2,2'-(2-methylpropylidene)bis[4,6-xylenol]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 March 2016 to 18 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study performed in accordance with OECD & EU test guidelines in compliance with GLP and reported with a valid GLP certificate.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(2-methylpropylidene)bis[4,6-xylenol]

EC Number:

251-394-8

EC Name:

2,2'-(2-methylpropylidene)bis[4,6-xylenol]

Cas Number:

33145-10-7

Molecular formula:

C20H26O2

IUPAC Name:

2-[1-(2-hydroxy-3,5-dimethylphenyl)-2-methylpropyl]-4,6-dimethylphenol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: Lowinox® 22IB46IUPAC: 2,2’-(2-methylpropylidene)bis[4,6-xylenol]Appearance: White to cream coloured powderBatch: WB44L0016Purity/Composition: 99.7%Test item storage: At room temperatureStable under storage conditions until: 18 November 2018 (retest date)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Normal human-derived epidermal keratinocytes

Source strain:

other: EpiDerm Reconstructed Human Epidermis, EPI-200, EPI-212, LOT: 23662

Details on animal used as source of test system:

EpiDerm Skin Model (EPI-200, Lot no.: 23662 kit DD and EE).The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
Source: MatTek Corporation, Ashland MA, U.S.A.
Tissues: On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

Reference items
Negative control: Milli-Q water (Millipore Corp., Bedford, Mass., USA).
Positive control: Potassium hydroxide (KOH; Merck, Darmstadt, Germany), an 8.0 normal solution was prepared.
Test item preparation: No correction was made for the purity/composition of the test item. The solid test item was applied directly on top of the skin tissue. Lowinox® 22IB46 was spread to match the size of the tissue.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

32.8 to 34.4 mg

Duration of treatment / exposure:

3 minutes & 1 hour

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

2 tissues per test group (3 minutes exposure, 1 hour exposure, negative control, positive control).

Test animals

Species:

other: Human

Strain:

other: EpiDerm Skin Model (EPI-200, Lot no.: 23662 kit DD and EE).

Details on test animals or test system and environmental conditions:

Environmental conditions: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 67 - 86%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.3°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, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Type of coverage:

open

Preparation of test site:

other: See details on Study Design

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Duration of treatment / exposure:

32.8 to 34.4 mg

Observation period:

3 minutes & 1 hour

Number of animals:

2 tissues per test group (3 minutes exposure, 1 hour exposure, negative control, positive control).

Details on study design:

Test for the interference of the test item with the MTT endpoint: A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Test for colour interference by the test item: Lowinox® 22IB46 was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, at least 25 mg of Lowinox® 22IB46 or 50 μl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.

Test for reduction of MTT by the test item: Lowinox® 22IB46 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 mg of Lowinox® 22IB46 was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple colour change or a blue / purple precipitate was observed.

Application/Treatment of the test item: The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before Lowinox® 22IB46 was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to Lowinox® 22IB46 and two for a 1-hour exposure. The skin was moistened with 25 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and 32.8 to 34.4 mg of the solid test item was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively. After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

Cell viability measurement: The DMEM medium was replaced by 300 μl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application, mean tissue viability

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Not considered to be corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application, mean tissue viability

Value:

90

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean tissue viability obtained after 3-minute and 1-hour treatments with Lowinox® 22IB46 compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Lowinox® 22IB46 compared to the negative control tissues was 98% and 90% respectively. Because the mean relative tissue viability for Lowinox® 22IB46 was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment Lowinox® 22IB46 is considered to be not corrosive.
Lowinox® 22IB46 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that Lowinox® 22IB46 did not interfere with the MTT endpoint.

Any other information on results incl. tables

Mean absorption in the in vitro skin corrosion test with Lowinox® 22IB46

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)	SD		A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.720	1.832	1.776	±	0.079	1.971	1.891	1.931	±	0.057
Lowinox® 22IB46	1.765	1.704	1.734	±	0.043	1.752	1.717	1.734	±	0.025
Positive control	0.185	0.185	0.185	±	0.000	0.202	0.122	0.162	±	0.056

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0418). Isopropanol was used to measure the background absorption.

Mean tissue viability in the in vitro skin corrosion test with Lowinox® 22IB46

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Lowinox® 22IB46	98	90
Positive control	10	8

 

Coefficient of Variation between tissue replicates

	3 minute	1 hour
Negative control	6.1	4.1
Lowinox® 22IB46	3.4	2.0
Positive control	0.1	39

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

INDIVIDUAL OD MEASUREMENTS AT 570 NM

	3-minute application (OD570) A B		1-hour application (OD570) A B
Negative control
OD570measurement 1	1.7293	1.8643	2.0186	1.9594
OD570measurement 2	1.7764	1.8906	2.0188	1.9330
OD570 measurement 3	1.7795	1.8673	2.0018	1.9065
Lowinox® 22IB46
OD570 measurement 1	1.8329	1.7691	1.8182	1.7949
OD570measurement 2	1.7870	1.7502	1.7930	1.7290
OD570measurement 3	1.7997	1.7185	1.7706	1.752
Positive control
OD570measurement 1	0.2320	0.2262	0.2617	0.1636
OD570measurement 2	0.2217	0.2293	0.2356	0.1650
OD570measurement 3	0.2276	0.2263	0.2332	0.1635

OD = Optical density

Duplicate exposures are indicated by A and B.

HISTORICAL CONTROL DATA FOR IN VITRO SKIN CORROSION STUDIES

	Negative control		Positive control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (% viability)	1-hour treatment (% viability)
Range	1.324 – 2.615	1.361 – 2.352	0.172 – 0.56	0.057 – 0.277	6 – 22	3 – 12
Mean	1.86	1.86	0.18	0.13	10.67	7.17
SD	0.24	0.22	0.10	0.05	3.9	2.36
n	65	67	64	61	30	30

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of December 2012 to December 2015.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that this test is valid and that Lowinox® 22IB46 is not corrosive in the in vitro skin corrosion test under the conditions of this study.

Executive summary:

The objective of this study was to evaluate Lowinox® 22IB46 for its ability to induce skin corrosion. For this purpose Lowinox® 22IB46 was topically applied on a human three dimensional epidermal model. The possible corrosive potential of Lowinox® 22IB46 was tested through topical application for 3 minutes and 1 hour.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 431: In Vitro Skin Corrosion: reconstructed human epidermis (RHE) test method (adopted 26 September 2014).

- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.

 

Batch WB44L0016 of Lowinox® 22IB46 was a white to cream coloured powder with a purity of 99.7%. Skin tissue was moistened with 25 μl of Milli-Q water and at least 25 mg of Lowinox® 22IB46 was applied directly on top of the skin tissue.

 

The positive control had a mean relative tissue viability of 10% after 3 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 7%, indicating that the test system functioned properly.

 

Skin corrosion is expressed as the remaining cell viability after exposure to the test item after 3 minutes and 1 hour. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Lowinox® 22IB46 compared to the negative control tissues was 98% and 90%, respectively.

 

Because the mean relative tissue viability for Lowinox® 22IB46 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment Lowinox® 22IB46 is considered to be not corrosive.

 

It is concluded that this test is valid and that Lowinox® 22IB46 is not corrosive in thein vitroskin corrosion test under the experimental conditions of this study.