[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

Endpoint summary

Description of key information

Skin irritation/corrosion (OECD 439): not irritating

Eye irritation (OECD 437): not irritating

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:

28 Apr - 22 May 2015

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:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200-SIT Kit
- Tissue batch number: 21671
- Production date: not specified
- Shipping date: not specified
- Delivery date: 19 May 2015
- Date of initiation of testing: 19 May 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C for 35 min and room temperature for 25 minutes
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material, after the rinsing the inserts were submerged in DPBS at least three times.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1)
- Wavelength: 570 ± 1 nm
- Filter: 570 ± 1 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 2.57 ± 0.089 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 5.96 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce MTT, an additional test with freeze-killed tissues was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 60 minutes exposure is less than or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after 60 minutes exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30µL
- Concentration: 100%

NEGATIVE CONTROL
- Amount applied: 30 µL
- Concentration: 100%

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5%

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

40.5 hours

Number of replicates:

triplicates for each treatment and control group

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 min / test material

Value:

114.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not specified
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes: After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval.
- Acceptance criteria met for positive control: yes: Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.1%.
- Acceptance criteria met for variability between replicate measurements: yes: The relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were below 11% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%)

Table 2. Results after 60 min incubation time.

Test group	Absorbance at 570 nm			Mean absorbance	Rel. absorbance (%) Tissue 1, 2 and 3	Rel. SD (%)	Mean rel. absorbance (% of negative control)
	Tissue 1*	Tissue 2*	Tissue 3*
Negative control	1.993	2.064	2.064	2.040	97.7 101.1 101.1	2.0	100.0
Positive control	0.063	0.069	0.056	0.063	3.1 3.4 2.7	10.6	3.1
Test substance	2.118	2.390	2.484	2.331	103.8 117.2 121.7	8.2	114.2

* Mean of 3 replicate wells after blank correction

** Relative absorbance per tissue (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

*** Relative absorbance per treatment group (rounded values): 100 × (mean absorbance test item/positive control) / (mean absorbance negative control)

The optical pre-experiment (colour interference pre-experiment) to investigate the test substance’s colour change potential in water did not led to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test substance after 1 h incubation with MTT-reagent did not show blue colour.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the Reconstructed Human Epidermis test the test substance does not possess any skin irritating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

17 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in Hank's Buffered Salt Solution (HBSS) at ambient temperature.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Applied volume: 0.75 mL

POSITIVE SUBSTANCE
- Applied volume: 0.75 mL
- Purity: 99%

NEGATIVE CONTROL
- Applied volume: 0.75 mL

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

triplicates for each treatment and control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each cornea was mounted in a specially designed cornea holder.

QUALITY CHECK OF THE ISOLATED CORNEAS:
At the end of the equilibration period, the basal opacity was determined (t0). Each cornea with a value of a basal opacity >7 was discarded.

TREATMENT METHOD:
The cornea holder consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on the top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. The anterior compartment received the test substance or the controls on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath for 10 minutes.

REMOVAL OF TEST SUBSTANCE:
The test substance was rinsed off from the application side with saline.

- POST-EXPOSURE INCUBATION: 2 h in a vertical position

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP_KiT opacitometer, Electro Design, France).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microplate reader (Versamax Molecular Devices) at 490 nm.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value of 3 corneae

Value:

1.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae was observed.
The positive control (2-ethoxyethanol) showed clearly increased opacity and distinctive permeability of the corneae fullfilling the criteria as severe irritating/corrosive.
Relative to the negative control, the test substance did not cause a relevant increase of the corneal opacity or permeability.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control resulted in opacity and permeability values that were less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: The positive control resulted in an IVIS which was within two standard deviations of the current historical mean.

Table 2. Results after 10 min incubation time.

Test group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS
		Mean		Mean
Negative control	0	0.00	0.052	0.063	0.78	0.95
	0		0.082		1.23
	0		0.056		0.84
Positive control	65.00*		1.943*		94.14	89.31
	64.00*		1.877*		92.15
	54.00*		1.843*		81.64
Test substance	1.00*		0.002*		1.03	1.80
	4.00*		0.014*		4.21
	0.00*		0.012*		0.18

*: corrected values

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the BCOP assay the test substance was not irritating to the eye. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 1.80.

Executive summary:

Relative to the negative control, the test item MENTHYL METHYL ETHER did not cause an relevant increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 1.80. According to OECD 437 (see table in chapter 3.8.3) the test item is not categorized (GHS).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

Currently there are 3 studies on skin corrosion/irritation available.

1) In vitro skin irritation: Reconstructed Human Epidermis test (2015)

The skin irritation potential of the test substance was determined by an in vitro skin irritation test in human keratinocytes according to OECD Guideline 439 and in compliance with GLP (2015). The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes. Each 30 μL of the test item, of the negative control (DPBS) or of the positive control (5% SLS) were applied to each tissue and spread to match the surface of the tissue. After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system. After treatment with the test substance the mean relative absorbance value did not decrease (114.2%) compared to the relative absorbance value of the negative control. This value does not affect the threshold for irritancy of ≤ 50%. Therefore, the test substance is not considered to possess an irritant potential.

2) Transcutaneous Electrical Resistant Test Method (2001)

The skin corrosion potential of the test substance was determined by an in vitro Transcutaneous Electrical Resistant (TER) test similar to OECD Guideline 430 (study was performed prior to the release of the OECD Guideline 430) and in compliance with GLP (2001). The test material was applied to the epidermal surface of three skin discs (obtained from the pelt of a young Wistar rat) per contact period, 1, 4, and 24 hours. At the end of the contact period the test material was removed using a jet of warm tap water. Corrosive substances produce an irreversible loss of normal stratum corneum integrity and function, this is measured as a reduction in the inherent TER. A threshold value has been established (5 kΩ) below which materials are considered likely to be corrosive in vivo. The TER measurements after 1, 4 and 24 hours exposure resulted in 19.2, 4.7 and 1.8 kΩ, respectively. Due to this result the test substance has to be classified as corrosive to skin. But according to the todays test guideline 430 also obvious damage of skin and skin disk dye content should be tested and included in the evaluation. This was not done in available test. These controls are considered as essential and therefore, these result are questioned and disregarded.

3) In vivo skin irritation test (2001)

The skin irritation potential of the test substance was determined by an in vivo skin irritation test in rabbits according to OECD Guideline 404 adopted in 1992 and in compliance with GLP (2001). A dose of 0.5 mL of the test substance at concentrations of 100%, 50%, 25%, 10%, 1% and 0% (vehicle only) was applied to the skin of four female rabbits for 4 hours. Whether occlusive or semi-occlusive conditions were used is not specified in the report. At the concentration of 100% the exposure time was cut short early after 5 min because of the disturbing behaviour of the animals, which showed well-defined signs of distress or pain. In none of the four animals a five-minute exposure time caused a full thickness destruction of skin tissue. For all other concentrations, after the exposure period of 4 hours the patch was removed and the skin sites were evaluated. Scores were taken 60 minutes, 24, 48, 72 hours and 7, 14 and 21 days after patch removal. Very well-defined skin reactions were observed among the rabbits. Under the experimental conditions the mean erythema scores (24/48/72 hours) for all animals were 2.0, 1.6, 1.5, 0.0, 0.0 and 0.0 at 100%, 50%, 25%, 10%, 1% and 0% (vehicle), respectively. The mean edema scores for all animals were 2.0, 0.3, 0.0, 0.0, 0.0 and 0.0 at 100%, 50%, 25%, 10%, 1% and 0% (vehicle), respectively. Based on the results, the test substance was not irritating to the skin up to a concentration of 50%. Since 100% test substance was not tested for the full 4 hours and no full thickness destruction was observed after 5 minutes of exposure it is not possible to give a statement about a potential classification according to irritation or corrosion. However, the test substance is a cooling substance which could be an explanation for the distressed behaviour of the animals and thus the observed effects are considered not to be an indication of irritating potential of the test substance.

Further studies to be considered for evaluation of the endpoint:

An in vitro eye irritation test is available (see description below) which gave no evidence that the test substance has eye irritating potential and thus is not classified as eye irritant.

The test substance was tested up to 100% regarding its skin sensitizing potential (LLNA – see section skin sensitisation). In this test all animals appeared normal which indicates that the test substance has no corrosive potential.

In an acute dermal toxicity study (see section for acute toxicity) in rats tested with 2000 mg/kg bw test substance no signs of dermal irritation were observed. All scores for erythema and oedema were 0 at all reading time points.

In an in vitro reconstructed skin micronucleus assay (see section for genotoxicity) no cytotoxicity was observed in any tested concentration.

Conclusion:

Taking into account all available data described above, the test substance is not considered to be a skin irritant because the most reliable test (OECD 439) shows that the test substance does not have irritating potential. The two other skin irritation tests (TER and OECD 404) have major deficiencies which do not allow a clear classification. Further studies, e.g. eye irritation study, LLNA, acute dermal toxicity and in vitro reconstructive skin micronucleus assay, show no indication for a skin irritating potential of the test substance.

Eye

The eye irritation potential of the test substance was determined in a bovine corneal opacity and permeability test (BCOP test) according to OECD Guideline 437 and in compliance with GLP (2016). After a first opacity measurement of the fresh bovine corneae, 750 µL of the neat test substance was applied directly to the epithelial surface of three cattle corneae in an incubation chamber in horizontal position for 10 min at 32 ± 1 °C. After the incubation phase the test substance was rinsed from the corneae. Further, the corneae were incubated for another 120 min at 32 ± 1 °C in a vertical position, while the anterior chamber contained incubation medium as well. Afterwards, opacity was measured a second time. In addition, the permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 min at 32 ± 1 °C. The results of the opacity and permeability measurement of the test substance were used to calculate an in vitro irritation score (IVIS) of 1.80. With the negative control saline neither an increase of opacity nor permeability of the corneae could be observed. The mean IVIS of the negative control was 0.95. The mean IVIS of the positive control (2-ethoxyethanol) was 89.31. Based on the results, the test substance was not irritating to the eye under the conditions of the test.

Justification for classification or non-classification

The available data on skin and eye irritation of the test substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.