[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 26 April 2017 and 08 June 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:

bis(cyclohexylmethyl) ether

Cas Number:

14315-63-0

Molecular formula:

C14H26O

IUPAC Name:

bis(cyclohexylmethyl) ether

Test material form:

liquid

Remarks:

Clear, colorless

Specific details on test material used for the study:

Identification: FRET 13-0460
CAS No.: 14315-63-0
Appearance: Colourless, liquid
Storage Conditions: In the refrigerator

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes (NHEK)

Cell source:

other: derived from human keratinocytes

Source strain:

other: not applicable

Details on animal used as source of test system:

The In vitro Skin Corrosion: Human Skin Model Test is based on the observation that skin corrosion (necrotic damage of viable skin cells) shows a high correlation with skin cell cytotoxicity, occurring rapidly after brief exposure of the skin barrier (stratum corneum) to a corrosive chemical. It is designed to predict and classify the skin corrosivity potential of a chemical by using a three-dimensional human epidermis model. The epidermis model (e.g. EpiDermTM) is derived from human keratinocytes and consists of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK, which are cultured on specially prepared cell culture inserts using serum free medium, attain levels of differentiation at the cutting edge of in vitro skin technology. Ultrastructurally, the skin models closely parallel human skin. The In vitro Skin Corrosion: Human Skin Model Test consists of topical application of the test material to the tissue for 3 minutes and 1 hour, followed by immediate determination of the cytotoxic effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the exposure period.

EpiDerm™ Kit, Lot No.: 25817

Justification for test system used:

Validation studies have shown that tests employing human skin models are able to discriminate between known skin corrosives (Optional sub-category 1A, optional sub-category 1B and 1C) and non-corrosives. The test protocol may also enable the distinction between severe and less severe skin corrosives.

Vehicle:

unchanged (no vehicle)

Details on 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 on cool packs and on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 06 June 2017. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl

NEGATIVE CONTROL, Deionised water
- Amount(s) applied (volume or weight): 50 µl

POSITIVE CONTROL, Potassium Hydroxide
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

duplicate

Test system

Type of coverage:

other: dispensed directly onto duplicate EpiDermTM tissue surface.

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on study design:

Test for Direct MTT Reduction and Colour Interference
A test item may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT. The MTT assay is affected only if the test item is present in the tissues when the MTT viability test is performed.
Some non-coloured test items may change into coloured test items in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility should be performed (step 1).
Step 1
50 µL of the test item were added to 0.3 ml of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If the solution changed colour significantly, the test item is presumed to have the potential to stain the tissue. An additional test on viable tissues (without MTT addition) should be performed (step 2).
Since the test item did not dye water when mixed with it, step 2 did not have to be performed.
Step 3
All test items (including those already evaluated in step 1 and step 2) should be further evaluated for their potential to interfere with MTT. To test if an item directly reduces MTT, 50 µL of the test item were added to 1 ml of a MTT/DMEM solution (1 mg/mL) and were incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. If the MTT/DMEM solution (1 mg/mL) turns blue/purple, the test item reduces MTT and an additional test on freeze-killed tissues (step 4) must be performed.
Since the test item did not prove to be a MTT reducer, step 4 did not have to be performed.


EXPERIMENTAL PERFORMANCE
Pre-warming of EpiDerm™ Tissues 18 to 19 hours before dosing, EpiDerm™ tissues were unpacked, and the inserts were transferred into 6-well plates containing the pre-warmed assay medium under sterile conditions using sterile forceps. 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.

Treatment
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 with 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.

MTT Assay
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 nearly 20 hours without 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 x 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.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: Relative absorbance (percentage of negative control)

Run / experiment:

3 minute treatment

Value:

96.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

other: Relative absorbance (percentage of negative control)

Run / experiment:

60 minute treatment

Value:

103

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a relevant change in colour.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The test item is considered to be non-corrosive to skin:
• since the viability after 3 minutes exposure is greater than 50% and
• the viability after 1 hour exposure is greater than 15%.

The acceptance criteria are met:
• the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.455 to 1.575)
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (3.2%)
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 0.2% to 4.6%)

Any other information on results incl. tables

Results after treatment with FRET 13-0460 and the controls

Dose Group	Ex-posure Inter-val	Absor-bance Well 1 (Tissue 1/2)	Absor-bance Well 2 (Tissue 1/2)	Absor-bance Well 3 (Tissue 1/2)	Mean Absor-bance (Tissue 1/2)	Mean Ab-sorbance (OD) of 3 Wells minus Blank	Mean Ab-sorbance (OD) of 2 Tissues	Absor-bance [% of Negative Control]*	CV [%]	Rel. Absor-bance [% of Negative Control]*
Blank		0.037	0.037	0.037	0.037	0.000
Negative Control	3 minutes	1.562	1.522	1.526	1.537	1.500	1.502	99.8	0.2	100.0
		1.536	1.546	1.543	1.542	1.505		100.2
Positive Control		0.392	0.377	0.382	0.384	0.347	0.339	23.1	3.3	22.6
		0.368	0.368	0.368	0.368	0.331		22.0
Test Item		1.557	1.500	1.493	1.517	1.480	1.448	98.5	3.1	96.4
		1.472	1.448	1.440	1.453	1.416		94.3
Blank		0.036	0.036	0.041	0.038	0.000
Negative Control	1 hour	1.575	1.528	1.524	1.542	1.505	1.469	102.5	3.5	100.0
		1.481	1.476	1.455	1.470	1.433		97.5
Positive Control		0.082	0.082	0.082	0.082	0.044	0.047	3.0	8.1	3.2
		0.088	0.087	0.088	0.087	0.049		3.4
Test Item		1.635	1.584	1.581	1.600	1.562	1.512	106.4	4.6	103.0
		1.526	1.487	1.489	1.501	1.463		99.6

* relative absorbance [rounded values]: (100 x (absorbance _{test item / positive control})) / (absorbance _{negative control})

This in vitro study was performed to assess the corrosive potential of FRET 13-0460 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 EpiDerm^TMwere 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 nearly 20 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 (22.6%) and for the 1 hour exposure period (3.2%) thus confirming the validity of the test system and the specific batch of tissue models.

After exposure to the test item FRET 13-0460 the relative absorbance value decreased slightly to 96.4% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was not reduced (103.0%). Both values did not fall below 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:

The test item, FRET 13-0460, was classified as non-corrosive to the skin. The following classification criteria apply:
EU CLP (1272/2008/EC)/UN GHS: Not classified for corrosivity.
UN Packing Group: Non-Corrosive.

Executive summary:

The skin corrosivity of the test substance was determined according to OECD Guideline 431 using the EpiDerm™ Reconstructed Human Epidermis Model. The relative absorbance value after 3 and 60 minutes exposure were above 50% and 15% respectively in relation to the control. Therefore the substance is not corrosive to skin, according to EU CLP criteria.