2-[(2-methyl-1-oxoallyl)oxy]ethyl hydrogen 3-chloro-2-hydroxypropylphthalate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2018 - 06 July 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:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8

Version / remarks:

February 2009

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-methyl-1-oxoallyl)oxy]ethyl hydrogen 3-chloro-2-hydroxypropylphthalate

EC Number:

259-130-3

EC Name:

2-[(2-methyl-1-oxoallyl)oxy]ethyl hydrogen 3-chloro-2-hydroxypropylphthalate

Cas Number:

54380-33-5

Molecular formula:

C17H19ClO7

IUPAC Name:

2-(3-chloro-2-hydroxypropyl)-6-({2-[(2-methylprop-2-enoyl)oxy]ethoxy}carbonyl)benzoic acid

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult human donors

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. It showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin(TM)SM, EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: approximately 25 mL PBS 1 x solution; rest of the PBS was removed from epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL stock solution; 2 mL of 0.3 mg/mL per well
- Incubation time: 3 hours
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is less than or equal to 50 % of the negative control.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is greater than 50 % of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 µL

VEHICLE
- Amount applied: 10 μL

POSITIVE CONTROL
- Amount applied: 10 μL
- Concentration: 5 % aq. solution

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1st run

Value:

87

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method the laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Table 1: OD values and viability percentages of the negative control

Substance		Optical Density (OD)	Viability (%)
Negative Control (1xPBS)	1	1.319	101
	2	1.431	110
	3	1.169	89
	mean	1.306	100
	Standard deviation (SD)		10.08

Table 2: OD values and viability percentages of the positive control

Substance		Optical Density (OD)	Viability (%)
Positive Control (SDS, 5% aq.)	1	0.091	7
	2	0.082	6
	3	0.058	4
	mean	0.077	6
	Standard deviation (SD)		1.28

Table 3: OD values and viability percentages of the test item

Substance		Optical Density (OD)	Viability (%)
Test item	1	1.035	79
	2	1.185	91
	3	1.197	92
	mean	1.139	87
	Standard deviation (SD)		6.89

Table 4: OD values and NSC % of additional control

Substance		Optical Density (OD)	Non Specific Colour % (NSC %)
Test item (test item treated tissues without MTT incubation)	1	0.063	3.1
	2	0.017
	mean	0.040

Table 5: Historical Control Data (Period of 2011 - 2018 July)

	Negative Control Data	Positive Control Data	Positive Control Data
	Phosphate Buffered Saline (1xPBS)	Sodium Dodecyl Sulphate (SDS) 5% aq. solution	Viability (% control)
Mean OD	0.858	0.110	13
Minimum OD	0.555	0.015	2
Maximum OD	1.431	0.299	39

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions.

Executive summary:

A study according OECD TG 439 to determine the skin irritation potential of the test on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 ± 1 °C for 42 hours (± 1h) in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37 ± 1 °C in 5 ± 1 % CO2, ≥ 95 % humidified atmosphere and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

The test item has an intrinsic colour (light yellow), therefore two additional test item treated tissues were used for the non-specific OD evaluation. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 87 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.