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Diss Factsheets

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 September 2015 to 25 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: (EC) No. 440/2008, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
1639131-79-5
Cas Number:
1639131-79-5
IUPAC Name:
1639131-79-5
Constituent 2
Reference substance name:
Phenol, 1,1-dimethylpropyl derivs.
IUPAC Name:
Phenol, 1,1-dimethylpropyl derivs.
Test material form:
other: Solid
Details on test material:
Identification: Phenol, 1,1-dimethylpropyl derivs.Appearance: Colourless to pale yellow solidBatch: OP: C605E003.1Purity/Composition: 100% Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB)Test item storage: At room temperature protected from light container flushed with nitrogenStable under storage conditions until: 31 July 2016 (expiry date)Purity/composition correction factor: No correction factor requiredTest item handling: Use amber glassware or wrap container in aluminum-foilChemical name (IUPAC), synonym or trade name: Phenol, 1,1-dimethylpropyl derivs.CAS Number: 1639131-79-5pH (1% in water, indicative range): 7.30 – 6.91 (determined by WIL Research Europe)

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Amount/concentration applied:
EpiDerm Skin Model (EPI-200, Lot no.: 22676 kit K and J).
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.

Test animals

Species:
other: EpiDerm Skin Model
Strain:
other: Not applicable
Details on test animals or test system and environmental conditions:
Test system: EpiDerm Skin Model (EPI-200, Lot no.: 22676 kit K and J). 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. Rationale: Recommended test system in international guidelines (OECD and EC). Source: MatTek Corporation, Ashland MA, U.S.A.Cell culture Tissues On the day of receipt the tissues were kept on agarose gel 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. Freeze-killed tissues (EPI-200, Lot no.: 22268 kit H and 22299 kit O) Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 ml DMEM medium. Further use of killed tissues was similar to living tissues. DMEM (Dulbecco’s Modified Eagle’s Medium) Supplemented DMEM medium, serum-free supplied by MatTek Corporation. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) medium MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation. 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 58 - 90%), 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: Not applicable
Vehicle:
water
Controls:
yes
Amount / concentration applied:
approximately 25 mg of the test item
Duration of treatment / exposure:
3 minutes & 1 hour
Observation period:
No observation period required for the study design
Number of animals:
4 tissues
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 The test item 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, approximately 25 mg of the test item 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. In case the test item induces colour interference in aqueous conditions, in addition to the normal procedure, two tissues must be treated with test item for 3 minutes and two tissues for 1-hour. Instead of MTT solution these tissues will be incubated with DMEM medium. Test for reduction of MTT by the test item Phenol, 1,1-dimethylpropyl derivs. was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, approximately 25 mg of the test item 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 ± 1.0°C. A negative control, sterile Milli-Q water was tested concurrently.In case the test item reacts with the MTT medium in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed non treated tissues must be used for the cytotoxicity evaluation with MTT. At the end of the exposure time it was checked if a blue / purple colour change 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 is started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The level of the DMEM medium is just beneath the tissue (see fig 1). The plates were incubated for approximately 1.5 hour at 37.0 ± 1.0°C. The medium was replaced with fresh DMEM medium just before the test item 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 Phenol, 1,1-dimethylpropyl derivs. 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 an excessive amount 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. In addition for the 3 minute and 1 hour exposure two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT. 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

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: mean tissue viability
Value:
52
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 minute. Reversibility: no data. (migrated information)
Irritation / corrosion parameter:
other: other: mean tissue viability
Value:
11
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 1 hour. Reversibility: no data. (migrated information)

In vivo

Irritant / corrosive response data:
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 the test item compared to the negative control tissues was 52% and 11% respectively. Because the mean relative tissue viability for the test item was below 15% after 1 hour treatment it is considered to be corrosive.
Other effects:
Phenol, 1,1-dimethylpropyl derivs. was checked for colour interference in aqueous conditions and for possible direct MTT reduction by adding the test item to MTT medium. Because a colour change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint. In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each timepoint. The non-specific reduction of MTT by the test item was 0.29% and 23% of the negative control tissues after 3 minutes and 1 hour respectively. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues.The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 12%. The maximum inter-tissue variability in viability between two tissues treated identically was less than 16% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 8% in the range of 20 – 100% viability. In the range of <20% viability, the maximum inter-tissue variability in viability between two tissues treated identically was less than 49% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 33%, however since the viabilities were below 20% the acceptability criteria were met. It was therefore concluded that the test system functioned properly.

Any other information on results incl. tables

Mean absorption in the in vitro skin corrosion test with Phenol, 1,1-dimethylpropyl dervis.

 

3-minute application

1-hour application

 

A (OD570)

B (OD570)

Mean (OD570)

 

SD

A (OD570)

B (OD570)

Mean (OD570)

 

SD

Negative control

2.108

2.211

2.160

±

0.073

2.144

2.220

2.182

±

0.053

Phenol, 1,1-dimethylpropyl dervis. (1)

1.223

1.039

1.131

±

0.130

0.164

0.319

0.241

±

0.110

Positive control

0.296

0.206

0.251

±

0.064

0.174

0.217

0.195

±

0.030

SD = Standard deviation

Duplicate exposure are indicated by A and B

(1) The values are corrected for the non-specific MTT reaction.

In this table the values are corrected for background absorption (0.0423). Isopropanol was used to measure the background absorption.

 

Mean tissue viability in the in vitro skin corrosion test with Phenol, 1,1-dimethylpropyl dervis.

 

3-minute application viability (percentage of control)

1-hour application viability (percentage of control)

Negative control

100

100

Phenol, 1,1-dimethylpropyl dervis.

52

11

Positive control

12

9

 

INDIVIDUAL OD MEASUREMENTS AT 570NM

 

3-minute application (OD570)

1-hour application (OD570)

 

A

B

A

B

Negative control

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

2.1091

2.1567

2.1865

 

2.2521

2.2835

2.2246

 

2.1831

2.2167

2.1602

 

2.2204

2.2843

2.2822

Phenol, 1,1-dimethypropyl dervis.

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

1.3610

1.2910

1.1627

 

1.1178

1.1074

1.0486

 

0.7161

0.7172

0.7151

 

0.8907

0.8467

0.8775

Negative control treated freeze killed tissue

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

0.1722

0.1670

0.1689

 

0.1747

0.1776

0.1724

 

0.1656

0.1697

0.1696

 

0.1631

0.1619

0.1599

Positive control

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

0.3182

0.3452

0.3513

 

0.2518

0.2487

0.2446

 

0.2180

0.2157

0.2153

 

0.2575

0.2559

0.2635

OD = Optical density

Duplicate exposure 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

1.076 – 2.167

1.361 – 2.203

0.017 – 0.29

0.063 – 0.226

6 – 16

4 – 12

Mean

1.78

1.78

0.16

0.12

10.6

7.0

SD

0.25

0.20

0.05

0.04

2.9

2.1

N

43

47

44

42

22

22

SD = Standard deviation

N = Number of observations

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

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
Phenol, 1,1-dimethylpropyl derivs. is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

In vitro skin corrosion test with Phenol, 1,1-dimethylpropyl derivs. using a human skin model.

This report describes the ability of Phenol, 1,1-dimethylpropyl derivs. to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test item 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.

 

Batch OP: C605E003.1 of the test item was a colourless to pale yellow solid. Skin tissue was moistened with 25 μl of Milli-Q water and an excess amount of the test item was applied directly on top of the skin tissue.

 

The positive control had a mean relative tissue viability of 12% 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. The acceptability criteria for the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues were met, indicating that the test system functioned properly.

The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point. The non-specific reduction of MTT by the test item was 0.29% and 23% of the negative control tissues after 3 minutes and 1 hour respectively. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues.

 

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 52% and 11%, respectively. Because the mean relative tissue viability for the test item was below 15% after the 1-hour treatment it is considered to be corrosive.

 

Finally, it is concluded that this test is valid and that Phenol, 1,1-dimethylpropyl derivs. is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.