irritation was observed in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. So, a sensitization reaction was noted in 10% (1/10) and in 20% (2/10) of the animals from the treated group 48 and 72 hours respectively after the challenge phase, on the area challenged with the test item at 100%. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (vehicle). In view to confirm or infirm these results, a new challenge phase was performed with the test item diluted at 50% in liquid paraffin and with liquid paraffin as control (vehicle), after a rest phase of 5 days. A slight erythema was recorded in 20% (2/10), in 20% (2/10) and in 60% (6/10) of the animals from the treated group, 24, 48, and 72 hours respectively after the challenge phase, on the treated area with the test item at 50%. A slight erythema was recorded in 20% (1/5), in 20% (1/5) and in 40% (2/5) of the animals from the negative control group, 24, 48, and 72 hours respectively after the challenge phase, on the treated area with the test item at 50%. As irritation was observed in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. So, no sensitization reaction was noted in animals from the treated group 24, 48 and 72 hours after the challenge phase, on the area challenged with the test item at 50%. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (vehicle).

As the result of the study was not unambiguously assessable due to the irritation observed in the control animals after challenge as well as after rechallenge, the study was discontinued. To further evaluate the sensitizing potential of the test substance a Buehler test in a seperate study was conducted (Phycher proj.-No.: SMB-3-PH-13/0038, BASF proj.-No.: 32H0072/12X446). This study is currently running.

 

Key study assignment:

As there a few information on sensitisation derived from all studies, all are integrated as weight of evidence.

 

Skin sensitisation assessment:

There are a few results available assessing the skin sensitizing potential in a weight of evidence. Information from in vitro test show partly negative [keratinocyte assay (66V0072/12A116)] and partly positive hints for sensitisation [DPRA (66V0072/12A114)]. But the the in vitro test battery could not be fully completed and can therefore not unequivocally deliver a result. Therefore a a guinea pig maximisation test was performed (30H0072/12X401). Unfortunately in this test a delayed irritation happened. This delayed irritation infects the test group as well as the negative controls. As both reactions are equal in strength there are hints that these reactions are only based on irritation. But to clear any concern a further guinea pig test (Bühler) is currently performed. Based on the current results there are doubts about sensitising effects of the substance based on a weight of evidence. Therefore the substance is considered to be not sensisitising.

The dossier will be update as soon as the results of the Bühler test are available.


Migrated from Short description of key information:
Skin sensitisation:
- in vitro skin sensitisation battery (DPRA, LuSens, MUSST): ambiguous / not valid (one test not valid due to solubility) (BASFSE, 2013, 67V0072/12A117 summary report)
- in vivo skin sensitisation, GPMT, OECD 406: ambiguous, delayed irritation observed in test group and controls in nearly same ratio, therefore result not unequivocal (BASFSE, 2013, 30H0072/12X401)
- in vivo skin sensitisation, Bühler, OECD 406: running (BASFSE, 2013, 32H0072/12X446 )

Respiratory sensitisation:
-No data available

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation:

According to GHS and DSD substances shall be classified as skin sensitiser if there are positive results from appropriate animal test. As in a weight of evidence approach the substance is considered to be not sensitising it needs not to be classified according to GHS (Regulation (EU) 1272/2008) as sensitising to skin and also not to be classified according to DPD (67/548/EEC).

 

Labelling for sensitisation:

GHS: no label

DSD: no label

 

Respiratory sensitisation:

No data available

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