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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(1-ethoxyvinyl)-3,3,5,5-tetramethylcyclohexanone
EC Number:
252-961-2
EC Name:
4-(1-ethoxyvinyl)-3,3,5,5-tetramethylcyclohexanone
Cas Number:
36306-87-3
Molecular formula:
C14H24O2
IUPAC Name:
4-(1-ethoxyethenyl)-3,3,5,5-tetramethylcyclohexan-1-one
impurity 1
Chemical structure
Reference substance name:
(3E,6E)-8-ethoxy-4,6,8-trimethylnona-3,6-dien-2-one
Molecular formula:
C14H24O2
IUPAC Name:
(3E,6E)-8-ethoxy-4,6,8-trimethylnona-3,6-dien-2-one
impurity 2
Reference substance name:
(E)-6-ethoxy-4,6,8-trimethylnona-3,7-dien-2-one
IUPAC Name:
(E)-6-ethoxy-4,6,8-trimethylnona-3,7-dien-2-one
impurity 3
Reference substance name:
(3E,6Z)-8-ethoxy-4,6,8-trimethylnona-3,6-dien-2-one
IUPAC Name:
(3E,6Z)-8-ethoxy-4,6,8-trimethylnona-3,6-dien-2-one
impurity 4
Reference substance name:
1,3-diethoxy-5,7,7-trimethylbicyclo[3.3.1]non-2-ene
IUPAC Name:
1,3-diethoxy-5,7,7-trimethylbicyclo[3.3.1]non-2-ene
impurity 5
Reference substance name:
3,5-diethoxy-1,7,7-trimethylbicyclo[3.3.1]non-2-ene
IUPAC Name:
3,5-diethoxy-1,7,7-trimethylbicyclo[3.3.1]non-2-ene
Test material form:
liquid
Specific details on test material used for the study:
Identification: Kephalis
Appearance: Pale yellow liquid
Batch: PE00134258
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light
Stable under storage conditions until: 08 July 2016 (expiry date)

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-011).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Source
SkinEthic Laboratories, Lyon, France.
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five μl of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/ml in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.
Amount/concentration applied:
The liquid test item was applied undiluted (25 μl) directly on top of the tissue.

Test animals

Species:
other: In vitro study

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
Kephalis was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Kephalis did not interact with the MTT endpoint.
The mean absorption at 570 nm measured after treatment with Kephalis and controls are presented in APPENDIX 1, Table 2.
The individual OD570 measurements are presented in APPENDIX 2.
Table 3 shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with Kephalis compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Kephalis compared to the negative control tissues was 14%. Since the mean relative tissue viability for Kephalis was below 50% it is considered to be irritant.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 44%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See APPENDIX 3). The standard deviation value of the percentage viability of three tissues treated identically was less than 8%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
It is concluded that this test is valid and that Kephalis is irritant in the in vitro skin irritation test under the experimental conditions described in this report.