4-(cyclohex-1-en-1-yl)-3-methylbutan-1-ol

Endpoint summary

Description of key information

The test substance is irritating to the skin and eye.

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

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

- Test substance storage In refrigerator (2-8°C) in the dark
- Description: Clear colourless liquid
- Expiry date: 19 March 2015

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

Test system
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 14-EKIN-018). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Preparation and preincubation:
- Tissues: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 26 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.
- MTT medium: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
- Environmental conditions: All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 72 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.0 - 36.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from humidity occurred that were caused by opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Study design
- Test for reduction of MTT by the test substance: The test substance was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 25 μl of the test substance was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.
- Application/Treatment of the test substance: The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Twenty five μl of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.
- Cell viability measurement: After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Interpretation
- Acceptability of the assay: The in vitro skin irritation test is considered acceptable if it meets the following criteria: a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18. b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18. c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.
- Data evaluation and statistical procedures: A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls. A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Irritation / corrosion parameter:

% tissue viability

Value:

9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 2 (irritant) based on GHS criteria

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

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)

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes

Specific details on test material used for the study:

- Test substance storage In refrigerator (2-8°C) in the dark
- Description: Clear colourless liquid
- Expiry date: 19 March 2015

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

Vehicle:

unchanged (no vehicle)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32±1°C. The corneas were incubated for the minimum of 1 hour at 32±1°C. After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

POSITIVE CONTROL USED
10% (w/v) Benzalkonium Chloride (Sigma-Aldrich Chemie GmbH, Germany) [CAS Number 63449-41- 2] solution prepared in physiological saline.

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (10% (w/v) Benzalkonium Chloride) or test substance was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test substance over the entire cornea. Corneas were incubated in a horizontal position for 10±1 minutes at 32±1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120±10 minutes at 32±1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

METHODS FOR MEASURED ENDPOINTS:
- Opacity measurement: The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Application of sodium fluorescein: Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck, Darmstadt, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90±5 minutes at 32±1°C.
- Permeability determinations: After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
- In vitro irritancy score: The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value). Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints. The IVIS cut-off values for identifying the test substances as inducing eye damage (UN GHS
Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are: ≤3: No category/Non irritant; >3; ≤55: No prediction can be made/Mild to moderate irritant; >55: Category 1/Severe irritant
- Acceptability of the assay: The assay is considered acceptable if: The positive control gives an in vitro irritancy score that falls within the laboratory historical mean value and the negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Irritation parameter:

in vitro irritation score

Value:

7.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls ranged between -0.1 and 0.1. The individual positive control in vitro irritancy scores ranged from 112 to 166 for Benzalkonium Chloride. The corneas treated with the positive control substance were turbid after the 10 minutes of treatment. The corneas treated with the test substance showed opacity values ranging from 5 to 6 and permeability values ranging from 0.091 to 0.237. The corneas were turbid with stripes after the 10 minutes of treatment with the test substance. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 6.4 to 9.6 after 10 minutes of treatment with the test substance.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion/irritation

No in vitro skin corrosion test has been performed as in the pre-screening test of the local lymph node assay, two mice were exposed for three consecutive days to either 10, 25, 50, or 100% test substance, respectively. Erythema and ear thickness measurements were determined up to 6 days. No erythema was observed and the small increase in ear thickness (<15%) observed after 3 days was restored to normal at day 6. Overall, it can be concluded that the substance is not corrosive. As a result, only an in vitro skin irritation test was performed.

In an in vitro skin irritation test using a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)), performed in accordance with OECD guideline 439 and following GLP, the skin irritation potential of the test substance was determined. The test substance was applied undiluted (25 μl) on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 9%. Since the mean relative tissue viability for the test substance was below 50% after 15 minutes treatment it is considered to be irritant. The positive control had a mean cell viability of 10% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly. Finally, it is concluded that this test is valid and that the test substance is irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Eye irritation

In an in vitro eye irritation test using the Bovine Corneal Opacity and Permeability test (BCOP test), the ocular irritation properties of the test substance was determined on isolated bovine corneas in accordance with OECD guideline 437 and following GLP. The test substance was applied as it is (750 μl) directly on top of the corneas for 10 ± 1 minutes. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 147 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test substance induced ocular irritation through both endpoints in the category mild to moderate, resulting in a mean in vitro irritancy score of 7.8 after 10 minutes of treatment. Since the test substance induced an IVIS > 3 ≤ 55.1, no prediction on the classification can be made based on the experimental conditions described in this report. Therefore as a worst-case approach, the test substance is considered to be irritating to the eye.

Justification for classification or non-classification

The test substance has to be classified as Skin Irrit 2: H315: Causes skin irritation and Eye Irrit 2: H319: Causes serious eye irritation according to Regulation (EC) No 1272/2008.