N-butyl 4-oxopentanoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 13th to 25th, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

adopted June 25th, 2018

Deviations:

yes

Remarks:

the reactivity check (RC) for the THP-1 cells used in this study wasn´t performed 2 weeks, but 4 weeks after thawing.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

N-butyl 4-oxopentanoate

EC Number:

218-143-4

EC Name:

N-butyl 4-oxopentanoate

Cas Number:

2052-15-5

Molecular formula:

C9H16O3

IUPAC Name:

butyl 4-oxopentanoate

In vitro test system

Details on the study design:

NON-GLP PRE-TESTS
The solubility of the test item was determined in a non-GLP pre-test in RPMI 1640 (100 mg/ml) and DMSO (500 mg/ml). The test item is soluble in DMSO at a concentration of 500 mg/ml but insoluble in RPMI1640 at a concentration of 100 mg/ml. Therefore, DMSO was used as solvent. A possible autofluorescence of the test item was determined using a 2475 Multi-λ Fluores-cence Detector and an excitation wavelength of 488 ± 5 nm. No emission was detected between 500 - 700 nm. Therefore, it is assumed that the test item has no influence on the result of the assay due to autofluorescence.

PREPARATION
First, a stock solution containing 500 mg/ml test item in DMSO was prepared. Subsequent dilutionl finally yielded a maximum concentration of 1000 µg/ml during treatment. The stock solution is used to prepare a geometric series of 7 dilutions (factor 2). Afterwards all concentrations were further diluted (1:250 fold) in culture medium (working solutions). Another 1:2 dilution was achieved by adding 1 part of each test item concentration to 1 part of the cell suspension in the treatment plate. In the end, the total dilution factor was then 1:500. The stock solution as well as the dilutions were freshly prepared on the day of treatment.

CONTROLS
In accordance to the OECD 442E the controls were tested at only one concentration. All stock solutions were prepared freshly on the day of treatment. The selected concentrations were given by the OECD 442E guideline
Medium Control: complete culture medium was used.
Solvent Control for the test item and positive control DNCB: Dimethyl sulfoxide (DMSO); purity: 99.5 %; final concentration:0.2 % in culture medium; to achieve this concentration, the dilution of the solvent was performed in the same way as for the test item
Positive Control: 2,4-dinitrochlorobenzene (DNCB); solvent: DMSO; final concentration: 4 µg/ml; for the experiment, a stock solution of 2 mg/ml of DNCB in DMSO was prepared and further diluted (1:250 fold) in complete culture medium (working solution). The working solution was afterwards diluted (1:2 fold) with the cells suspension to achieve the final test concentration of 4 µg/ml.

TEST SYSTEM
Reasons for the Choice of the THP-1 Cell Line: the OECD 442E indicates that the human monocytic leukaemia cell line, THP-1 should be used for the h-CLAT.
Cell Cultures: THP-1 cells are stored in liquid nitrogen in the cell bank to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproduci-ble characteristics of the cells. The THP-1 cells are routinely seeded every 2-3 days at the density of 0.1 – 0.2 * 10^6 cells/ml. They were maintained at densities from 0.1 to 1.0 * 10^6 cells/ml. Prior to using them for testing, the cells were qualified by conducting a reactivity check. For the pre-test 2 cells of passage 19 were used. For the main experiments cells of passage 20 were used. After thawing the cells were cultivated in RPMI 1640 complete culture medium in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
Reactivity Check: four weeks after thawing, a reactivity check of the cells was performed. For that, the two positive controls 2,4-dinitrochlorobenzene (DNCB) (CAS n. 97-00-7, ≥ 99% purity, test concentration: 4 µg/ml) and nickel sulfate (NiSO4) (CAS n. 10101-97-0, ≥ 99% purity, test concentration: 200 µg/ml) as well as the negative control, lactic acid (LA) (CAS n. 50-21-5, ≥ 85% purity, test concentration: 1000 µg/ml) were used. These substances as well as all additional information are given by the OECD 442E. The experimental procedure was identical to the experiments in this study. The two positive controls produced a clearly positive result of the two surface markers CD86 and CD54. The negative control produced a clearly negative result of both surface markers. Therefore, the cells were found to be suitable for the experiments. For the pre-test as well as the experiments only cells which have successfully passed the reactivity check were used.
Prior to use in the pre-test and the experiments, the proficiency of the cells was demonstrated in a reactivity check. Only the cells which passed the reactivity check were used for the pre-test and the experiments. The runs for Experiment I and II were performed on the same day, provided that for each run: a) independent fresh stock solutions and working solutions of the test item and antibody solutions were prepared and b) independently harvested cells were used (cells came from the same passage and were collected from different culture flasks)
 
PRE-TEST
A preliminary cytotoxicity test was performed to determine the concentrations to be used for the CD86/CD54 expression measurement in the main experiments. The following 8 concentrations of the test item were tested with an incubation time of 24 h in the pre-test: 1000 µg/ml, 500 µg/ml, 250 µg/ml, 125 µg/ml, 62.5 µg/ml, 31.3 µg/ml, 15.6 µg/ml and 7.8 µg/ml. For that 1.6 * 10^5 cells / well of a 96-well plate were exposed to each concentration of the test item for 24 h at 37.0 ± 1.0 °C and 5.0 ± 0.5 % CO2. During treatment the plate is closed with a sealing tape to avoid cross-contamination. Following treatment, the cells were transferred into sample tubes and centrifuged for 5 min at 250 * g. Afterwards the supernatant was removed and the remaining cells were suspended in 200 µl staining buffer and then 200 µl were transferred into one well of a 96-well plate. After that, the cells were washed three times with staining buffer (centrifugation: 250 * g for 5 min). After that, the cells were resuspended in 150 μl staining buffer and 7.5 µl PI solution (12.5 µg/ml) were added to achieve a final concentration of 0.595 µg/ml PI. The plate was incubated for 15 min on ice and 5 minutes at room temperature in the dark. Af-terwards, the PI uptake was analysed by flow cytometry with the acquisition channel PerCP (corresponds to the FL-3 channel of the predecessor model). In total 10 000 (± 10 %) viable cells (PI negative) were acquired. The cell viability was automatically calculated by the flow cytometer. Based on these results, the concentrations for the main experiments were defined.
None of the controls induced a cytotoxic effect. The cell viabilities of medium and solvent controls were higher than 90 %. Therefore, the pre-test 2 was valid. None of the tested concentrations induced a cytotoxic effect with DMSO as solvent
- Exposure Concentrations and Dose Selection
Since no CV75 could be determined (no reduction of viability below 75 %) in the pre-test 2, and the solvent of the test item is DMSO, the maximal test item concentration in the experiments was 1000 µg/ml. To achieve the highest test item concentration, a 500 x stock solution in DMSO was pre-pared and diluted (geometric series of 7 dilutions; factor 1.2).

MAIN TEST-EXPERIMENTS I AND II
The performance of experiment I and II was identical. Eight test item concentrations were tested in each experiment (279.1 µg/ml, 334.9 µg/ml, 401.9 µg/ml, 482.3 µg/ml, 578.7 µg/ml, 694.4 µg/ml, 833.3 µg/ml, 1000.0 µg/ml). 1 * 10^6 cells / well of a 24-well plate were exposed to each concentration of the test item for 24 h ± 0.5 h at 37.0 ± 1.0 °C and 5.0 ± 0.5 % CO2. During treatment the plates were closed with a sealing tape to avoid cross-contamination. Following treatment, the cells were transferred into sample tubes, collected by centrifugation (250 * g for 5 min) and then washed twice with 1 ml staining buffer. After that, the cells were resuspended in 600 µl blocking solution and incubated on ice for 15 min. Then, 180 µl of the cell suspension were added in three wells of a 96-well round bottom plate respectively. The plates were centrifuged at 250 * g for 5 minutes and the supernatant was discarded. After that, 50 µl of diluted FITC-labelled antibodies (anti-CD86, anti-CD54 or mouse IgG1) were added in one of the three wells respectively and the plate was incubated on ice for 30 min in the dark. Before use, the antibodies were diluted 3:25 v/v for CD86, 3:50 v/v, for CD54 and IgG1 with staining buffer. After the incubation on ice, the cells were washed 3 times with 200 µl staining buffer (centrifugation: 250 * g for 5 min) before 150 µl staining buffer as well as 7.5 µl PI working solution (12.5 μg/ml, final concentration: 0.595 μg/ml) were added to each well. Afterwards the plate was incubated for 15 min on ice and 5 min at room temperature in the dark before measurement at the flow cytometer (BD FACS LyricTM). FITC-Detection: (Ex-citation: 488 nm / Detection filter 527/32; corresponding to the FL-1 channel). In total 10000 viable cells (PI negative) were acquired and analysed. The viability was directly measured and calculated by the flow cytometer and is given as % values.

Results and discussion

Positive control results:

RFI of CD86 = 501 % (Exp.II); 443 (Exp.II)
RFI of CD54 = 346 % (Exp.I); 379 (Exp.II)

In vitro / in chemico

Results

Results 1

Run / experiment:

other: other: Experiment I and Experiment II

Parameter:

other: RFI of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A calculation of the EC150 was not possible since none of the RFI values was above 150 (for CD86) at any of the tested concentrations.

Results 2

Run / experiment:

other: other: Experiment I and Experiment II

Parameter:

other: RFI of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A calculation of the EC200 was not possible since none of the RFI values was above 200 at any of the tested concentrations.

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: prior to routine use, the validity of the h-CLAT test was demonstrated in a proficiency study. 12 proficiency substances were selected to represent the range of re-sponses for skin sensitisation hazards. The expected h-CLAT prediction as well as the reference range were correctly obtained for 10 substances. All values fell within the re-spective reference range (CV75, EC150, EC200). Therefore, the proficiency of the test system was demonstrated.

VALIDITY CRITERIA
The assay is considered acceptable if it meets the following criteria:
- The cell viabilities of medium and solvent/vehicle controls are higher than 90%.
- In the solvent/vehicle control, RFI values of both CD86 and CD54 do not exceed the positive criteria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %).
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to iso-type control should be > 105%.
- In the positive control, RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %) and cell viability should be more than 50 %.
- For the test item, the cell viability should be more than 50 % in at least four tested concentrations in each run.
If any of these criteria is not met, the experiment is considered not valid and needs to be repeated.
Negative results are acceptable only for test items exhibiting a cell viability of less than 90 % at the highest concentration tested. If the cell viability at 1.2 × CV75 is equal or above 90 % the negative result should be discarded. In such a case another Pre-test has to be performed to refine the dose selection. It should be noted that when 5000 µg/ml in PBS or medium, 1000 µg/ml in DMSO or the highest soluble concentration is used as the maximal test concentration of a test item, a negative result is acceptable even if the cell viability is above 90 %.
All validity criteria were met. Therefore, the study is considered as valid.

- Range of historical values if different from the ones specified in the test guideline: for all control substances historical data are available, which demonstrate the reliability and the validity of those substances.

Deviations from the Guideline
The following deviations from the guideline were observed: the reactivity check (RC) for the THP-1 cells used in this study wasn´t performed 2 weeks, but 4 weeks after thawing. Anyway, the RC was performed at least 2 weeks after thawing, the cells didn’t have a higher passage than 30 and were no longer in culture than 2 months. Therefore, this deviation is uncritical and doesn’t have any influence on the results of this study.

Deviations from the Study Plan
The following deviations from the study plan were observed:
1)The reactivity check (RC) for the THP-1 cells used in this study wasn´t performed 2 weeks, but 4 weeks after thawing. Anyway, the RC was performed at least 2 weeks after thawing, the cells didn’t have a higher passage than 30 and were no longer in culture than 2 months. Therefore, this deviation is uncritical and doesn’t have any influence on the results of this study.
2) In the first pre-test the stock solution was used to prepare a geometric series of 7 dilutions – the dilution factor was 1.2 instead of 2. This deviation was uncritical for the study and didn´t influence the results, because the first pre-test was repeated.

Applicant's summary and conclusion

Interpretation of results:

other: negative in the h-CLAT and is therefore considered not to have the potential to activate dendritic cells

Conclusions:

negative in the h-CLAT and is therefore considered not to have the potential to activate dendritic cells

Executive summary:

The sensitising potential of the test item was assessed in the in vitro assay by quantifying changes in the expression level of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells. The test was performed according to the OECD Guideline 442E. Two valid experiments with a treatment period of 24 hours were performed.

In the experiments, the highest nominal applied concentration (1000 µg/ml) was chosen based on the results obtained in the pre-test. A geometric series (factor 1.2) of 7 dilutions thereof was prepared and tested. As solvent control for the test item, DMSO was used in a final concentration of 0.2 % in culture medium. As positive control, 2,4-dinitrochlorobenzene (DNCB) was used. Prior to the study, the cells used in the experiments were checked in a reactivity check and were found to be suitable for the experiments. All acceptance criteria were met and therefore, the study was considered valid.

In both experiments all of the tested concentrations of the test item didn´t show any cytotoxicity. The RFI of CD86 was not ≥ 150 % as well as the RFI of CD54 was not ≥ 200 % at any tested concentration. Therefore, in accordance with the classification criteria the result of this study is negative.

In conclusion, it can be stated that under the experimental conditions of this study, the test item, was negative in the h-CLAT and is therefore considered not to have the potential to activate dendritic cells.