2-hydroxy-3-phenoxypropyl methacrylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - July 2018

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:

February 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:

2-hydroxy-3-phenoxypropyl methacrylate

EC Number:

240-994-5

EC Name:

2-hydroxy-3-phenoxypropyl methacrylate

Cas Number:

16926-87-7

Molecular formula:

C13H16O4

IUPAC Name:

2-hydroxy-3-phenoxypropyl 2-methylprop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
171512
- Expiration date of the lot/batch:
23 December 2019

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

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaVaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test.
Supplier: this cell line was provided by Givaudan
Batch: D1
Storage conditions: at -80ºC
Mycoplasm: absence of mycoplasm was confirmed

Results and discussion

Positive control results:

First run: all acceptance criteria were fulfilled for the positive and negative controls.
Second run: all acceptance criteria were fulfilled for the positive and negative controls. The criterion "the average induction (Imax) in the three replicate plates for the positive control at 64µM should be between 2 and 8" was not fulfilled (i.e. Imax = 9.65). However since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

62.99 µM

Cell viability:

viable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

49.38 µM

Cell viability:

viable

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

First run:
- no precipitate/emulsion was observed in an test item-treated wells at the end of the 48 hr period.
- a decrease in cell viability (<70%) was noted at concentrations >=1000µM
- the corresponding IC30 and IC50 were calculated to be 618.35 and 729.90µM, respectively
-statistically gene-fold inductions above the threshold of 1.5 were noted at concentrations >= 62.5 µM and up to 500µM with a dose-response
- The Imax was 21.91 and the calculated EC1.5 was 49.38µM

Second run:
- no precipitate/emulsion was observed in an test item-treated wells at the end of the 48 hr period.
- a decrease in cell viability (<70%) was noted at concentrations >=1000µM
- the corresponding IC30 and IC50 were calculated to be 595.33 and 825.59µM, respectively
-statistically gene-fold inductions above the threshold of 1.5 were noted at concentrations >= 125 µM and up to 500µM with a dose-response
- The Imax was 17.55 and the calculated EC1.5 was 62.99µM4

Any other information on results incl. tables

Results analysis

Data evaluation was performed using a validated Excel sheet. The generated raw data (luminescence data for the luciferase activity and absorbance data for the MTT test) were pasted into an Excel template, and all data procesing was performed automatically.

For the MTT and the luciferase data, the background value recorded in the empty well withour cells (blank) was substracted.

For MTT data, the % viability was calculated for each well in the test plate in relation to average of the six negative contol wells.

For the luciferase data, the average value of the six negative control wells was set to 1, and for each well in the plate, the fold induction was calculated in relation to this value.

For wells in which a statistically significant gene-induction (using a student test, also called T-test) over the 1.5 threshold was found, the following parameters were calculated from the processed raw data:

-I_max: maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured,

- EC_1.5: Concentration at which a 1.5 -fold luciferase gene induction is obtained,

- IC₅₀ and IC₃₀: concentrations effecting a reduction of cellular viability of 50% and 30%,

Indication whether significant 1.5 -fold gene induction occured below the IC₃₀

The data were plotted in graphs and the I_max and the EC_1.5 values were visually checked since uneven dose-response curves or large variation may lead to wrong extrapolations.

Also, the individual and overall geometric means IC₅₀ and IC₃₀ were calculated, when applicable.

Acceptance and Evaluation criteria

Acceptance criteria

Each run was considered valid if the following criteria were met:

- the positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations.

- the average EC_1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (I_max) in the three replicate plates for the positive control of 64 µM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of Cinnamic Aldehyde was carefully checked, and the run was accepted if there was a clear dose-response with increasing luciferase activity at increasing concentration for the positive control,

- the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be <20%.

Evaluation criteria of the test item

The results of each run are analyzed individually and if the test item is classified as positive in two runs, the final outcome is considered positive. If the test item is classified as negative in two runs, the final outcome is negative. In case, the first two runs were not concordant, a third run was performed and the final outcome was that of the two concordant runs.

The test item is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:

- I_max is > 1.5 -fold and statistically significantly different as compared to the negative control (as determined by a two-tail, unpaired Student's T-test),

- at the lower concentration with a gene induction> 1.5 -fold (i.e. at the EC_1.5 determining value), the cell viabillity is >70%,

- the EC_1.5 value is < 1000 µM (or < 200µg/mL for test item without MW),

- there is an appropriate overall dose-response for luciferase induction (or a reproducable biphasic response).

Results

Solubility Test

In the solubility test, the test item was found stable in DMSO at 200mM. Therefore this vehicle was selected for the preparation of the test item stock formulations.

No precipitate or emulsion was observed once the test item stock formulation was diluted in the treatment culture medium to a final concentration of 2000 µM.

KeratinoSens run (Appendices 2 and 3)

The I_max, IC₃₀, IC₅₀, EC_1.5 and viability values obtained for cells treated with the test item in each run as well as the mean and SD values are presented in Appendix 2. The viablity values (%), induction values, I_max, IC₃₀, IC₅₀ and EC_1.5 values obtained with the positive control are presented in Appendix 3. In addition the luminescence values of all negative control wells ant the %CV between these values for each run are also presented in Appendix 3.

First run

All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid.

This run was performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:

- no precipitate/emulsion was observed in any test item treated wells at the end of the 48-hour treatment period,

- a decrease in cell viability (i.e. cell viability <70%) was noted at concentration >= 1000µM,

- the coresponding IC30 and IC₅₀ were calculated to be 618.35 and 729.90 µM respectively,

- statistically gene-fold inductions above the threshold of 1.5 were noted at concentrations >= 62.5µM and up tp 500 µM with a dose-response,

- the Imax was 21.91 and the calculated EC_1.5 was 49.38 µM.

Second run

All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid. The citerion "the average induction (I_max) in the three replicate plates for the positive control at 64 µM should be between 2 and 8" was not fulfilled (i.e. I_max of 9.65). Howwever, since a clear dose-response with increasing luciferase activity at increasing concnetrations was obtained for the positive control, this was considered not the have any impact on the validity of the results of this run.

The same concentrations as thos in run 1 were used.

At these tested concentrations:

- no precipitate/emulsion was observed in ny test item-treated wells a the end of the 48 -hour treatment period,

- a decrease in cell viability (i.e. cell viability <70%) was noted at concentration >= 1000µM,

- the corresponding IC₃₀ and IC₅₀ were calculated to be 595.33 and 825.59 µM, respectively,

- statistically gene-fold inductions above the threshold of 1.5 were noted at concentrations of >125 µM and up to 500 µM with a dose-response,

- the I_max was 17.55 and the calculated EC_1.5 was 62.99 µM.

The evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the experimental conditions of this stuy, the test item, 3-phenoxy-2-hydroxypropyl methacrylate, was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.

Executive summary:

Summary

The objective of this study was to evaluate the potential of the test item, 3 -phenoxy-2 -hydroxypropyl methacrylate, to activate the Nrf2 transcription factor. This test is part of the tiered strategy for the evaluation of skin sensitiation potential. Thus, data generated with the present Test Guideline should be used to support the descrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

Principle

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherant HaCaT keratinocyte cell line. The KeratinoSens cell line is stabaly transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

Methods

The KeratinoSens cells were first plated on 96 -well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to difference concentraions of test item and of positive controls. The treated plates were then incubated for 48 hours at 37ºC. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescense. In parallel, the cytotoxicty was measured by MTT reduction test and was taken into consideration in the interpreatation of the sensitisation results. Two independant runs were performed.

Results

For each run, the test item was solubilised in DMSO at 200mM.

First run

All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid.

This run was performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.

At these tested concentrations:

- no precipitate/emulsion was observed in any test item-treated wells at the end of the 48 -hour treatment period,

- a decrease in cell viability (i.e. cell viability <70%) was noted at concnetrations >= 1000µM,

- the corresponding IC₃₀ and IC₅₀ were calculated to be 61.35 and 729.90 respectively,

- statistically gene-fold inductions above the threshold of 1.5 were noted at concentrations >= 62.5 µM and up to 500µM with a dose-response,

- the I_max was 21.91 and the calculated EC_1.5 was 49.38 µM

Second run

All acceptance critera were met for the positive and negative controls, this run was therefore considered to be valid. The criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8" was not fulfilled (i.e. Imax 9.65). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run.

The same concentrations as those in the run 1 were used.

At these tested concentrations:

- no precipitate/emulsion was observed in any test item-treated wells at the end of the 48 -hour treatment period,

- a decrease in cell viabilty (i.e. cell viability <70%) was noted at concentration >= 1000µM,

- the corresponding IC₃₀ and IC₅₀ were calculated to be 595.33 and 825.59 µM respectively,

- statistically gene-fold inductions above the threshold of 1.5 were noted at concentrations >=125 µM and up to 500µM with a dose-response,

- the I_max was 17.55 and the calculated EC_1.5 was 62.66 µM

Conclusion

Under the experimental conditions of this study, the test item, 3 -phenoxy-2 -hydroxypropyl methacrylate, was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.