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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:
2017-03-16 to 2017-05-05
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
23 July 2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-hydroxypropyl octanoate
Cas Number:
102731-54-4
Molecular formula:
C11H22O3
IUPAC Name:
3-hydroxypropyl octanoate

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: donors with stated consent
Justification for test system used:
Dermal irritation is generally defined as "the production of reversible inflammatory changes
in the skin". The potential for chemical induced skin irritation is an important consideration
in establishing procedures for the safe handling, packing and transport of chemicals. It is
usually determined in vivo in the Draize rabbit skin irritation test as described in OECD
guideline 404. 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 prevalidation study performed by ECVAM, the in vitro skin irritation test using
the human skin model EpiDerm™ and EpiSkin™ 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.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 SIT kits, EpiDerm™
- Tissue batch number: 25810
- Delivery date: 2017-05-03
- Date of initiation of testing: 2017-05-03 start of pre-incubation

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min, roomt temperature for 25 min
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: at least 15
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Microplate reader: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No MTT interference

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability is less or equal to 50 %.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50 %
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
each 30 μL
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42.2 h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
81.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
4.5
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent showed weakly coloured purple oily drops. The reaction was very poor, therefore, an additional test with freeze-killed tissues was not performed, since the outcome of the test would not have been influenced importantly.
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

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
- Range of historical values: positive control 4.37 +/- 21.6 % viability; negative control 1.74 +/- 9.4 Absorbance

Any other information on results incl. tables

Results




























































































































Dose
Group
Tissue
No.
Absorbance
570 nm
Well 1
Absorbance
570 nm
Well 2
Absorbance
570 nm
Well 3
Mean Absorbance
of
3 Wells
Mean-
Absorbance
of three
Wells
Blank
corrected
Mean
Absorbance
of 3
Tissues
after Blank
Correction
Rel.
Absorbance
[%]
Tissue
1, 2 + 3
Relative
Standard
Deviation
[%]
Mean Rel.
Absorbance
[% of
Negative
Control]
Blank 0.0380.0380.0360.0370    
Negative
Control
11.5671.5181.5371.5411.5041.38108.98.6100
21.4051.4071.4141.4091.37299.4
31.2941.3081.3071.3031.26691.7
Positive
Control
10.0960.1020.1010.10.0620.0624.55.14.5
20.0970.0950.0950.0960.0594.3
30.1020.1040.1010.1020.0654.7
Test Item11.2221.3111.3141.2821.2451.12990.210.881.8
21.0431.0441.0321.041.00272.6
31.1611.1721.1951.1761.13982.6

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the study according to OECD 439 the test item was determined to be not irritant to skin.
Executive summary:

The in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.


The colourless test item did not change colour when mixed with deionised water (pre-test for colour interference). An additional test with viable tissues (without MTT addition) was not necessary. In the pre-test for direct MTT reduction a few very weak purple coloured oily drops were observed indicating a slight MTT reducing potential of the test item. Since the reaction was extremely poor, it was relinquished to perform an additional test with freezekilled tissues to determine a correction factor for calculating the true viability in the main experiment. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SLS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system. After treatment with the test item BIO4307/2 the mean relative absorbance value decreased to 81.8 % compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin.

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