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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-24 - 2018-04-27 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD TG 442E
Version / remarks:
OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 09 October 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
An in vitro (or in chemico) study is the first step in a tiered testing approach under REACH.

Test material

Constituent 1
Reference substance name:
Reaction mass of 2-O-α-L-Rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid and α-L-Rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid
EC Number:
604-610-3
Cas Number:
147858-26-2
IUPAC Name:
Reaction mass of 2-O-α-L-Rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid and α-L-Rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 °C; in closed packaging

In vitro test system

Details on the study design:
A skin sensitiser refers to a substance that will lead to an allergic response following skin contact as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS)]. The potential to induce skin sensitisation is an important consideration included in procedures for the safe handling, packaging and transports of chemicals.
The assessment of skin sensitisation typically involves the use of laboratory animals. Classical methods comprise the Magnusson Kligman Guinea Pig Maximisation Test, the Buehler Test (TG 406) as well as the local lymph node assay, in its radioactive and non-radioactive form (TG 429, TG 442A/B). In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the human cell line activation test (h-CLAT) showed evidence of being a reliable and relevant method to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute the animal test currently in use.
The h-CLAT is supposed to address the third key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), the activation of dendritic cells (DC) typically accompanied by expression of specific cell surface markers, chemokines and cytokines. The h-CLAT quantifies the expression of the two surface markers CD86 and CD54 which are considered to be associated with the process of DC activation by using the human monocytic leukemia cell line THP-1 as a surrogate. The expression level of CD86 and CD54 following exposure to test chemicals are used for supporting the discrimination between sensitisers and non-sensitisers.
This test may be used for supporting the discrimination between skin sensitisers and non-sensitisers in accordance with UN GHS “Category 1”. It does not allow the classification of chemicals to the subcategories 1A and 1B as defined by UN GHS nor predict potency for safety assessment decisions. Therefore, all substances giving a positive result in the h-CLAT will be classified into UN GHS “Category 1”

This in vitro method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its potential to upregulate cell surface markers using fluorescence-activated cell sorting (FACS).

The correlation of upregulation of immunological relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event in the skin sensitisation process as described by the AOP. This method that measures the markers of DC activation, based on DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals.

This test method is able to detect chemicals that cause skin sensitisation and allows hazard identification in accordance with UN GHS “Category 1”. Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as IATA, combining them with other complementary information e.g., derived from in chemico or in vitro assays addressing other key events of the AOP

Cell line: The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 10E6 cells/mL.
Cells were cultured in 75 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 µg/mL streptomycin at 37 ± 1°C and 5% CO2.

Results and discussion

Positive control results:
The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86.

In vitro / in chemico

Results
Key result
Remarks on result:
positive indication of skin sensitisation
Remarks:
see "overall remarks, attachments"
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not stated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The study was performed according to OECD TG 442E under GLP on the registered substance itself. Positive and negative control were valid.
The h-CLAT addresses the third key event of the skin sensitisation adverse outcome pathway (AOP) that was defined by the OECD in 2012 and is a part of the AOP-based “two out of three” skin sensitisation integrated testing strategy for hazard identification.
In this study under the given conditions the test item did upregulate the cell surface markers in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser in accordance with UN GHS category 1.
The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA. However, depending on the regulatory framework, positive results generated with in vitro sensitization methods may be used on their own to classify a chemical into UN GHS category 1.
However, as the current study examines only one of the three key events which are part of the skin sensitisation adverse outcome pathway (AOP), the registrant concludes that further studies are required to draw definitive conclusions on the classification of the substance, and so the absolute necessity for classification as skin sensitizer is not given.
Executive summary:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study according to OECD TG 442E under GLP, Decanoic acid, 3-[[6-deoxy-2-O-(6-deoxy-a-L-manno-pyranosyl)-a-L-mannopyranosil-1-(carboxymethyl)octyl ester, mixture with 1-(carboxymethyl)octyl 3-[(6-deoxy-a-L-mannopyranosyl)oxy]decanoate was dissolved in 0.9% NaCl.

A CV75 of 157.42 ± 20.15 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps:

52.72, 63.27, 75.92, 91.10, 109.32, 131.19, 157.42, 188.91 µg/mL

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 0.0% (CD86), 0.0% (CD54) and 0.0% (isotype IgG1 control) in both experiments because of an absence of measureable cells.

In the first experiment, the expression of the cell surface marker CD86 was increased up to 321% at a concentration of 63.27 µg/mL. Increased CD86 expression was observed starting at a concentration from 52.72 up to 188.91 µg/mL. In the second experiment, the expression of the cell surface marker CD86 was increased up to 457% at a concentration of 188.91 µg/mL. Increased CD86 expression was observed starting at a concentration of 52.72 µg/mL and 75.92 up to 188.91 µg/mL.

In the first experiment, the expression of the cell surface marker CD54 was increased up to 3879% at a concentration of 91.10 µg/mL. Increased CD54 expression was observed starting at a concentration from 52.72 up to 188.91 µg/mL. In the second experiment, the expression of the cell surface marker CD54 was increased up to 5066% at a concentration of 91.10 mg/mL. Increased CD54 expression was observed starting at a concentration from 52.72 up to 188.91 µg/mL.

Since the expression of both cell surface markers clearly exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.

The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (335% experiment 1; 355% experiment 2) and 200% for CD54 (367% experiment 1; 416% experiment 2) were clearly exceeded.

Conclusion: In this study under the given conditions the test item did upregulate the cell surface markers in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser in accordance with UN GHS category 1.

The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA. As the current study examines only one of the three key events which are part of the skin sensitisation adverse outcome pathway (AOP), the registrant concludes that further studies are required to draw definitive conclusions on the classification of the substance, and so the absolute necessity for classification as skin sensitizer is not given.

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