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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
Study period:
1st December 2020-7th December 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
[1S-(1α,3aβ,4α,8aβ)]-decahydro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene
EC Number:
207-491-2
EC Name:
[1S-(1α,3aβ,4α,8aβ)]-decahydro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene
Cas Number:
475-20-7
Molecular formula:
C15H24
IUPAC Name:
4,8,8-trimethyl-9-methylenedecahydro-1,4-methanoazulene
Test material form:
liquid
Specific details on test material used for the study:
Longifolene Coeur
Batch: hh-20190720-11
Purity: 94.6%
Expiry Date: 01 October 2021
Sponsor Appearance: Light yellow clear liquid
Covance Appearance: Clear colorless liquid
Storage Conditions: Room temperature, under nitrogen

In vitro test system

Test system:
human skin model
Source species:
other: The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen
Cell type:
non-transformed keratinocytes
Cell source:
other: The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
Source strain:
not specified
Details on animal used as source of test system:
N/A
Justification for test system used:
Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin models EpiSkin™ and EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential .

The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. 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 (see OECD TG 439) and is used for the purpose of classification as irritating or non-irritating according to chemicals law (EU CLP, UN GHS). The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- EpiSkinTM Tissues (0.38cm2) lot number : 20-EKIN-049
- Maintenance Medium lot number : 20-MAIN3-040
- Assay Medium lot number : 20-ESSC-039
- Purity 94.6%
- Delivery date: 01 December 2020
- Date of initiation of testing: 01/12/20

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time:
- Spectrophotometer:
- Wavelength:
- Filter:
- Filter bandwidth:
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function:
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied:
10 µL

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL

TEST ITEM PREPARATION
10 µL (26.3 µL/cm^2) of the undiluted test item were applied to each of the triplicate tissues
Duration of treatment / exposure:
15 minute
Duration of post-treatment incubation (if applicable):
42-Hour post-exposure incubation period.
Number of replicates:
3, triplicate tissues were treated with test substance, positive control or negative control.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15-Minute exposure period followed by the 42-Hour post-exposure incubation period
Value:
118.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The MTT solution containing the test item did not turn blue or purple which indicated that the
test item did not directly reduce MTT.

The solution containing the test item was colorless. It was therefore unnecessary to run color
correction tissues.

Any other information on results incl. tables

The individual and mean OD570 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.
The relative mean viability of the test item treated tissues was 118.2% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.
It was considered unnecessary to perform IL-1α analysis on the retained maintenance medium as the results of the MTT test were unequivocal.


Table 1:


































































Item



OD570 of


tissues



Mean OD570


of triplicate


tissues



± SD of


OD570



Relative


individual


tissue


viability (%)



Relative


mean


viability (%)



± SD of


Relative


mean


viability (%)



Negative


Control Item



0.867



 


0.844



 


0.025



102.7



 


100



 


2.9



0.818



96.9



0.846



100.2



Postive


Control Item



0.061



 


0.045



 


0.018



7.2



 


5.4



 


2.1



0.049



5.8



0.026



3.1



Test item



0.995



 


0.997



 


0.040



117.9



 


118.2



 


4.7



0.959



113.6



1.038



123.0



 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study and under the experimental conditions reported, the test item was classified as
non-irritant. The following classifications apply:

EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).
Executive summary:

Introduction


The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.


Method


Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm.
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).


 


Results


The relative mean viability of the test item treated tissues was 118.2% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.
Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.


Conclusion
In this study and under the experimental conditions reported, the test item was classified as non-irritant. The following classifications apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).