Reaction mass of 3,4,5-trichlorophthalic acid, strontium salt and 3,4,6-trichlorophthalic acid, strontium salt and 3,4-dichlorophthalic acid, strontium salt and 3,5-dichlorophthalic acid, strontium salt and 3,6-dichlorophthalic acid, strontium salt and 3-chlorophthalic acid, strontium salt and 4,5-dichlorophthalic acid, strontium salt and 4-chlorophthalic acid, strontium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-10-02 to 2019-11-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. Therefore, the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Test material

Test material information

Constituent 1

Reference substance name:

Strontium dichlorophthalate

EC Number:

304-292-3

EC Name:

Strontium dichlorophthalate

Cas Number:

94248-20-1

Molecular formula:

C8H4Cl2O4.Sr

Test material form:

solid

Details on test material:

Strontium phthalate (HPLC): 0.51 w/w %
Strontium 3-chlorophthalate (HPLC): 0.73 w/w %
Strontium 3,6-dichlorophthalate (HPLC) / Strontium 4-chlorophthalate (HPLC): 10.59 w/w %
Strontium 3,4-dichlorophthalate (HPLC): 20.56 w/w %
Strontium 3,5-dichlorophthalate (HPLC): 0.66 w/w %
Strontium 3,4,6-trichlorophthalate (HPLC): 0.18 w/w %
Strontium 4,5-dichlorophthalate (HPLC): 60.59 w/w %
Strontium 3,4,5-trichlorophthalate (HPLC): 4.39 w/w %
Strontium tetrachlorophthalate (HPLC): 0 w/w %

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Dynamit Nobel GmbH Explosivstoff- und Systemtechnik; Batch: WE: 50352354
- Expiration date of the lot/batch: 28 November 2019
- Purity test date: 2 September 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction was made for the composition/purity of the test item. In the main experiments the test item was suspended in DMSO at 200 mM (white suspension). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate.

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study 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, Switzerland). Upon receipt, cells are 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 the appropriate maintenance medium.

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

CELL CULTURE

Basic medium:
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).

Maintenance medium:
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 μg/mL).

Exposure medium:
Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal 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 51 – 94 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 – 36.7°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.

SUBCULTURING

Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock and were not cultured for more than 25 passages from the frozen stock (P+25).

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 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+9 in experiment 1 and P+13 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±1.0°C in the presence of 5% CO2. In total 2 valid 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 5 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 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; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37°C± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) 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 the luciferase activity. The Imax was 2.22 and the EC1.5 55 μM in experiment 1 and in experiment 2 the Imax was 2.24 and the EC1.5 75 μM.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Mean of Experiment 1 and 2

Parameter:

other: max. luciferase activity (Imax) induction

Value:

1.23

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: max. luciferase activity (Imax) induction

Value:

1.2

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No significant luciferase induction >1.5-fold in tested concentration range; no EC1.5 value could be calculated

Results 3

Key result

Run / experiment:

other: experiment 2

Parameter:

other: max. luciferase activity (Imax) induction

Value:

1.26

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No significant luciferase induction >1.5-fold in tested concentration range; no EC1.5 value could be calculated

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Two independent experiments were performed. The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 2000 μM (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.
For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.
The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC1.5 determining concentration.

ACCEPTANCE OF RESULTS:
Both tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was within two standard deviations of the historical mean (55 μM and 75 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.22-fold and 2.24-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (12% in experiment 1 and 2).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

For individual results see tables in box "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Overview Luminescence Induction and Cell Viability of Strontium dichlorophthalate in Experiment 1 and 2

Concentration (µM)	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Exp 1 luminescence	1.13	1.20	1.14	1.16	1.12	1.11	1.10	0.99	0.93	0.91	0.89	0.67
Exp 1 viability (%)	110.2	101.5	97.2	99.8	98.5	96.8	94.7	97.2	90.9	84.3	72.6	54.1
Exp 2 luminescence	1.22	1.26	1.06	1.15	1.16	1.15	1.01	0.88	0.92	0.74	0.59	0.56
Exp 2 viability (%)	112.6	113.3	107.2	105.3	111.9	108.4	104.1	97.9	96.7	86.1	78.2	64.0

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

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	1.09	1.18	1.28	1.57***	1.85***	2.22***
Exp 1 viability (%)	112.6	108.6	102.3	104.2	99.4	98.6
Exp 2 luminescence	1.13	1.15	1.20	1.44	1.74***	2.24***
Exp 2 viability (%)	115.7	122.6	115.8	108.2	106.2	95.3

*** p<0.001 Student’s t test

Table 3: Overview EC_1.5, I_max, IC₃₀and IC₅₀Values

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test item Experiment 1	NA	1.20	1140	NA
Test item Experiment 2	NA	1.26	1576	NA
Pos Control Experiment 1	55	2.22	NA	NA
Pos Control Experiment 2	75	2.24	NA	NA

NA = Not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Test item is considered a non-sensitiser

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, test item can be considered to be a non-sensitizer.

Executive summary:

In a dermal sensitisation study conducted according to OECD 442D with strontium dichlorophthalate in DMSO, the sensitisation potential of the test item was assessed on the basis of the activation of keratinocytes using the in vitro KeratinoSens™. Cells were incubated with the test item for 48 h at 37 °C and later checked for luciferase activity.

Sensitisation was scored by measuring maximum luciferase activity induction (Imax), cell viability and EC1.5. For both Experiment 1 and 2, Imax was less than a 1.5 fold increase and no biologically relevant induction of the luciferase activity was measured at any of the test concentrations in both experiments, thus the EC1.5 could not be calculated.

Therefore, in this study, the test item strontium dichlorophthalate is considered to be a skin non-sensitiser.