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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:
2018-01-15 to 2018-01-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz (8.4.2015)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
additive
Test material form:
liquid
Details on test material:
- State of aggregation: clear liquid
Specific details on test material used for the study:
The test item was stored in the test facility in a closed vessel at room temperature (20 ± 5°C) and kept under inert gas.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.
Details on animal used as source of test system:
not applicable
Justification for test system used:
This in vitro study was performed in order to evaluate the potential of hexanal to evoke skin irritation in a Reconstructed human Epidermis (RhE) test method.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm TM
- Tissue batch number(s): 25874
- Delivery date: 2018-01-16
- Date of initiation of testing: 2018-01-15 (pre-test)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C (35 min) room temperature (25 min)
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsing after exposure, after post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-well plate. Then the inserts were transferred into the lower row of the 6-well plate

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Microtiter plate photometer Anthos Reader 2010 Flexi (Anthos Microsysteme GmbH)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.71 ± 0.09
- Barrier function: 5.28 h
- Morphology: presence of functional stratum corneum, a viable basal layer, and intermediate spinous and granular layers
- Contamination: sterile (no bacteria, yeast, other fungi, no HIV-1, Hepatitis B, Hepatitis C virus)

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 60 minutes exposure is less than or equal to 50%
- The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than 50%
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): 100 %

VEHICLE
no vehicle used

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 100 %

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 %
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42.5 h
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
2.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
2.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
2.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
2.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
The OECD Guideline 439 addresses the human health endpoint skin irritation and does not allow a discrimination between skin irritation and skin corrosion.
Conclusions:
The test item hexanal is considered as at least irritant to skin. After the treatment, the mean value of relative tissue viability was reduced to 2.5 %. This value is well below the threshold for skin irritation (50 %). The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8. The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18 %). For these reasons, the result of the test is considered valid.
Executive summary:

One valid experiment according to OECD Guideline 439 following GLP with three tissues and two independent MTT measurements was performed. The tissues of the human skin model EpiDermTM were treated with hexanal for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier). DPBS-buffer was used as negative control and 5 % SDS solution was used as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.8. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 1.9 % (required: <= 20 %). The variation within the tissue replicates of negative, control, positive control and test item was acceptable (well below 18 %). Thus, confirming the validity and sensitivity of the test system. After the treatment with the test item, the mean value of relative tissue viability was only 2.5 % (reduction by 97.5 %). This value is well below the threshold for a skin irritation potential (50 % viability). Test items that induce viability values below the threshold of 50 % are considered to be at least irritant to skin. Therefore, hexanal is considered at least irritant to skin in the Reconstructed human Epidermis (RhE) Test Method. The OECD Guideline 439 addresses the human health endpoint skin irritation and does not allow a discrimination between skin irritation and skin corrosion.