Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 November 2016 to 13 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Original Guideline adopted July 28, 2015
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
6-tert-butyl-1,1-dimethylindan-4-yl methyl ketone
EC Number:
236-114-4
EC Name:
6-tert-butyl-1,1-dimethylindan-4-yl methyl ketone
Cas Number:
13171-00-1
Molecular formula:
C17H24O
IUPAC Name:
1-(6-tert-butyl-1,1-dimethyl-2,3-dihydro-1H-inden-4-yl)ethan-1-one
Test material form:
solid: crystalline

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories (69007 Lyon, France)
Vehicle:
water
Remarks:
The test item tissues were wetted with 5 µL of deionised water before applying the test item.
Details on test system:
TEST SYSTEM: human skin model EpiSkin™
EpiSkin™ Kit
Supplier: SkinEthic Laboratories (69007 Lyon, France)
Date received: 10 January 2017
EpiSkin™ Kit Lot No.: 17-EKIN-002

The EpiSkin™ tissue 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 EpiSkin™ tissues (surface 0.38 cm2) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 10 January 2017. On the day of experiment the pre-incubation phase of the EpiSkin™ tissues started.

Test for Direct MTT Reduction and Colour Interference:
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose the test item (approximately 10 mg) was mixed with 90 µL of deionised water in a pre-experiment. The test item/water mixture was gently shaken for 15 minutes at room temperature.
The colour of the test item/water mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, an additional test with viable tissues (without MTT addition) was not necessary.
For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 10 mg of the test item was added to 2 mL of MTT solution (0.3 mg/mL) and the mixture will be incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution with 10 µL of DMEM was used as the control.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.

Pre-warming of EpiSkin™ Tissues:
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for about 22.5 hours.

Treatment:
The test item tissues were wetted with 5 µL of deionised water. The negative control, positive control and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues. The 12-well plates were treated for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

IL-1 α Immunoassay:
Samples of all treatment groups were taken from the wells. The plates were shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well was taken and was stored in the freezer at –20 °C. Since the results derived from the MTT assay were not unclear or borderline, the IL-1 α concentration in the medium was not determined and the taken samples will be discarded after report finalization.

MTT Assay:
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues were blotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for nearly 3 hours while shaking at room temperature.
Per tissue sample 2 x 200 µL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate. Isopropanol was used for blank measurement. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

DATA EVALUATION:
The mean OD of the three tissues per group (negative control, positive control and test item) was calculated after blank correction. The mean OD of the negative control corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = (OD test item or positive control / mean OD negative control) *100
For the test item and the positive control, the mean relative viability +/- rel. standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:
in vitro result : in vivo prediction
mean tissue viability =< 50%: irritant
mean tissue viability > 50%: non-irritant

Acceptability of the Assay:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is >= 0.6 till <= 1.5.
The rel. standard deviations between tissues of the same treatment group should be <= 18%.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is <= 40%.
The data of the quality control (determined by SkinEthic Laboratories, 69007 Lyon, France) of the respective EpiSkin™ lot is mentioned in the report (the acceptance limit of the IC50 should be between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS).
The historical data (means, rel. standard deviation, and ranges) of the positive control as well as the negative control obtained with the EpiSkin™ model at Envigo CRS GmbH are mentioned in the report.



Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
approximately 10 mg test item:
5 µL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item were applied to and spread on the wetted triplicate tissues.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
approximately 42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: Mean relative tissue viability (%)
Value:
115.2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Consequently, additional tests with freeze-killed or viable tissues were not necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Quality control data (determined by SkinEthic Laboratories, 69007 Lyon, France) of the respective EpiSkinTM lot: the acceptance limit of the IC50 should be between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (actual value: 2.2 mg/mL)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is >= 0.6 till <= 1.5 (actual value: 1.053).
- Acceptance criteria met for positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is <= 40% (actual: 8.1%).
- Acceptance criteria met for variability between replicate measurements: The rel. standard deviations between tissues of the same treatment group should be <= 18% (actual: <= 15.9%).
- Range of historical values: The actual values for the positive and negative control lie within the Envigo CRS GmbH historical data.

Any other information on results incl. tables

IL-1 α Immunoassay:

Since the results derived from the MTT assay were not unclear or borderline, the IL-1 α concentration in the medium was not determined.

Results after treatment with the test item and controls:

Test Group

Absorbance 570 nm
Tissue 1*

Absorbance 570 nm
Tissue 2*

Absorbance 570 nm
Tissue 3*

Mean Absorbance of 3 Tissues

Stand. Dev.

Relative Absorbance [%] Tissue 1, 2 + 3**

Relative Standard Deviation [%]***

Rel. Absorbance
[% of Negative Control]****

Negative Control

1.111

1.094

0.954

1.053

0.086

105.5
103.9
90.6

8.2

100.0

Positive Control

0.074

0.082

0.101

0.086

0.014

7.0
7.8
9.6

15.9

8.1

Test Item

1.178

1.222

1.242

1.214

0.033

111.8
116.0
117.9

2.7

115.2

* Mean of two replicate wells after blank correction

** Relative absorbance per tissue [rounded values]: (100 * (absorbance tissue)) / mean absorbance negative control

*** Rel. standard deviation: (Standard deviation absorbance / mean absorbance of 3 tissues) * 100

**** Relative absorbance per treatment group [rounded values]: (100 * (mean absorbance test item)) / mean absorbance negative control

Applicant's summary and conclusion

Interpretation of results:
other: not classified, criteria not met
Remarks:
according to EU CLP Regulation (EC) No. 1272/2008 and its amendments.
Conclusions:
The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissues was 115.2%. The relative tissue viability was not reduced, the value is above the threshold for irritancy of <= 50%. Therefore, the test item is not considered to be a skin irritant under the conditions of this study.
Executive summary:

The possible skin irritation potential of the substance was tested in compliance with OECD TG 439 and GLP principles. Three tissues of the human skin model EpiSkin™ were topically applied with 5 µL deionized water in order to improve contact between the solid test item and the epidermis. Each approximately 10 mg of the solid test item were applied to and spread on the wetted triplicate tissues. In addition, triplicate tissues were applied with 10 µL of the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate). Each three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes. After approximately 42 hour post-exposure incubation period, determination of the cell viability was performed. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison with untreated negative controls is used to predict skin irritation potential. The validity criteria for the positive and negative control were met. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissues was 115.2%. This value is above the threshold for irritancy of <= 50%. Therefore, the test item is not considered to be a skin irritant under the conditions of this study.