Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 - 23 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

Cas Number:

30199-25-8

Molecular formula:

C11H20O

IUPAC Name:

(4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

Constituent 2

Reference substance name:

cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane

Cas Number:

69680-38-2

Molecular formula:

C12H24O2

IUPAC Name:

cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane

Test material form:

liquid

Details on test material:

- Physical State: Colourless liquid

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: N/A

In vitro test system

Test system:

human skin model

Source species:

other: MatTek EpiDerm Reconstructed Epidermis Model Kit (EpiDerm tissues Lot no.: 23356 and Assay medium Lot no.: 091516SLC)

Cell type:

other: epithelial

Cell source:

other: MatTek EpiDerm Reconstructed Epidermis Model Kit (EpiDerm tissues Lot no.: 23356 and Assay medium Lot no.: 091516SLC)

Source strain:

not specified

Details on animal used as source of test system:

MatTek EpiDerm Reconstructed Epidermis Model Kit
Date recieved: 20 Sep 2016
EpiDermTM Tissues (0.63cm2) lot number: 23356
Assay Medium lot number: 091516SLC
Stored refrigerated until use.

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: Tissues were incubated for 1 hour at 37 °C, 5% CO2 in 0.9 mL of pre-warmed assay medium.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 60 minute samples were incubated at 37 °C, 5% CO2 during the exposure phase. 3 min samples were not incubated (as the exposure period was too short).
- Temperature of post-treatment incubation (if applicable): Tissues were incubated at 37 °C, 5% CO2 for 3 hours post test item rinsing in the presence of MTT.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Trays were filled and emptied under a constant soft stream of DPBS until test item was removed.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: N/A
- Filter bandwidth: N/A

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
6 tissues were prepared for each of the 3 minute and 60 minutes exposure groups (2 x test item, 2 x negative control and 2 x positive control samples prepared per testing group).

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60‑Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean aborbency of test item / mean absorbency of negative control) x 100
Classification of corrosivity potential is based on relative viabilities for both exposure times according to Table 1.

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
- Concentration (if solution): N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): N/A

Duration of treatment / exposure:

Tissues exposed to the test item, positive control or negative control for 3 minutes or 60 minutes.

Duration of post-treatment incubation (if applicable):

Tissues incubated for 3 hours post-rinsing.

Number of replicates:

Duplicate replicates for each test group.

Test animals

Species:

other: N/A

Strain:

other: N/A

Test system

Type of coverage:

other: N/A

Preparation of test site:

other: N/A

Vehicle:

other: N/A

Amount / concentration applied:

N/A

Duration of treatment / exposure:

N/A

Observation period:

N/A

Number of animals:

N/A

Details on study design:

A 24‑well plate was prepared for use as a “holding plate” for both the 3‑Minute and 60‑Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24‑well plate was prepared for the MTT loading. 300 µL of either pre‑warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre‑incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3‑Minute exposure period was returned to the incubator, while the other was being dosed for the 60‑Minute exposure. For the 60‑Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60‑Minute exposure period.
When dosing for the 60‑Minute exposure period was complete, the same procedure was repeated for the 3‑Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24‑well plate prepared for MTT loading. The plate was incubated (37 C, 5% CO2) for 3 hours. Once the 60‑Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3‑Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24‑well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre‑labeled 96‑well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure period

Value:

107.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 % viability

Positive controls validity:

valid

Remarks:

4.0 % viability

Remarks on result:

other: no indication of corrosivity

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure period

Value:

72.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 % viability

Positive controls validity:

valid

Remarks:

3.4 % viability

Remarks on result:

other: no indication of corrosivity

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No.

- Direct-MTT reduction: 50 µL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.

The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

- Colour interference with MTT: 50 µL of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 oC, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

The solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 2.060 for the 3‑Minute exposure period and 2.017 for the 60‑Minute exposure period. The negative control acceptance criteria were therefore satisfied.

- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 3.4% relative to the negative control following the 60‑Minute exposure period. The positive control acceptance criterion was therefore satisfied.

- Acceptance criteria met for variability between replicate measurements:In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

- Range of historical values if different from the ones specified in the test guideline: N/A

In vivo

Irritant / corrosive response data:

Details of cell viability after 3 and 60 minutes exposures are described in Table 2.

Any other information on results incl. tables

Table 2: Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	MeanOD562of individual tissues	Mean OD562of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	2.017	2.060	0.061	3.0	100*
		2.103
	60 Minutes	2.082	2.017	0.093	4.6
		1.951
Positive Control	3 Minutes	0.091	0.082	0.013	na	4.0
		0.073
	60 Minutes	0.075	0.069	0.009	na	3.4
		0.062
Test Item	3 Minutes	2.235	2.217	0.025	1.1	107.6
		2.199
	60 Minutes	1.373	1.471	0.139	9.4	72.9
		1.569

OD = Optical density

*=  The mean % viability of the negative control tissue is set at 100%

na= Not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study the test substance is not considered to be corrosive in the in vitro skin corrosion test using the EpiDerm Skin Model.

Executive summary:

The skin corrosivity potential of reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP.

Duplicate tissues were treated with the test item for exposure periods of 30 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 562 nm of each well was measured using a Anthos 2001 microplate reader.

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 107.6 and 72.9 %, respectively. The quality criteria required for acceptance of the resuts was met.

Under the conditions of this study the test substance is not considered to be corrosive to the skin.