2-hydroxy-3-phenoxypropyl methacrylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 17 May and 18 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 02-04 June 2015 Date on Certificate: 22 September 2015

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: Room temperature (15-25°C, ≤70 RH%), protected from humidity (tight closed container).

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: no data

Source strain:

not specified

Details on animal used as source of test system:

Not applicable. EPISKINTM(SM) is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum.

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM, Manufactuer: SkinEthic, France
- Tissue batch number(s): 18-EKIN-020
- Expiry Date: 21 May 2018
Kit Contents
Units: EPISKINTM(SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKINTM(SM) biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium”
(Batch No.: 18 MAIN3 024; Exp. Date: 23 May 2018)
A flask of sterile “Assay Medium”
(Batch No.: 18 ESSC 020; Exp. Date: 23 May 2018)

Kit Reception
The pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable

The kits were found to be in good order at reception.
Storage
The EPISKINTM(SM) kits were kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 22.1-24.6°
- Temperature of post-treatment incubation (if applicable): not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
After the incubation time (4 hours), all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly.
The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: No damage noted


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL - stock solutiopn. Diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL
- Incubation time: 3 hours (±15 minutes)
- Spectrophotometer: Thermo Fisher Scientific, Catalogue Number: 24072800, Serial Number: 0920-14
- Wavelength: 570 nm


NUMBER OF REPLICATE TISSUES:
Two replicates for test item were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : killed tissues
- Procedure used to prepare the killed tissues:
Living epidermis units (Manufacturer: SkinEthic, France, Batch No.:17-EKIN-048, Expiry Date: 01 December 2017) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the epidermis units were frozen on 01 December 2017 (the frozen units could be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Maintenance Medium). Further use of killed tissues was similar to living tissues.
- N. of replicates : Two
- Method of calculation used:
The measured OD value is corrected by the result of the additional controls before calculation of viability% as follows:
Data calculation for test items having MTT-interacting potential:

Non Specific MTT reduction calculation (NSMTT%):
NSMTT% = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD
ODKT: test item treated killed tissues OD
ODNC: negative control OD

If NSMTT% is ≤ 50%, then true MTT metabolic conversion (TODTT) has to be undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues
– The % relative viability (RV%) for each test item replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test item is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2

If NSMTT% is > 50% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

Data calculation for test items having colouring potential

For test items detected as able to stain the tissues the non-specific OD is evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability % as detailed below:

Non Specific Colour % (NSC living %):
NSC living % = (mean ODCTV / mean ODNC)×100
ODCTV: test item treated viable tissue (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)

If NSC living % is ≤ 5 % then the normal calculation mode is used.

If NSC living % is > 5 % and ≤ 50 %, then additional correction (TODTT) has to be undertaken as follows:

TODTT = [ODTV - ODCTV]
ODTT: test item treated viable tissue (incubated with MTT)
ODCTV: test item treated viable tissue (not incubated with MTT)

– The % relative viability (RV%) for each test item replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test item is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2

If NSC living % is > 50 % relative to the negative control, additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.


Data calculation for test items having both MTT-interacting and colouring potential


For test items detected as able to both stain the tissues (3.7.2) and interfere with MTT (3.7.1) may also require a third set of controls before calculation of the “true” viability %.

Non Specific Colour % with killed tissues (NSCkilled %):
NSCkilled % = (mean ODCTK / mean ODNC)×100
ODCTK: test item treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)

In that case the true MTT metabolic conversion (TODTT) is undertaken as follows:

TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV + mean ODCTK]
ODTT: test item treated viable tissues (incubated with MTT)
ODKT: test item treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test item treated viable tissues (not incubated with MTT)
ODCTK: test item treated killed tissues (not incubated with MTT)

The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
The mean value of the 2 individual relative viability % for test item is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2


DECISION CRITERIA
- The test substance is considered to be corrosive to skin if, for 2 disks, both disks have mean viability of <35%
- The test substance is considered to be non-corrosive to skin if, for 2 disks, both disks have mean viability of ≥35%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 0.9% w/v

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): ca. 100%

Duration of treatment / exposure:

4 hours (±10 minutes)

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

In this assay, two replicates for test item were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. Furthermore, as the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used in. To avoid a possible double correction for colour interference, for non-specific colour in killed tissues, two additional disks were used.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

145.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system:
- Direct-MTT reduction: Results of the additional controls on killed epidermis are shown in Table 2 (below) Based on these observed mean OD difference (0.017), the calculated NSMTT% is 1.9%. As the test item was showed being an MTT-interacting substance and the test item had an intrinsic colour, two additional test item-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.011, Non Specific Colour % (NSCkilled%) was calculated as 1.2% (see Table 3). However, because the NSCliving% was not used, therefore correction with NSCkilled% was not necessary.
- Colour interference with MTT: As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.007, Non Specific Colour % (NSCliving%) was calculated as 0.8% (see Table 1). This is below the threshold of 5%, therefore correction due to colouring potential of the test item was not necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Met

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - Mean OD Values 0.90
- Acceptance criteria met for positive control: Yes - Mean viability 0.2%
- Acceptance criteria met for variability between replicate measurements: Yes - difference of viability <30%

Any other information on results incl. tables

ADDITIONAL CONTROLS

 

As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.007, Non Specific Colour % (NSC_living%) was calculated as 0.8% (see Table 1). This is below the threshold of 5%, therefore correction due to colouring potential of the test item was not necessary.

Table 1: Optical Density (OD) and the calculated Non Specific Colour % (NSC_living%) of the Additional Control Tissues

Additional control	Optical Density (OD)			NSC % (living)
		Measured	Blank Corrected
Treated with PHPM	1	0.050	0.004	0.8
	2	0.058	0.011
	Mean	--	0.007

Notes:

1.      Mean blank value was 0.047.

2.      Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

As colour change (purple) was observed after three hours of incubation of the test item in MTT working solution, thus the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Results of the additional controls on killed epidermis are shown in Table 2. Based on these observed mean OD difference (0.017), the calculated NSMTT% is 1.9%.

Table 2: Optical Density (OD) and the calculated Non-Specific MTT Reduction (NSMTT%) of the Additional Control Tissues (Killed Epidermis)

Additional control (killed epidermis)	Optical Density (OD)			NSMTT	NSMTT %
		Measured	Blank Corrected
Treated with PHPM	1	0.075	0.029	0.009	1.9
	2	0.091	0.044	0.025
	Mean	--	0.036	0.017
Treated with physiological saline (0.9% (w/v) NaCl)	1	0.064	0.017	--
	2	0.068	0.022
	Mean	--	0.019

Notes:

1.      Mean blank value was 0.047.

2.      Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

As the test item was showed being an MTT-interacting substance and the test item had an intrinsic colour, two additional test item-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.011, Non Specific Colour % (NSC_killed%) was calculated as 1.2% (see Table 3). However, because the NSC_living% was not used, therefore correction with NSC_killed% was not necessary.

 

Table 3: Optical Density (OD) and the calculated Non Specific Colour % (NSC_killed%) of the Additional Control Tissues (Killed Epidermis)

Additional control	Optical Density (OD)			NSC % (living)
		Measured	Blank Corrected
Treated with PHPM	1	0.058	0.011	1.2
	2	0.057	0.011
	Mean	--	0.011

Notes:

1.      Mean blank value was 0.047.

2.      Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

VIABILITY RESULTS

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 4. The mean OD value for the test item treated skin samples showed a 145.5% relative viability compared to the negative control.

Table 4: Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical Density (OD)			TODTT	Viability (% RV)
		Measured	Blank Corrected
Negative Control: Physiological saline (0.9% (w/v NaCl)	1	0.948	0.901	--	100.1
	2	0.945	0.899	--	99.9
	mean	--	0.900	--	100.0
Positive Control: Glacial acetic acid	1	0.049	0.002	--	0.2
	2	0.049	0.002	--	0.2
	mean	--	0.002	--	0.2
Test Item: PHPM	1	1.381	1.335	1.318	146.4
	2	1.365	1.318	1.301	144.6
	mean	--	1.326	1.309	145.5

Notes:

1.    Mean blank value was 0.047.

2.    Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

  

3.      TOD_TT: the measured values were corrected for non-specific MTT reduction (by subtracting the NSMTT value of 0.017)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results indicate that the test item is non-corrosive to the skin.

Executive summary:

An in vitro skin corrosivity test of PHPM test item was performed in a reconstructed human epidermis model.EPISKIN^TM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Disks of EPISKIN^TM(SM) (two units) were treated with PHPM test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSC_living%) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSC_killed), two additional disks were used. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with PHPM, the mean cell viability was 145.5% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test with PHPM (Batch number: 171512), the results indicate that the test item is non-corrosive to the skin.