Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 February 2017-08 March 2017

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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

EC Number:

268-582-0

EC Name:

Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

Cas Number:

68130-25-6

Molecular formula:

C10 H20 O2 . x C8 H16 O2 . x C8 H16 O2 . x C5 H12 O4

IUPAC Name:

Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

Test material form:

liquid

Details on test material:

Identification: H-32
CAS No.: 68130-25-6
Batch: 64296
Purity: > 99%
Appearance: Pale yellow liquid
Expiry Date: 30 September 2018
Storage Conditions: At room temperature
Stability in Solvent: Not relevant
Purpose of Use: Industrial chemical

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

Epi-200 kits and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia). The EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ∅).
EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 07 March 2017. On day of receipt the preincubation phase of the EpiDerm™ tissues started.

Vehicle:

unchanged (no vehicle)

Details on test system:

At least 1 hour before dosing, EpiDerm™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed assay medium. A 24-well plate was prepared as holding plate containing 300 μL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.

Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control for the following exposure times:
• Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
• Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
• Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes
After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle / multipipette containing DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 μL) was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required.
Following rinsing, the tissues were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the tissues were rinsed three times with DPBS and carefully dried with blotting paper. The inserts were transferred into new 24-well plates. The tissues were each immersed in 2 mL of extractant solution (isopropanol) pipetted in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for 2 hours while shaking at room temperature. After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24- well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour. 3 × 200 μL aliquots of the blue formazan solution were transferred from each tissue into a 96- well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL (79.4 μL/cm2 according to guideline) of the test item were dispensed directly onto duplicate EpiDermTM tissue surface.
Concurrent controls were used for several Envigo CRS GmbH studies performed simultaneously. Each 50 μL were applied to each set of duplicate tissues for the 3 min and 1 hour exposure periods.

Duration of treatment / exposure:

3 minutes and 60 minutes

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: relative absorbance value %

Run / experiment:

3 mins exposure

Value:

95.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

other: relative absorbance value %

Run / experiment:

60 mins exposure

Value:

90.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

This in vitro study was performed to assess the corrosive potential of H-32 by means of the Human Skin Model Test with EpiDerm™ tissues models. The test item passed the MTT- and the Colour Interference pre-tests. Independent duplicate tissues of EpiDermTM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour,

respectively. Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then

aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for 2 hours at room temperature.

The required acceptability criteria were met. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (25.8%) and for the 1 hour exposure period (2.3%) thus confirming the validity of the test system and the specific batch of tissue models. After exposure to the test item H-32 the relative absorbance value only decreased slightly to 95.9% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 90.7%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, H-32 is non corrosive to skin according to EU CLP and UN GHS.

Executive summary:

This in vitro study was performed to assess the corrosive potential of H-32 by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not dye deionised water or changed colour when mixed with it (pre-test for colour interference).

Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the

acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus

the validity of the test system and the specific batch of the tissue models is confirmed. After exposure of the tissues to the test item the relative absorbance value decreased to 95.9% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 90.7%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item H-32 is non corrosive to skin according to EU CLP and UN GHS.