cis-2-tert-butylcyclohexan-1-ol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Test material

Test material information

Constituent 1

Reference substance name:

cis-2-tert-butylcyclohexan-1-ol

EC Number:

230-601-5

EC Name:

cis-2-tert-butylcyclohexan-1-ol

Cas Number:

7214-18-8

Molecular formula:

C10H20O

IUPAC Name:

cis-2-tert-butylcyclohexan-1-ol

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes (NHEK)

Cell source:

other: EpiDerm tissues were provided as kits (MatTek).

Justification for test system used:

This in vitro method allows the identification of corrosive and non-corrosive substances and mixtures in accordance with UN GHS. It further allows a partial sub-categorisation of corrosives in optional subcategory 1A or a combination of optional sub-categories 1B and 1C.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm(MatTek)
- Procedure: Upon receipt of the EpiDerm the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37 ±1 °C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 0.9 mL fresh assay medium, and the surface was dried using a sterile cotton tip. The 6-well plate used for the 3 min experiment was placed back into the incubator. The other plate was used for the 60 min treatment. About 1 h before the end of the first treatment period the MTT solution was prepared and pre-warmed in the incubator.

60 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of e.g. 20 sec. was kept between dosing. Then the 6-well plate was incubated at 37 ±1 °C, 5.0% CO2 / 95% air.
3 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 sec. was kept between dosing.

REMOVAL OF TEST MATERIAL AND CONTROLS: After the exposure period, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate” containing 300 μL prewarmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate” containing 300 μL pre-warmed MTT solution. The plate was incubated for 3 h at 37±1 °C, 5.0% CO2 / 95% air.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE: After the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. Then the inserts were transferred into 12-well “extraction plates”. 2 mL of isopropanol were pipetted into each insert: thus, the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out either over night without shaking at room temperature or, alternatively, at least 2 h with shaking at room temperature. After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min. Per each tissue 3 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

NUMBER OF REPLICATE TISSUES: 4 tissues per dose group, 2 replicates for each treatment period
(3 min and 60 min exposure time).

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

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Not applicable

PREDICTION MODEL / DECISION CRITERIA:
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 60 min exposure is less than 15% (combination of optional sub-categories 1B and 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 60 mins exposure is greater than or equal to 15%.
- The test substance is considered to be sub-category 1A (optional) to skin if the viability after 3 minutes exposure is less than 25%.
- The test substance is considered to be sub-category 1B and 1C (a combination of optional) if the viability after 3 minutes exposure is greater than or equal to 25%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg / 25 µl H2O

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

The test was performed on a total of 4 tissues per dose group, 2 replicates for each treatment period
(3 min and 60 min exposure time).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min treatment

Value:

96.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min treatment

Value:

67.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues was ≥0.8 and ≤ 2.8 for each exposure period (1.887, 1.861). The mean relative tissue viability (% negative control) of the positive control was ≤ 15% (7.3%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤30% (0.7% - 4.4%).

Any other information on results incl. tables

Pre-Experiments: The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixture of 25 mg test item per 300 μL Aqua dest. and per 300 μL isopropanol showed no colouring as compared to the solvent. 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.

Results of 3 min Experiment:

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.925	1.878	0.168	0.285	1.834	1.729
	1.870	1.876	0.168	0.280	1.886	1.808
	1.893	1.877	0.168	0.282	1.874	1.804
Mean Absolute OD570	1.887****		0.225		1.823
OD570 - Blank Corrected	1.879	1.832	0.122	0.239	1.788	1.683
	1.824	1.830	0.122	0.234	1.840	1.762
	1.847	1.831	0.122	0.236	1.828	1.758
Mean OD570 of 3 Aliquots (Blank Corrected)	1.850	1.831	0.122	0.237	1.819	1.734
SD OD570  of 3 Aliquots	0.028	0.001	0.000	0.003	0.027	0.044
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.841*		0.179		1.777
SD OD570 of 2 Replicate Tissues	0.014		0.081		0.060
Mean Relative Tissue Viability [%]	100.0		9.7		96.5
Coefficient Of Variation [%]^***	0.7		45.3		3.4

^*               corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability

^***             coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is £ 30%

^****            The mean absolute OD₅₇₀ of the negative control is ≥ 0.8 and ≤ 2.8

Results of 60 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.769	1.793	0.216	0.129	1.233	1.241
	1.866	1.939	0.233	0.130	1.340	1.241
	1.867	1.935	0.233	0.130	1.334	1.196
Mean Absolute OD570	1.861****		0.178		1.264
OD570 - Blank Corrected	1.723	1.747	0.170	0.083	1.187	1.196
	1.821	1.893	0.188	0.084	1.295	1.195
	1.821	1.889	0.187	0.084	1.289	1.150
Mean OD570 of 3 Aliquots (Blank Corrected)	1.788	1.843	0.182	0.084	1.257	1.180
SD OD570  of 3 Aliquots	0.057	0.083	0.010	0.001	0.060	0.026
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.816*		0.133		1.219
SD OD570 of 2 Replicate Tissues	0.039		0.069		0.054
Mean Relative Tissue Viability [%]	100.0		7.3**		67.1
Coefficient Of Variation [%]^***	2.1		52.2		4.4

^*               corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability

^**              mean relative tissue viability of the 60 min positive control is £ 15%

^***             coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is £ 30%

^****            The mean absolute OD₅₇₀ of the negative control is ≥ 0.8 and ≤ 2.8

Historical Data:

	Mean	SD	Range of LCL – UCL	n
OD570 of NC (3 min Experiment)	1.754	0.223	1.308– 2.199	63
OD570 of NC (60 min Experiment)	1.806	0.200	1.407 – 2.205	65
Relative Tissue Viability [%] of PC (60 min experiment)	5.9	2.3	1.4 – 10.4	65
CV [%] (in the range of 20 – 100% viability)	7.0	10.4	0.0 – 27.9	319

LCL:       Lower control limit (95%, mean – 2*SD)

UCL:      Upper control limit (95%, mean + 2*SD)

n:            number of control values

 

Applicant's summary and conclusion

Interpretation of results:

other: Not skin corrosive in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

In this study under the given conditions the test item showed no corrosive effects.

Executive summary:

The potential of Verdol to cause dermal corrosion was assessed in the EpiDerm skin corrosivity test according to OECD guideline 431. 25 mg of the test item (moistened with 25 μL H2O) were applied directly atop the EpiDerm tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 min and 60 min exposure period and compared to those of the concurrent negative controls. The test item has no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary. The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥50% (96.5%) after 3 min treatment and ≥15% (67.1%) after 60 min treatment.