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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:
2015-06-25 to 2015-07-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methylpropyl-(R)-2-hydroxypropanoate
EC Number:
407-770-0
EC Name:
2-methylpropyl-(R)-2-hydroxypropanoate
Cas Number:
61597-96-4
Molecular formula:
C7H14O3
IUPAC Name:
2-methylpropyl-(R)-2-hydroxypropanoate
Test material form:
other: colourless liquid
Details on test material:
- Name of test material (as cited in study report): Isobutyl-R-lactate
- Physical state: liquid
- Analytical purity: 99.7 %
- Lot/batch No.: 1411000032
- Expiration date of the lot/batch: 2016-11-09
- Storage condition of test material: at room temperature
- Other: colourless

Test animals

Species:
other: human epidermis model:Episkin
Details on test animals or test system and environmental conditions:
Test system:
The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal (non-cancerous), adult human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Test system

Type of coverage:
not specified
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit: 10 µL (26.3 µL/cm²)

PREPARATION OF THE TEST ITEM:
The test item was applied undiluted

CONTROLS:
Controls were set up in parallel to the test item in order to confirm the validity of the test.
Negative control: DPBS (Gibco, Cat. No. 14040-091, Lot No.: 1672813).
Positive control: 5 % sodium dodecyl sulfate (SDS; AppliChem, Art.-No. A1112,0500, CAS No.: 151-21-3, Lot No.: 1V010396) in aqua dest.
Duration of treatment / exposure:
15 ± 0.5 min
Details on study design:
TEST SITE
- Area of exposure: EPISKIN-SM tissue
- size per reconstructed epidermis unit: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing (if done): after 15 ± 0.5 min with DPBS

STUDY DESIGN:
Upon receipt of the EPISKIN-SM tissues, the tissues were transferred into 12-well plates containing 2 mL pre-warmed maintenance medium per well, and were incubated in a humidified incubator at 37 ± 1 °C, 5.0 % CO2 for at least 24 h.
After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the tissues were incubated at room temperature for 15 ± 0.5 min. The positive control was re-spread after 7 min contact time. Afterwards, the tissues were washed with DPBS to remove any residual test item. Excess DPBS was removed by blotting bottom with blotting paper. The inserts were placed in a prepared 12-well plate containing 2 mL pre-warmed fresh maintenance medium and post-incubated at 37 ± 1 °C, 5.0% CO2 for 42 ± 1 h.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 12-well plate containing 2 mL pre-warmed MTT medium and further incubated for 3 h ± 5 min. at 37 ± 1 °C, 5.0 % CO2.
After the 3 h MTT incubation period the tissues were placed on blotting paper to dry. Afterwards a total biopsy of the epidermis using the special biopsy punch was performed and the epidermis was separated from the collagen matrix with the aid of forceps. Both parts (epidermis and collagen matrix) were transferred into suitable tubes and 500 μL of acidic isopropanol were added. The plugged tubes were mixed using a vortex mixer. Extraction was carried out protected from light over the weekend at 2-8 °C.
At the end of the formazan extraction period the tubes were mixed by vortexing until solution colour became homogeneous.
If any visible cell/tissue fragments were in suspension, the tubes were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
Per each tissue 2 × 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer.

SCORING SYSTEM:
The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 [18] and UN GHS [16], [17] “Category 2”, if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50 %. The test substance may be considered as non-irritant to skin in accordance with UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is higher than 50 %.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: mean relative tissue viability
Value:
ca. 88.5
Remarks on result:
other:
Remarks:
Basis: mean (of triplicates). Time point: 15 min treatment and 42h post-incubation. Max. score: 100.0. Remarks: Max. score: 100 % viability obtained by the negative control. (migrated information)

Any other information on results incl. tables

Table 1: Results of the test item isobutyl-R-lactate

Name Negative Control Positive Control Test Item
Tissue 1 2 3 1 2 3 1 2 3
Absolute OD 570 0.815 0.726 0.989 0.083 0.093 0.070 0.650 0.911 0.693
0.835 0.725 1.008 0.085 0.106 0.073 0.673 0.898 0.715
OD570 (Corrected) 0.775 0.687 0.950 0.043 0.053 0.030 0.610 0.872 0.653
0.796 0.686 0.968 0.045 0.067 0.033 0.633 0.859 0.675
Mean OD 570 (Corrected) 0.810* 0.045 0.717
SD OD 570 0.138 0.014 0.130
Relative Tissue Viabilities [%] 97.0 84.7 118.4 5.5 7.4 3.9 76.8 106.8 82.0
Mean Relative Tissue Viability [%] 100 % 5.6** 88.5
SD Tissue Viability [%]*** 17 1.7 16.0

*Corrected mean OD 570 of the negative control corresponds to 100 % absolute tissue viability

**Mean relative tissue viability of the three positiv control tissues is ≤ 40 %

***Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18 %.

Table 2: Test acceptance criteria

  Value Cut off pass/fail
Mean OD570 nm Blank 0.039 < 0.1 pass
Mean Absolute OD570 nm NK 0.85 0.6 ≤ NK ≤1.5 pass
Mean Relative Viability [%] PC 5.6 ≤ 40 % pass
SD of % Viability [%] 1.7–17.0 ≤ 18 % pass

NK: negative control

PC: positive control

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, in the human skin model EPISKIN-SM (SkinEthic) no irritant effects after treatment with isobutyl-R-lactate were observed.
Executive summary:

In the present study 10 µL of undiluted isobutyl-R-lactate (99.7 % purity) was applied directly atop the EpiSkin-SMTM tissue for 15 min followed by 42 h post incubation period and immediate determination of cytotoxic effects via MTT reduction assay in accordance to OECD guideline 439.

Irritant potential from the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control. No irritant effects were observed. The mean relative tissue viability (% negative control) was > 50 % (88.5 %) after 15 min treatment and 42 h post-incubation. The controls confirmed the validity of the study.