2-hydroxy-3-phenoxypropyl methacrylate

Endpoint summary

Description of key information

Testing was performed in accordance with the requirements for skin sensitisation testing (11 Oct 2016). THe following studies are submitted:

- DPRA Assay (OECD 442C)

- KeratinoSens (OECD 442D)

- Local Lymph Node Assay (OECD 429)

All studies are performed under the conditions of GLP.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

June - July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

no

Principles of method if other than guideline:

Deciations to the study plan:
Due to technical reasons, the temperature values (maximum of 25.7°C) outside the expected range of 19-25°C and relative humidity values (minimum of 27% and maximum of 80%) outside the expected range of 30-70% were recorded occasionally times during the study. The observed differences in the environmental parameters were considered not to adversely affect the results or integrity of the study.

Due to unscheduled delay of reporting the Draft Report issued later than indicated in the Study Plan. This fact had no impact on the study on the results or integrity of the study.

To minimise animal use, the positive control group was part of a concurrent study where the ear thickness and biopsy weights were measured, therefore the ear thickness and biopsy weights were determined in case of positive control group, however this data is not reported, but all data are kept in the raw data binder. This fact had no impact on the study on the results or integrity of the study.

Due to technical reasons the measurement of the samples was completed later than indicated in the Study Plan. Based on the half-life of the 3H isotope (over 12 years), this fact would not influence the activity of the samples in a measurable manner. This fact was considered not to adversely affect the results or integrity of the study.

Due to technical reasons the incubation time was longer than indicated in the Study Plan. This fact was considered not to adversely affect the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Name: PHPM
Batch/Lot number: 171512
CAS number: 16926-87-7
Appearance: Light yellow to slightly amber liquid
Purity*: 97.9 %
Expiry date: 23 December 2019
Storage conditions: Room temperature (15-25°C, ≤70 RH%), protected from humidity (tight closed container).
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 12 weeks (age-matched within 1 week)
- Weight at study initiation: 19.7 – 22.7 grams (The weight variation in animals in the study did not exceed +/- 20 % of the mean weight.)

- Housing: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II polypropylene / polycarbonate
Bedding/Nesting: Bedding and certified nest building material was available to animals during the study

- Diet (e.g. ad libitum):
Animals received ssniff® SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable “Complete feed for Rats and Mice – Breeding and Maintenance” (Batch number: 883 29966, Expiry date: October 2018 and Batch number: 79746230, Expiry date: August 2019) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), and Gel diet Transport (Batch Number: 60181080030101, Expiry date: 19 April 2019 and Batch Number: 60191350010101, Expiry date: 15 May 2020), Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France) ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Water (e.g. ad libitum):
Animals received tap water from the municipal supply from 500 mL bottles, ad libitum. Water quality control analysis was performed at least once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8200 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at Charles River Laboratories Hungary Kft.
- Acclimation period: 35 days
- Indication of any skin lesions: None identified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-25.7°C
- Humidity (%): 27 - 80 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

The highest achievable concentration based on the regulatory requirements of OECD 429 and the physical characteristics of the test item was 100% (undiluted). The formulations at 50% (w/v) 25% and 2% (w/v) in AOO was also suitable for treatment. The formulations appeared to be solution by visual examination.

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:

- Irritation:
Preliminary Irritation/Toxicity Test

The Preliminary Irritation/Toxicity Test was started according to the Study Plan on
CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of
100% (undiluted) and 50%(w/v) in AOO , plus 2% (since it was significantly below the highest available) was tested (2 animals/dose).

The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.

The maximum concentration of test item in an acceptable vehicle was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 100% (undiluted).

In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored.

- Systemic toxicity:
Marked body weight loss (>5% reduction of body weight) was observed in one animal in the 2% (w/v) dose group, therefore the mean body weight of this group was above the limit, however no marked body weight loss (>5% reduction of body weight) was observed on the other animal of this dose group. Furthermore, no marked body weight loss (>5% reduction of body weight) was observed in the animals of the higher dose groups (100% (undiluted) and 50% (w/v) dose groups) in the preliminary test. It is considered that the body weights were no adversely affected by treatment.
The draining auricular lymph nodes of the animals were visually examined:
they were larger than normal in all animals of the 100% (undiluted) and in one animal of the 50%(w/v) dose group, they were slightly enlarged than normal on the other animal of the 50% (w/v) dose group and one animal of the 2% (w/v) dose group, they were normal in the other animal of the 2% (w/v) dose group (subjective judgement by analogy with observations of former experiments).


- Ear thickness measurements:
Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6. The ear thickness values and ear punch weights were within the acceptable range in all animals.

- Erythema scores:
Very slight erythema (score 1) was observed in all animals of the 100% (undiluted) dose group on Day 3.

Based on the in vitro results and the observations (lymph node size) of the preliminary experiment, positive results were expected in the 50% (w/v) concentration. Based on the available information 50% (w/v) was the highest test concentration in the main study. In this case, where the 1A vs 1B classification is probably required, a dose concentration of 2% was included in the selected test concentrations to allow full classification without use of additional animals, for animal welfare reasons.

MAIN STUDY
During the preliminary experiment and the main study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:

Evaluation of Results
The number of radioactive disintegrations per minute (DPM) was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.

Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

Interpretation of Results


The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
 

Main study

Clinical Observations


During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
Both ears of each mouse were observed for erythema and scored using Table 2 [1,2,3]. Individual records were maintained.


Measurement of Body Weight


Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of 0.1 g.
 


Ear Thickness Measurements


The positive control group was a part of a concurrent study where ear thickness was measured in the study using a thickness gauge and additional quantification of the ear thickness was also performed by ear punch weight determination.

Key result

Parameter:

EC3

Value:

5.3

Test group / Remarks:

All groups

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

PHPM 2% (w/v) in AOO

Key result

Parameter:

SI

Value:

6.2

Test group / Remarks:

PHPM 25% (w/v) in AOO

Key result

Parameter:

SI

Value:

12.4

Test group / Remarks:

PHPM 50% (w/v) in AOO

Cellular proliferation data / Observations:

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group. Larger than normal lymph nodes were observed in the 50% (w/v) dose group. Normal lymph nodes were observed in the 25% and 2% (w/v) dose group. Larger than normal lymph nodes were observed in the positive control group.

BODY WEIGHT MEASUREMENT
Marked body weight loss (≥5%) was observed for one animal of the 25% (w/v) dose group. However, the mean body weight changes of these groups were within the acceptable range and therefore considered as individual variability.
No evidence of test item related effects observed on the mean body weight changes in the main study.

Individual Body Weights for all Animals with Group Means

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change^# (%)
8141	1	Negative (vehicle) control	22.6	23.0	1.8
8153	2	AOO	21.3	22.5	5.6
8140	3		20.4	21.3	4.4
8147	4		19.7	18.9	-4.1
		Mean	21.0	21.4	1.9
8150	5	PHPM	21.9	22.4	2.3
8148	6	50% (w/v in AOO	21.3	20.8	-2.3
8144	7		20.0	19.5	-2.5
8139	8		20.8	21.6	3.8
		Mean	21.0	21.1	0.3
8156	9	PHPM	22.7	21.0	-7.5
8143	10	25% (w/v) in AOO	21.8	23.0	5.5
8155	11		20.3	21.2	4.4
8152	12		20.6	19.7	-4.4
		Mean	21.4	21.2	-0.5
8146	13	PHPM	22.5	22.7	0.9
8157	14	2% (w/v) in AOO	21.0	22.1	5.2
8154	15		20.3	20.2	-0.5
8149	16		21.0	19.6	-6.7
		Mean	21.2	21.2	-0.3
8151	17	Positive control	22.7	22.3	-1.8
8142	18	25% (w/v) HCA	20.7	20.1	-2.9
8158	19	in AOO	19.9	20.0	0.5
8145	20		19.8	19.1	-3.5
		Mean	20.8	20.4	-1.9

Notes:

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

DPM. DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5% (w/v) TCA)	36 38	-	-	-	-
Negative control (AOO)	4844	4807.0	8	600.9	1.0
PHPM 50% (w/v) in AOO	59760	59723.0	8	7465.4	12.4
PHPM 25% (w/v) in AOO	29964	29927.0	8	3740.9	6.2
PHPM 2% (w/v) in AOO	4822	4785.0	8	598.1	1.0
Positive control (25% (w/v) HCA in AOO	69044	69007.0	8	8625.9	14.4

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Remarks:

The test item was liquid, which was formulated in AOO for the main test. Since there were no confounding effects of excessive irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these exaggerated test conditions were considered as good evidence that PHPM is a sensitizer. Based on the results, the test item is classified as Category 1 (sub-category 1B) according to the GHS or CLP. The EC3 value of PHPM is 5.3% (w/v).

Conclusions:

In conclusion, under the conditions of the present assay, PHPM, tested in a suitable vehicle, was shown to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay. The EC3 value of PHPM is 5.3% (w/v).

The following classification/labelling is triggered:
Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017: Category 1 (sub-category 1B).

Executive summary:

 

The aim of this study was to determine the skin sensitisation potential of
PHPM following dermal exposure in mice. The study is being performed with vertebrate animals as the applied regulatory in vitro alternative tests indicated a positive result, but did not allow full regulatory classification. Therefore, an in vivo study is being run to provide reliable information about the skin sensitisation potential of the test item for regulatory acceptance. In this case, where the 1A vs 1B classification is required, a dose concentration of 2% was included in the selected test concentrations.

 

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline [1], the best vehicle for the test item was AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration was 100% (undiluted). The formulations at 50% (w/v), 25% (w/v) and 2% (w/v) in AOO were also suitable for treatment. The formulations appeared to be solutions by visual examination.

 

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose) 100% (undiluted), 50% and 2% (w/v) in AOO. Based on the observations recorded in the preliminary test, the 50% (w/v) dose was selected as top dose for the main test.

 

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

-    three groups received PHPM (formulated in AOO) at 50% (w/v), 25% (w/v) and 2% (w/v) concentrations,

-    the negative control group received the vehicle (AOO) only,

-    the positive control group received 25 % (w/v) HCA (dissolved in AOO).

 

The test item formulations were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring the incorporation of tritiated methyl thymidine (³HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

 

No mortality or systemic toxicity was observed during the main study. No test item residue was observed on the ears of all animals. Erythema (score 1) was observed in three animals at 50% (w/v) and in two animals at 25% (w/v) on Day 3. No test item related effects were observed on the mean body weight changes in the main study.

 

The stimulation index values were 12.4, 6.2 and 1.0 at concentrations of 50% (w/v),
25% (w/v) and 2% (w/v), respectively.

 

 

 

The result of the positive control substance (a-Hexylcinnamaldehyde, HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay [1]. A lymphoproliferative response (SI = 14.4) in harmony with historical control data was noted for the positive control chemical, this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay, PHPM, tested in a suitable vehicle, was shown to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay. The EC3 value of PHPM is 5.3% (w/v).

 

The following classification/labelling is triggered:

Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017: Category 1 (sub-category 1B).

 

Reference 2

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

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

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

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

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

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

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

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.

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.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

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:
Room temperature
- Solubility and stability of the test substance in the solvent/vehicle:
Acetonitrile

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

Test System
Cysteine peptide
Peptide sequence: AC-RFAACAA-COOH
Peptide sequence synonyms: Ac RFAACAA-OH or RFAACAA-COOH
Molecular weight: 750.88 g/mol
Supplier: JPT Peptide Technologies GmbH
Batch No: 11016HS_MHeW1017
Storage condition: At -20°C
Description: White powder
Specific handling conditions: none

Batch number and any information relating to the characterization and integrity of the test system are documented in a certificate of analysis, which is archived in Citoxlab France files.
The cysteine peptide solution was freshly prepared at 0.667 mM in an aqueous phosphate buffer (pH 7.5) solution. The detailed preparation method is described in a Citoxlab France analytical method, specific to the DPRA test.

Lysine peptide
Peptide sequence: AC-RFAAKAA-COH
Peptide sequence synonyms: Ac RFAAKAA-OH or RFAAKAA-COOH
Molecular weigh: 775.91 g/mol
Supplier: JPT Peptide Technologies GmbH
Batch No: 120514HSDWW1017
Storage condition: At -20ºC
Description: White powder
Specific handling conditions: none

Batch number and any information relating to the characterization and integrity of the test system are documented in a certificate of analysis, which is archived in Citoxlab France files.
The lysine peptide solution was freshly prepared at 0.667 mM in an aqueous ammonium acetate buffer (pH 10.2) solution. The preparation method is described in a Citoxlab France analytical method, specific to the DPRA test.

Equipment, reagents and computer systems (indicative list)
Equipment list
- High Performance Liquid Chromatography (HPLC) systems with a UV detector (220 nm),
- analytical chromatography columns (Zorbax SB C18, 100 x 2.1 mm, 3.5µm HPLC analytical column),
- precision electronic scales,
- pH meter,
- small laboratory equipment [micropipettes, vortex, etc],
- centrifuge,
- fridge and freezer.

Reagents
Only reagents with high purity were used throughout this study. Their purity grade and the identification of the Suppliers are described in the raw data.
The list of reagents used in this study is described in a Citoxlab France analytical method procedure, specific to the DPRA test.

Computer systems
The Citoxlab France's computer systems used in the study are detailed in the following table:
Software Version Application function
CITPharma (CITAC) 3 Test item receipt and inventories, reagents, matrix
PANORAMA E2 2.60.0000 Acquisition of temperature and humidity in study rooms (study and laboratory, cold chambers)
CITAC-CITMaster 3 CIT Application Center: Web business portal Master schedule sheet (including Study note) Master schedule sheet- Study event
Empower 2 Build 2154 Acquisition and management of chromatographic data
CITAC-CITEquipment 1 CIT Application Center: Web business portal Management of the equipments

Design of the direct peptide reactivity assay
The test item was tested in one run. The run was processed as described below.
Preparation of the samples
The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepare per peptide buffer.

Co-elution control samples preparation
For the col-elution control with cysteine peptide:
50 µL of the test item formulation was incubated with 750 µL of cysteine peptide dilution buffer (without cysteine peptide) and 200 µL of acetonitrile.
For the co-elution control with lysine peptide:
In parallel, 250 µL of test item formulation was incubated with 750 µL of lysine peptide dilution buffer (without lysine peptide).

Reference control samples preparation
Reference control A and B samples
In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.
Reference control C samples
Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.

For the reference contrl C prepared with cysteine peptide:
50 µL of vehicle (acetonitrile) was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
For the reference control C prepared with lysine peptide:
In parallel, 250 µL of vehicle (acetonitrile) was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

Cinnamaldehyde (positive control) depletion control samples preparation
For the reactivity of cinnamaldehyde with cysteine peptide:
50 µL of cinnamaldehyde at 100 mM in aceonitrile was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
For the reactivity of cinnamaldehyde with lysine peptide:
In parallel, 250 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

20.22 %

At concentration:

100 mM

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

66.44 %

At concentration:

100 mM

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

high reactivity [in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes

Results

Solubility results

The test item was found soluble at 100 mM in acetonitrile with sonication. Therefore, this vehicle was retained.

Evaluation of the presence of precipitate at the end of the incubation with peptides

At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test item and positive control samples) was performed prior to HPLC analysis.

As precipitate was observed in positive samples incubated with the cysteine or lysine peptides, these vials were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Only supernatants were then injected into the HPLC/UV system.

For the other samples, the vials were directly transferred into the HPLC/UV system.

Evaluation of the results

Results for the test item and the positive control are presented in Tables 1 and 2, respectively. The peak areas for calibration curve samples are presented in Tables 3.1 and 3.2, the peptide concentrations and peak areas in reference control samples are presented in Table 4.1 and 4.2.

Representative chromatograms of the co-elution, reference control C and test item samples are presented for each peptide in Figures 1 to 6.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples (figure 1 and 4) indicated that the test item did not co-elute with either lysine or cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in Data analysis and calculation:

- for the cysteine peptide, the mean depletion value was 66.44%,

- for the lysine peptide, the mean depletion value was 20.22%.

The mean of the percent cysteine and percent lysine depletions was equal to 43.33%. Accordingly, the test item was considered to have high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrates approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitization potential of the test item.

Table 4.1 Concentrations and peak areas of cysteine peptide in reference control samples

Determination of cysteine peptide concentration in reference control A samples

Date	Sample number	Cysteine peptide
		Area (µV/sec)	Concentration (mM)	% Dev
19 April 2018	Reference control A.1 Reference control A.2 Reference control A.3	2606461 2607751 2610579	0.494 0.494 0.494	(-1.3) (-1.2) (-1.1)
Mean SD % CV		- - -	0.494 0.000 0.1	(-1.2) - -

- : not applicable

Cystiene peptide peak areas in reference control B and C samples prepared in acetonitrile

Date	Sample Number	Peak areas (µV/sec)
19 April 2018	Reference control B.1 Reference control B.2 Reference control B.3 Reference control B.4 Reference control B.5 Reference control B.6 Reference control C.1 Reference control C.2 Reference control C.3	2613916 2635353 2616544 2571622 2585168 2598878 2650746 2637776 2623054
Mean SD % CV		2614784 25764 1.0

Table 4.2 Concentrations and peak areas fo lysine peptide in reference control samples

Determination of lysine peptide concentration in reference contrl A samples

Date	Sample number	Lysine peptide
		Area (µV/sec)	Concentration (mM)	% Dev
23 April 2018	Reference control A.1 Reference control A.2 Reference control A.3	1599744 1560922 1539183	0.507 0.495 0.488	(1.4) (-1.1) (-2.5)
Mean SD % CV		- - -	0.496 0.010 2.0	(-0.7) - -

- : not applicable

Lysine peptide peak areas in reference control B and C samples prepared in acetonitrile

Date	Sample number	Peak areas (µV/sec)
23 April 2018	Reference control B.1 Reference control B.2 Reference control B.3 Reference control B.4 Reference control B.5 Reference control B.6 Reference control C.1 Reference control C.2 Reference control C.3	157233 1633536 1590851 1562631 1580046 1563485 1570610 1577601 1571560
Mean SD % CV		1580173 21795 1.4

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the experimental conditions of this study , the test item, 3-phenoxy-2-hydroxypropyl methacrylate, was considered to have high peptide reactivity. The test item is considered positive in the DPRA assay.

Executive summary:

Summary

The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.

Methods

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24 -hr contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.

Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in three relevant reference control C samples (in the appropriate solvents).

Results

The test item was dissolved at 100 mM in acetonitrile without sonication.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in Data analysis and calculation:

- for the cysteine peptide, the mean depletion value was 66.44%,

- for the lysine peptide, the mean depletion value was 20.22%.

The mean of the percent cysteine and percent lysine depletions was equal to 43.33%. Acordingly, the test item was considered to have high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

This positive result can be used to suport the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitization potential of the test item.

Conclusion

Under the experimental conditions of this study, the test item 3 -phenoxy-2 -hydroxypropyl methacrylate, was considered to have high peptide reactivity. The test item is considered positive in the DPRA assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance is classified as a skin sensitiser Cat 1B in accordance with EU CLP (Regulation (EC) No. 1272/2008).