Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Jan 2012 to 6 Feb 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

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:
Reaction Mass of (1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl formate and (1R,2S)-2,6,6-trimethylcycloheptyl formate
EC Number:
939-618-9
Cas Number:
25225-08-5
Molecular formula:
C11H20O2
IUPAC Name:
Reaction Mass of (1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl formate and (1R,2S)-2,6,6-trimethylcycloheptyl formate
Test material form:
liquid

In vitro test system

Test system:
human skin model
Remarks:
EpiDerm™ (EPI-200) skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
The EpiDerm™ (EPI-200) skin model consists of normal human epidermal keratinocytes from one single donor, derived from neonatal-foreskin tissue. The keratinocytes are plated on chemically modified, collagen-coated, 9 mm ID cell culture inserts (surface area 0.64 cm2). The skin models are commercially available and were obtained from MatTek Corporation, USA. Upon arrival, the skin models were pre-incubated in 0.9 mL normal maintenance medium (NMM) provided with the kits in a 6-well plate for 1 h in a humidified incubator (ca. 37ºC and 5% CO2). At the end of the first pre-incubation period, the tissues were further pre-incubated overnight in fresh NMM for 21 h in a humidified incubator (ca. 37ºC and 5% CO2).

Preliminary tests:
The potential of the test substance to stain the skin membrane was investigated by incubating 30 μL of the test substance in 300 μL demineralized water for ca. 1 h (at ca. 37ºC and 5% CO2). At the end of the exposure period the presence and intensity of the staining (if any) was evaluated visually. The test substance did not significantly change the colour of the solution. Therefore, it was concluded that the test substance would not have the potential to stain the skin membrane. Some chemicals are known to non-specifically reduce MTT, resulting in a blue precipitate. Therefore, prior to the start of the study, 30 μL of the test substance was incubated in 1 mL 1 mg/mL MTT in Dulbecco’s Modified Eagle Medium for ca. 3 h at ca. 37ºC and 5% CO2 and the formation of any blue/purple formazan product was assessed visually. During incubation the test substance solution did not turn blue/purple and therefore it was concluded that the test substance did not reduce MTT. A nylon mesh was applied to facilitate equal distribution of the liquid test substance. To test if the test substance interacts with a nylon mesh, 30 μL of the test substance was applied to a nylon mesh. After ca. 60 min incubation at ambient temperature the possible interaction with the mesh was checked under a microscope. The test substance did not show any interaction with a nylon mesh. Therefore, the nylon mesh was used to facilitate equal distribution of the test substance.

Exposure to study substances:
Following the second pre-incubation, the skin models were exposed to 30 μL of the negative control, test substances or positive control. Immediately after application a nylon mesh (provided with the EPI-200 kit) was placed on the skin model surface to facilitate an equal distribution of liquid substances. Exposure was initiated at ambient temperature in the laminar down flow cabinet. After dosing the last skin membrane, all 6-well plates were transferred to a humidified incubator (ca. 37ºC and 5% CO2). After 35 min, the plates were removed from the incubator and placed in the laminar down flow cabinet until the period of 60 min was completed for the first dosed skin membrane. When the exposure period was completed, the skin membranes were removed from the well and the skin surface was carefully washed using excess of phosphate buffered saline (PBS). Subsequently, the skin membrane was blotted dry and the skin membranes were carefully dried using a sterile cotton swab. The skin membranes were then transferred to a clean 6-well plate containing fresh medium (0.9 mL/well) and incubated in a humidified incubator (ca. 37ºC and ca. 5% CO2). Medium was refreshed after 24 h. Following an additional 18 h incubation period, viability was determined using the MTT assay.

MTT assay:
The MTT solution of 1 mg/mL was prepared by diluting MTT concentrate five times in MTT diluent (provided with the MTT-100 assay kit). The bottoms of the inserts were blotted dry, and the inserts were transferred to a 24-well plate containing 300 μL of MTT solution per well. After 180 min incubation in a humidified incubator at ca. 37ºC and 5% CO2, the skin membranes were rinsed three times with PBS. The formazan product was extracted from the tissue using 2 mL MTT extractant (provided with the MTT-100 assay kit). Extraction was performed in the dark for 2 days at 2-10ºC. Following 2 days of extraction, the skin membrane was pierced with a needle to allow the extract to run into the well from which the skin membrane was then taken. The optical density was measured in triplicate in 200 μL sub-fractions using a spectrophotometer set at 570 nm. Extractant solvent alone was used as blank and optical density of the skin membrane extract was correct for the blank. The mean optical density was calculated and expressed as the percentage viability compared to the negative control (mean tissue viability).

Interpretation of the results:
The test is considered valid if the optical density of the negative control is ≥ 1.0 and ≤ 2.5, if the optical density of the extractant solvent alone is < 0.1, if skin membranes treated with the positive control demonstrate a mean tissue viability ≤ 20% compared to the negative control, and if the standard deviation (SD) calculated from individual skin membrane viability percentages of the three replicates is < 18%. The test is considered invalid if the test does not meet one or more of these acceptance criteria. The in vitro irritation potential of the test substance is determined from the relative mean skin membrane viabilities compared to the negative control tissues, using the following prediction model: Mean skin membrane viability in vitro (% of negative control) ≤ 50 %. The substance is an irritant (I), UN GHS Category 2; Mean tissue viability > 50 %. The substance is a non-irritant (NI), UN GHS No Category.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 μL
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Optical density of the negative control (PBS) and positive control (5% SDS) were within the acceptable ranges and correctly indicated non irritancy and irritancy, respectively. In the range between 20% and 100% mean viability the SD was < 18%. This demonstrated validity of the study.
The relative mean skin membrane viabilities compared to the negative control tissues were as follows (% of control):
Negative control (PBS): 100 ± 1
Substance: 93 ± 10
Positive control (5% SDS): 6 ± 0

Applicant's summary and conclusion

Interpretation of results:
other: Not irritating
Remarks:
In accordance with EU CLP (EC no 1272/2008 and its amendments)
Conclusions:
The substance does not cause skin irritation.
Executive summary:

In a study, performed according to OECD TG 439 and in compliance with GLP, the test substance was examined for its potential to cause in vitro skin irritation using the EpiDerm™ reconstructed skin membranes. The EpiDerm™ skin membranes were topically exposed to one dose level of the test substance (30 μL undiluted) for 1 h. After culturing for 42 h the viability of the epidermal cells was assessed using the MTT test. The general principle for the detection of viability via the MTT test is the conversion of the yellow tetrazolium salt (MTT) to the blue/purple coloured product formazan by mitochondrial enzymes. The formation of formazan was measured using a spectrophotometer. Phosphate-buffered saline and 5% sodium dodecyl sulphate were used as positive and negative control, respectively. Mean tissue viability of the positive control was 6%. Tissue viability following exposure to the substance was 93± 10% (mean ± SD) compared to the negative control. As this value was above the limit value of 50%, the substance was considered to be non-irritating to human skin.