Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 09 August 2018 to 31 August 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Updated information. Study now available and provided ahead of extended submission date of 31/05/2019.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™. (Adopted March, 2018).

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin sensitisation tests is the KeratinoSens™ assay, which is recommended in international guidelines (e.g. OECD 442D).

Test material

Test material information

Constituent 1

Reference substance name:

Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

EC Number:

271-672-2

EC Name:

Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

Cas Number:

68603-74-7

Molecular formula:

The substance is a UVCB so there is no molecular formula

IUPAC Name:

Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

Test material form:

solid

In vitro test system

Details on the study design:

EXPERIMENTAL DESIGN

Test System
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, the cells were propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using an appropriate maintenance medium.

Exposure Medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Environmental Conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 79 – 99%), containing 5.0% CO2 in air in the dark at 37°C. Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Replicates
Two independent experiments were performed.

Test Substance Preparation and Vehicle
No correction was made for composition/purity of the test substance. The test substance was dissolved in ethanol to prepare a stock solution from which spike solutions were prepared for dilution into the relevant test cocnentrations.

Vehicle Control
The vehicle control was 1% DMSO for the positive control or 0.25% EtOH for the test item in exposure medium. Eighteen wells were tested per plate.

Positive Control
The positve control was Ethylene dimethacrylate glycol. It was prepared in DMSO and diluted in test medium final concentration range of 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.


Plating of Cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+13 in experiment 1 and P+4 in experiment 2.

Treatment of Cells
The medium was removed and replaced with fresh culture medium (150 µL culture medium containing serum but without Geneticin) to which 50 µL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37 ±1°C in the presence of 5% CO2. In total 2 experiments were performed.

Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment
For the KeratinoSens™ cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; and cells were incubated for 3 - 4 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Results and discussion

Positive control results:

The positive control, Ethylene dimethacrylate glycol, caused a dose related induction of luciferase activity and passed the acceptance criteria in both experiments.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Luciferase activity induction (fold increase)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: 0.94 fold increase

Results 2

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Luciferase activity induction (fold increase)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: 0.99 fold increase

Other effects / acceptance of results:

OTHER EFFECTS:
In experiment 1 precipitation was observed at test concentrations of 25 µg/mL and upwards at the start and end of the incubation period in the 96-well plates.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions used in the study.

Executive summary:

Introduction

The objective of this study was to evaluate the ability of N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens™ assay. The study was designed to meet the requirements of the following guidelines:

• OECD Guideline TG 442D. In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method. (Adopted June, 2018).

• EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™. (Adopted March, 2018).

Method

Two independent experiments were performed.

The test item was dissolved in ethanol at 80 mg/mL. From this stock 11 spike solutions in ethanol were prepared. The stock and spike solutions were diluted 400-fold in the assay resulting in test concentrations of 0.10 - 200 µg/mL (2-fold dilution series).The test item precipitated at dose concentrations of 25 µg/mL and upwards.  The highest test concentration was considered to be the limit of solubility. In the second experiment, a more detailed dose-response analysis was performed using a lower maximum concentration (13 µg/mL). 

Positive, negative and vehicle controls were also run.

 

Results

Both experiments passed the acceptance criteria.

The test item showed toxicity (IC₃₀ values of 0.5µg/mL and 0.84µg/mL and IC₅₀ values of 0.79µg/mL and 1.1 µg/mL in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 0.94-fold and 0.99-fold in experiment 1 and 2 respectively. The test itemis classified as negative in the KeratinoSens™ assay since negative results (<1.5-fold induction) were observed at test concentrations up to and beyond a concentration showing cytotoxicity.

Conclusion

In conclusion, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates is classified as negative(noactivation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions used in the study.