4-methylcyclohexanone

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 July 2017 to 28 July 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 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-methylcyclohexanone

EC Number:

209-665-3

EC Name:

4-methylcyclohexanone

Cas Number:

589-92-4

Molecular formula:

C7H12O

IUPAC Name:

4-methylcyclohexan-1-one

Test material form:

liquid

Details on test material:

- Other: Colourless liquid

In vitro test system

Test system:

isolated skin discs

Source species:

rat

Source strain:

Wistar

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Envigo RMS (UK) Limited, Oxon, UK.
- Sex: Female
- Age at study initiation (in days): 21-23 days
- Housing: Suspended solid-floor polypropylene cage furnished with woodflakes.
- Diet: 2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK ad libitum.
- Water: Mains drinking water ad libitum)

Justification for test system used:

Corrosive substances produce an irreversible loss of normal stratum corneum integrity and function, this is measured as a reduction in the inherent Transcutaneous Electrical Resistance below a corrosive threshold level (5 kΩ). The TER was measured using a low voltage, alternating current, electronic databridge.
Rats are the preferred species of choice due to the development of quantitative methods for the measurement of skin corrosivity in the rat and are specified in the appropriate test guidelines.

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: Following an acclimatization period of 2 days the animal was shaved to remove hair from the dorsal surface. The shaved area was washed using an antibiotic wash. After 3 days a second antibiotic wash was performed. Three days later the animal was killed using ascending concentrations of carbon dioxide followed by cervical dislocation. The animal was in the telogen phase of hair growth and little or no hair growth was visible.
When the animal had been humanely killed the dorsal skin was removed from the rat as a single pelt. Care was taken during the procedure to avoid unnecessary damage to the pelt. Excess fat was removed and the pelt mounted, epidermal side uppermost, onto a polytetrafluoroethylene (PTFE) tube. The tissue was secured in place using a rubber “O” ring. Excess tissue was trimmed away and the “O” ring/PTFE interface sealed with soft paraffin wax. The tube was supported by a clamp inside a labelled 30 mL glass receptacle containing 10 mL electrolyte solution (154 mM MgSO4).

- Quality control for skin discs: Two skin discs of approximately 0.79 cm2 were taken from the pelt and the TER measured as a quality control procedure. Each disc had to give a resistance value of greater than 10 kΩ in order for the remainder of the pelt to be used in the assay. If either disc fell below the 10 kΩ threshold, the pelt was discarded. The quality control discs were then discarded and new discs from the acceptable pelt were mounted on the PTFE tubes.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 20-23°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: At the end of the exposure period, the test item was removed by washing the skin disc with a jet of warm tap water until no further test item could be removed.

DETERMINATION OF TRANSCUTANEOUS ELECTRICAL RESISTANCE
The TER was measured using a Wheatstone Bridge with a low voltage alternating current. Prior to measurement of the resistance, the surface tension of the skin disc was reduced by adding a sufficient volume of 70% ethanol to cover the epidermis. The ethanol was removed by inverting the tube after approximately 3 seconds. The PTFE tube was then placed in the labelled receptor chamber and the tissue was hydrated by the addition of 3 mL MgSO4 solution (154 mM) to the inside of the PTFE tube. Any air bubbles present were dislodged by tapping the tube.

The stainless steel electrodes of the databridge were placed on either side of the skin disc. The measurement was taken and a value in Ω/kΩ per skin disc was displayed on the databridge display. The mean TER for the skin discs was calculated.

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3 skin discs per treatment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean TER value recorded for the 24 hour contact period is 5 kΩ or lower and the skin disc is obviously damaged or if the mean TER value is 5 kΩ or lower, showing no obvious damage but the mean disc dye content is greater than or equal to the mean disc dye content for the 10M Hydrochloric acid.
- The test substance is considered to be non-corrosive to skin if the mean TER value recorded for the 24 hour contact period is greater than 5 kΩ or if the mean TER value is 5 kΩ or lower showing no obvious damage, with the mean disc dye content below the mean disc dye content for 10M Hydrochloric acid.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 150 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 150 μL distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 150 μL
- Concentration (if solution): 10M hydrochloroc acid (approximately 36%)

Duration of treatment / exposure:

24 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

Mean of three replicates

Value:

1.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

standard deviation ± 0.3

Any other information on results incl. tables

Table 1: Individual and Mean Transcutaneous Electrical Resistance Measurements

Treatment	Tissue Number	TER	Mean TER	Standard Deviation
Test item	1 2 3	1.9 kΩ 1.3 kΩ 1.7 kΩ	1.6 kΩ	± 0.3
Positive Control (10M hydrochloric acid (approximately 36%))	1 2 3	- 845 Ω 852 Ω	848.5 Ω	± 4.9
Negative Control (Distilled water)	1 2 3	22.1 kΩ 15.6 kΩ 25.6 kΩ	21.1 kΩ	± 5.1

 

A visual inspection of the three skin discs treated with the test item showed all three to be very pale (bleached) and thin. These reactions were considered indicative of dermal corrosion and the dye binding/penetration assessment was not required.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

The test substance was considered to have the potential to cause corrosion in vivo.

Executive summary:

The skin corrosivity potential of the test substance was assessed using the Transcutaneous Electrical Resistance (TER) Assay. The integrity of the pelt was confirmed using a Quality Control test. The test item was applied to the epidermal surface of three skin discs mounted on a polytetrafluoroethylene (PTFE) tube for a contact period of 24 hours at 20-23°C. At the end of the contact period the test item was removed using a jet of warm tap water until no further material can be removed. Corrosive substances produce an irreversible loss of normal stratum corneum integrity and function, this is measured as a reduction in the inherent TER. Test items that give a mean electrical resistance of 5 kΩ or less are considered likely to be corrosive in vivo. The TER was measured using a low voltage alternating current electronic databridge.

The mean electrical resistance ± SD for the test substance, positive control and negative control was 1.6 kΩ (±0.3), 848.5 Ω (±4.9) and 21.1 kΩ (±5.1), respectively.

The test substance was considered to have the potential to cause corrosion in vivo.