4-methylcyclohexanone

Endpoint summary

Description of key information

Four studies addressing skin irritation/corrosion were available.

In a non-GLP, non-guideline study, undiluted test substance was applied to the skin of guinea pigs in a concentration range of 1.0 to 10.0 cc/kg (Eastman, 1959). Erythema, necrotic areas were observed. The study was assigned a Klimisch reliability rating of 4, due to the very minimal study details reported.

In a non-GLP, non-guideline study, undiluted test substance was applied to the skin of guinea pigs in a concentration range of 5.0 to 20.0 cc/kg (Eastman, 1960). Slight to gross edema, erythema and small to large necrotic areas were observed. The study was assigned a Klimisch reliability rating of 4, due to the very minimal study details reported.

Due to the age and insufficient documentation of the available information for the purposes of classification, and potential impact on permissible testing, modern in vitro studies were carried out.

The skin corrosivity potential of the test substance was assessed using the EpiDerm™ Human Skin Model, conducted according to guideline OECD 431 (Warren, 2017). The mean percent cell viability for the test substance at 3 and 60 minutes was 88.9% and 10.9%, respectively. Therefore under the conditions of this assay the test substance was considered to be Category 1B or 1C, corrosive to the skin. The guideline study conducted under GLP was assigned a Klimisch reliability rating of 1.

Due to potential false positive results in the EpiDerm study observed in an analogous substance, the skin corrosivity potential of the 4-methylcyclohexanone was confirmed using the in vitro Transcutaneous Electrical Resistance (TER) Assay, according to guideline OECD 430 (Pooles, 2017). 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. Test items that give a mean electrical resistance of 5 kΩ or less are considered likely to be corrosive in vivo. The mean electrical resistance ± SD for the test substance was 1.6 kΩ (±0.3). As a result, the test substance was considered to have the potential to cause corrosion in vivo. The guideline study conducted under GLP was assigned a Klimisch reliability rating of 1.

The two available in vitro studies for skin corrosion are considered to be adequate, relevant, and reliable for the purposes of classification and risk assessment. The data is considered conclusive, and no further in vivo testing is required.

According to Regulation (EC) No 1907/2006 (REACH) Annex VII Item 8.2.1 column 2, due to classification for skin corrosivity, the study on in-vitro eye irritation does not have to be performed.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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 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

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

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.

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.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 August 2016 to 25 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell source:

other: EpiDerm™ reconstructed human epidermis model kit

Source strain:

not specified

Justification for test system used:

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to predict the corrosivity potential of the test item.
This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ reconstructed human epidermis model kit (supplied by MatTek)
- EpiDermTM Tissues (0.63cm2) lot number: 23351
- Assay medium lot number: 081816TMA
- Delivery date: 24 August 2016
- Date of initiation of testing: 24 August 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

APPLICATION OF TEST MATERIAL AND RINSING
-Before pre-incubation was complete, 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 only 3 minutes, 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 Dulbecco’s phosphate buffered saline (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. Tissues 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 extract (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.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : Not required

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the relative mean tissue viability after 3 minutes exposure is less than 50% (Category 1A), or if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15% (Category 1B or 1C)
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

Duration of treatment / exposure:

Pre-incubation (37°C, 5% CO2) approx 1 h before dosing.
After pre-incubation of EpiDerm™ tissues, medium aspirated and replaced with 0.9 mL fresh assay medium. The 6-well plate for the 3-Min exposure returned to the incubator, while the other was being dosed for the 60-Min exposure. For the 60-min exposure period, 50 μL sterile distilled water (negative control) was added to the 1st two tissues. Tissues dosed at regular intervals allowing time to rinse each tissue following exposure and to ensure each tissue gets an equal exposure time. 50 μL of test item and 50 μL of 8.0 N potassium hydroxide (positive control) also applied to the corresponding tissues in turn. Plate returned to incubator (37°C, 5% CO2) for 60-min exposure period.
When dosing 60-Min exposure period complete, same procedure repeated for the 3-min exposure period. As exposure time was only 3 mins, tissues were dosed at regular intervals to ensure each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s phosphate buffered saline (DPBS) to gently remove any residual test item. Excess DPBS removed by blotting the bottom of the tissue insert with tissue paper. Each tissue placed into prepared holding plate until all tissues were rinsed. Tissues then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37°C, 5% CO2) for 3 h. Once 60-min exposure period complete, the same rinsing and MTT loading procedure was repeated.
After 3-h MTT incubation was complete, inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extract (isopropanol) used to completely immerse each insert and the plate covered with plate sealer to prevent isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure period

Value:

88.9

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure period

Value:

10.9

Negative controls validity:

valid

Positive controls validity:

valid

Table 1:Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Time	Exposure period (mins)	Mean OD562of individual tissues	Mean OD562of duplicate tissues	Standard deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative control	3	2.728 2.505	2.617	0.158	6.0	100
Negative control	60	2.629 2.639	2.634	0.007	0.3	100
Positive control	3	0.131 0.129	0.130	0.001	na	5.0
Positive control	60	0.134 0.133	0.134	0.001	na	5.1
CA5110	3	2.370 2.282	2.326	0.062	2.7	88.9
CA5110	60	0.286 0.288	0.287	0.001	0.5	10.9

 

Interpretation of results:

other: Category 1B (corrosive) or 1C (corrosive)

Conclusions:

The mean percent cell viability for the test substance at 3 and 60 minutes was 88.9% and 10.9%, respectively, therefore under the conditions of this assay the test substance was considered to be Category 1B or 1C, corrosive to the skin.

Executive summary:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean variabilities for each treatment group were as follows:

3 minute exposure: 100, 5.0 and 88.9% viability for negative control, positive control and test substance, respectively

60 minute exposure: 100, 5.1 and 10.9% viability for negative control, positive control and test substance, respectively

The quality criteria required for acceptance of results in the test were satisfied.

In conclusion, the mean percent cell viability for the test substance at 3 and 60 minutes was 88.9% and 10.9%, respectively, therefore under the conditions of this assay the test substance was considered to be Category 1B or 1C, corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study need not be conducted because the available information indicates that the criteria are met for classification as corrosive to the skin or irritating to eyes

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Two in-vitro skin corrosion studies according to OECD 430 and 431 gave positive results for corrosivity.

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a TER assay (Pooles, 2017), the mean electrical resistance for the test substance was 1.6 kΩ (±0.3). Test items that give a mean electrical resistance of 5 kΩ or less are considered likely to be corrosive in vivo. As a result 4-methylcyclohexanone is classified as Corrosive Category 1.

In an EpiDerm™ Human Skin Model assay (Warren, 2017), the mean percent cell viability for the test substance at 3 and 60 minutes was 88.9% and 10.9%, respectively. Therefore under the conditions of this assay the 4-methylcyclohexanone was considered to be Category 1B or 1C, corrosive to the skin.

Both studies are considered valid and in agreement. The EpiDerm assay has been validated to further differentiate between Category 1A and Category 1B or 1C, and as a result, 4-methylcyclohexanone is classified as Category 1B or 1C.