2-methylcyclohexanone

Endpoint summary

Description of key information

To assess Irritation/corrosion potential of 2-MCH, several studies were performed:

In vitro Skin Corrosion: Human Skin Model Test (EpiDerm^TM), In vitro Skin Irritation: Human Skin Model Test (EpiDerm^TM) and Eye Irritancy Potential using the Bovine Corneal Opacity and Permeability Assay according to OECD Guidelines 431, 439 and 437 respectively.

1. OECD 431: The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 50% (96.3%) after 3 min treatment and ≥ 15% (66.3%) after 60 min treatment.

2. OECD 439: In this study under the given conditions the test item showed irritant effects. The test item is therefore classified as “irritant” in accordance with UN GHS “Category 2”.

3. OECD 437: Under the conditions of this experiment - 2-methylcyclohexanone is not eye damaging (Cat. 1). However, no prediction can be made regarding whether it is eye irritating or not classifiable for eye effects.

 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date: 29 October 2019
Experimental Starting Date: 14 November 2019
Experimental Completion Date: 06 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Final

Deviations:

no

Remarks:

There were no deviations from the study plan.

GLP compliance:

yes

Specific details on test material used for the study:

Prior to treatment, all EpiDerm™ tissues were gently blotted to remove moisture. The test item was applied undiluted. 30 µL (47 µL/cm2) of the test item were dispensed directly atop the EpiDerm™ tissue. The test item was gently spread to match size of the tissue using a bulb-headed Pasteur pipette.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

The test was carried out with the reconstituted three-dimensional human skin model EpiDerm™
(MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have
been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (MillicellR). The EpiDerm™ epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was applied undiluted. 30 µL (47 µL/cm2) of the test item were dispensed directly atop the EpiDerm tissue.

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

All plates were incubated for 25 ± 1 min under the sterile flow and for the remaining time of 35 ± 1 min transferred to the incubator. Then the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, in one-minute intervals. Subsequently, the inserts were completely submerged 3 times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper. The inserts were placed in prepared new 6-well plates containing 0.9 mL pre-warmed fresh assay medium per well and the tissue surface was dried using a sterile cotton tip. The plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h. After this post-incubation period the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a 24-well plate containing 300 µL pre-warmed MTT medium.
This plate was incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%.
After the MTT incubation, the tissues were rinsed 3 times with DPBS and afterwards placed on blotting paper to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature for at least 2 h with gentle shaking on a plate shaker.
Before using the extracts, the plate had been shaken for 15 min and the inserts were pierced with an injection needle.The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate.OD was measured at 570 nm in a plate
spectrophotometer using isopropanol as a blank

Number of replicates:

The test was performed on a total of 3 tissues per dose group.

Irritation / corrosion parameter:

% tissue viability

Value:

4.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The test item showed irritant effects.

Pre-Experiments
The mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the
solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
The mixture of 30 µL of the test item per 300 µl aqua dest. and/ per 300 µL isopropanol showed no
colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.
The test item showed no non-specific reduction of MTT and no relevant colouring potential after
mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of
possible false-negative results were necessary.

Table 1. Result of the Test Item 2-MCH

Name	Negative Control					Positive Control					Test Item
Replicate Tissue	1		2		3	1		2	3		1		2	3
Absolute OD570	1.316		1.328		1.317	0.096		0.097	0.095		0.093		0.094	0.096
	1.312		1.315		1.315	0.094		0.097	0.100		0.097		0.101	0.103
Mean Absolute OD570	1.317****					0.097					0.097
OD570 – (Blank Corrected)	1.271		1.282		1.271	0.050		0.051	0.050		0.048		0.049	0.051
	1.266		1.270		1.270	0.049		0.051	0.055		0.051		0.055	0.057
Mean OD570 of the Duplicates (Blank Corrected)	1.268		1.276		1.270	0.049		0.051	0.052		0.050		0.052	0.054
Total Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	1.271*					0.051					0.052
SD of Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	0.004					0.002					0.002
Relative Tissue Viability [%]	99.8	100.3		99.9		3.9	4.0			4.1	3.9	4.1			4.2
Mean Relative Tissue Viability [%]	100.0					4.0**					4.1
SD of Relative Tissue Viability [%]***	0.3					0.1					0.2
CV [% Viabilities]	0.3					3.0					4.3

*
Blank-corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.
**
Mean relative tissue viability of the three positive control tissues is ≤ 20%.
***
Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.
****
The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed irritant effects. The test item is therefore classified as “irritant” in accordance with UN GHS “Category 2”.

Executive summary:

In the present study the skin irritant potential of 2-MCH was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404, [7]) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.
The mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
The mixture of 30 µL of the test item per 300 µl aqua dest. and/ per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.
The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.
The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (4.1%) after 60 min treatment and 42 h post-incubation.

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.316	1.328	1.317	0.096	0.097	0.095	0.093	0.094	0.096
	1.312	1.315	1.315	0.094	0.097	0.100	0.097	0.101	0.103
Mean Absolute OD570	1 317****			0.097			0.097
OD570 (Blank Corrected)	1.271	1.282	1.271	0.050	0.051	0.050	0.048	0.049	0.051
	1.266	1.270	1.270	0.049	0.051	0.055	0.051	0.055	0.057
Mean OD570 of the Duplicates (Blank Corrected)	1.268	1.276	1.270	0.049	0.051	0.052	0.050	0.052	0.054
Total Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	1.271*			0.051			0.052
SD of Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	0.004			0.002			0.002
Relative Tissue Viability [%]	99.8	100.3	99.9	3.9	4.0	4.1	3.9	4.1	4.2
Mean Relative Tissue Viability [%]	100.0			4.0**			4.1
SD of Relative Tissue Viability [%]***	0.3			0.1			0.2
CV [% Viabilities]	0.3			3.0			4.3

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April-May 2019

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Final

Deviations:

no

Remarks:

There were no deviations from the study plan

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The test item was used as provided by the sponsor.

Species:

cattle

Details on test animals or tissues and environmental conditions:

Room temperature, protected from light

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

750 µL of the test substance or control

Duration of treatment / exposure:

10 minutes incubation at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 hours incubation at 32 ± 1 °C

Number of animals or in vitro replicates:

3 replicates for each group

Irritation parameter:

in vitro irritation score

Value:

25.89

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

In Vitro Irritation Score

Cornea No.  Test Item     Corrected            Corrected          IVIS
                                  Opacity Value      OD490 Value

     1                                 0.39                 0.005

     2          Negative          0.53                 0.006

     3          Control            0.35                 0.001

    MV                               0.43                 0.004              0.49

     4                              18.66                 1.821

     5         Positive          23.47                 1.044

     6         Control           21.82                 1.756

    MV                             21.32                 1.540             44.42

     7                               8.62                  1.035

     8        Test item          7.69                  1.261

     9                               8.29                  1.243

    MV                             8.20                  1.180             25.89

 

MV=mean value
                                  

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this experiment - 2-methylcyclohexanone is not eye damaging (Cat. 1). However, no prediction can be made regarding whether it is eye irritating or not classifiable for eye effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation: The results of the in vitro test according to OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method) indicate that the 2-methylcyclohexanone showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (4.1%) after 60 min treatment and 42 h post-incubation. The test item is therefore classified as “irritant” in accordance with UN GHS “Category 2”.

Skin corrosion: The results of the in vitro test according to OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method) indicate that the 2-methylcyclohexanone showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 50% (96.3%) after 3 min treatment and ≥ 15% (66.3%) after 60 min treatment. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.2.

Eye irritation: The results of the in vitro OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage) show that 2-methylcyclohexanone is not eye damaging (Cat. 1). However, no prediction can be made regarding whether it is eye irritating or not classifiable for eye effects.