Adenosine 5'-(trihydrogen diphosphate), monopotassium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 May - 2 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Adenosine 5'-(tetrahydrogen triphosphate), disodium salt

EC Number:

213-579-1

EC Name:

Adenosine 5'-(tetrahydrogen triphosphate), disodium salt

Cas Number:

987-65-5

Molecular formula:

C10H14N5O13P3.2Na

IUPAC Name:

disodium 9-{5-O-[hydroxy({[(hydroxyphosphinato)oxy]phosphinato}oxy)phosphoryl]pentofuranosyl}-9H-purin-6-amine

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

impurity 2

Reference substance name:

Unknown impurities.

Molecular formula:

Not available as unknown impurities.

IUPAC Name:

Unknown impurities.

Test material form:

solid: crystalline

Details on test material:

Storage conditions: 2-8°C
Batch No: 11678500

In vitro test system

Details on the study design:

- Test concentrations: final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.977 µM (final concentration vehicle DMSO of 1%)
- All concentrations of the test item were tested in triplicate.
- Positive control: Ethylene dimethacrylate glycol, final concentration 7.81 to 250 µM (final concentration DMSO of 1%)
- Negative control: vehicle: 1% DMSO in exposure medium

- 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, Switzerland).

- Cell culture:
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Manteinance Medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/ml).
Exposure medium: Dulbecco’s minimal 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 63 – 86%), containig 5.0 ± 0.5% CO2in air in the dark at 37.0 ± 1.0°C (actual range 36.7 - 37.4 °C).

EXPERIMENTAL DESIGN
- 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 test item, one plate was used for the luciferase activity measurements, and one parallel plate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was 25 in experiment 1 and 16 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 substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0 oC 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 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 luminometer to assess the quantity of luciferase (integration time one second).
- Cytotoxicity assessment
For the KeratinoSensTM 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 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 with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 µM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
- Data analysis
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

- Data interpretation
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 µM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Results and discussion

Positive control results:

• Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.69 and the EC1.5 13.7 µM.
• Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.23 and the EC1.5 46.0 µM.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

Imax [442D]

Value:

7.53

Positive controls validity:

valid

Remarks:

Imax: 2.69

Remarks on result:

other: maximal average fold induction of luciferase activity (Imax): 7.53 fold

Results 2

Key result

Run / experiment:

other: 2

Parameter:

Imax [442D]

Value:

6.18

Positive controls validity:

valid

Remarks:

Imax: 4.23

Remarks on result:

other: maximal average fold induction of luciferase activity (Imax): 6.18 fold

Results 3

Key result

Run / experiment:

other: 1

Parameter:

EC 1.5 [442D]

Value:

45.3 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

EC1.5: 13.7 µM

Results 4

Key result

Run / experiment:

other: 2

Parameter:

EC 1.5 [442D]

Value:

41 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

EC1.5 46.0 µM

Other effects / acceptance of results:

Acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was between 5 and 125 µM (13.7 µM and 46.0 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold in experiment 2 (4.23-fold). In experiment 1 the induction at 250 µM was with 1.98 just below 2 but a dose response was observed and the maximum induction was with 2.69 (125 µM) clearly above 2-fold.
• Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (9.1% and 9.6% in experiment 1 and 2, respectively).

Any other information on results incl. tables

ATP, Di-Na (CAS No 987-65-5) showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 45.3 and 41.0 µM in experiment 1 and 2, respectively) was measured in both experiments.  The maximum luciferase activity induction (Imax) was 7.53-fold and 6.18-fold in experiment 1 and 2 respectively.  ATP, Di-Na (CAS No 987-65-5) is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations of <1000 µM with a cell viability of >70% compared to the vehicle control.

Overview Luminescence Induction and Cell Viability of ATP, Di-Na in Experiment 1 and 2

Concentration (µM)	0.977	1.95	3.91	7.81	15.6	31.3	62.5	125	250	500	1000	2000
Exp 1 luminescence	0.70	0.83	0.83	1.02	1.25	1.20	1.87	2.77^**	4.03^***	7.40^***	7.53^***	5.16^***
Exp 1 viability (%)	100.4	94.1	90.8	81.2	83.3	95.7	83.3	95.5	87.0	77.4	67.2	72.1
Exp 2 luminescence	0.89	0.94	1.05	1.12	1.15	1.27	2.01^*	2.38^**	3.59^***	6.18^***	5.81^***	5.63^***
Exp 2 viability (%)	97.7	90.6	87.8	82.7	81.2	88.6	103.7	102.0	105.2	96.6	88.7	89.7

^*p< 0.05;^**p<0.01;^***p<0.001 Students t test

Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.81	15.6	31.3	62.5	125	250
Exp 1 luminescence	1.11	1.63^***	1.95^***	2.36^***	2.69^***	1.98^***
Exp 1 viability (%)	99.3	90.1	97.5	102.1	106.4	101.9
Exp 2 luminescence	0.95	1.03	1.33	1.69^***	2.60^***	4.23^***
Exp 2 viability (%)	97.6	103.0	108.8	121.7	132.0	135.2

^***p<0.001 Students t test

Overview EC_1.5, I_max, IC₃₀and IC₅₀Values

	EC1.5 (µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	45.3	7.53	NA	NA
Test item Experiment 2	41.0	6.18	NA	NA
Pos Control Experiment 1	13.7	2.69	NA	NA
Pos Control Experiment 2	46.0	4.23	NA	NA

Applicant's summary and conclusion

Interpretation of results:

other: activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes

Remarks:

Study will be used for classificatin in combination with other studies (Weight of Evidence)

Conclusions:

In conclusion, ATP, Di-Na (CAS No 987-65-5) is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of ATP, Di-Na (CAS No 987-65-5) to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 11678500 of ATP, Di-Na was a white solid with a purity of 89.2 % m/m.  

ATP, Di-Na was suspended in dimethyl sulfoxide at 200 mM. From the 200 mM stock 11 spike solutions in dimethyl sulfoxide were prepared. At concentrations of 6.25 mM and higher in experiment 1 and at 3.13 mM and higher in experiment 2, ATP, Di-Na formed a suspension in dimethyl sulfoxide whereas at 3.13 mM and lower in experiment 1 and at 1.56 mM and lower in experiment 2 it was fully soluble.  The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.977 – 2000 µM (2-fold dilution series).  The test item showed no precipitation at any of the test concentration.  Two independent experiments were performed.

Both experiments passed the acceptance criteria:

-The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.

-The EC1.5 of the positive control was between 5 and 125 µM (13.7 µM and 46.0 µM in experiment 1 and 2, respectively).  A dose response was observed and the induction at 250 µM was higher than 2-fold in experiment 2 (4.23-fold). In experiment 1 the induction at 250 µM was with 1.98 just below 2 but a dose response was observed and the maximum induction was with 2.69 (125 µM) clearly above 2-fold.

-Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (9.1% and 9.6% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

ATP, Di-Na showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 45.3 and 41.0 µM in experiment 1 and 2, respectively) was measured in both experiments.  The maximum luciferase activity induction (Imax) was 7.53-fold and 6.18-fold in experiment 1 and 2 respectively.  ATP, Di-Na is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations of =1000 µM with a cell viability of >70% compared to the vehicle control.

In conclusion, ATP, Di-Na (CAS No 987-65-5) is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.