3-(4,4-dimethylcyclohex-1-en-1-yl)propanal

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-03-2010 to 19-05-2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Conducted equivalent to or similar to guideline and performed under GLP. The results of the assay should not be utilised as a standalone assay.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: SkinEthic™ HCE EIT

Version / remarks:

Study conducted prior to the full validation and/or OECD acceptance of the test assay ; however conducted to equivalent methodology, scientifically valid with sufficient documentation suitable for assessment.
The development of the model is documented in : Nguyen D. et al., Three-dimensional construct of the human corneal epithelium for in vitro toxicology. In: H. Salem and S.A. Katz, Editors, Alternative Toxicological Methods, CRC Press (2003), pp.147-159.
The prevalidation was documented in: Van Goethem F., et al., Prevalidation of a new in vitro reconstituted human cornea model to assess the eye irritating potential of chemicals. Toxicology in vitro 20 (2006), pp.1-17.

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

SkinEthic™ HCE EIT

Deviations:

yes

Remarks:

Sodium Dodecyl Sulphate (SDS) 1%w/v was used as the positive control rather than Methyl Acetate. The expsoure duration was 10 minutes rather than 30 minutes in the modern guideline. The results of the assay should not be utilised as a standalone assay.

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: EURL ECVAM DB-ALM: Protocol no. 190 : SkinEthic HCE Eye Irritation Test Liquid (EITL)

Version / remarks:

SkinEthic™ HCE EIT

Deviations:

yes

Remarks:

Sodium Dodecyl Sulphate (SDS) 1%w/v was used as the positive control rather than Methyl Acetate. The expsoure duration was 10 minutes rather than 30 minutes in the modern guideline. The results of the assay should not be utilised as a standalone assay.

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2009 ; signature: November 2009

Test material

Test material information

Constituent 1

Reference substance name:

3-(4,4-dimethylcyclohex-1-en-1-yl)propanal

EC Number:

700-339-0

Cas Number:

850997-10-3

Molecular formula:

C11H18O

IUPAC Name:

3-(4,4-dimethylcyclohex-1-en-1-yl)propanal

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Refrigerator (between 0 and 10ºC under nitrogen)
- Other: colourless

Test animals / tissue source

Species:

other: human-derived epidermal keratinocyte tissues

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The test method, subsequent to the conduct of the test is indicated as a validated test method recommended by OECD and/or recognised internationally for the sequential testing strategy for the assessment of ocular irritation.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: SkinEthic RHC model (otherwise known as the SkinEthic HCE model) : consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. The supplier of the test tissues is SkinEthic Laboratories, France. Date of receipt by the test laboratory was on the < 24 hours prior to the conduct of the test.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL of the test item
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.

Duration of treatment / exposure:

10 mins at 37.0 ± 1.0°C, 5% CO2 in air

Duration of post- treatment incubation (in vitro):

3 hours at 37.0 ± 1.0°C, 5% CO2 in air post treatment / rinsing incubation
- Rinsing consisted of initially rinsing each tissue insert using DPBS. Rinsing was achieved by filling and emptying tissue Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 µl of maintenance medium (at room temperature) until all the tissues were rinsed.
- Following rinsing, the tissues (two per group) were transferred to a pre-labelled 24-well plate designated ‘MTT Loading plate’ containing 300 µl of a 0.5 mg/ml MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37°C, 5% CO2 in air.

Number of animals or in vitro replicates:

Two tissues were used for each treatment group (test item, negative control and positive control).

Details on study design:

- Details of the test procedure used: See below.
- RhCE tissue construct used, including batch number: SkinEthic RHC model (otherwise known as the SkinEthic HCE model) date of receipt provided in the full study report.
- Doses of test chemical and control substances used: 30 µL of the test item / 30 µL “Solution A” used as supplied by manufacturer (negative control) / 30 µL Sodium Dodecyl Sulphate (SDS) 1%w/v in sterile distilled water (positive control)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Duplicate tissues were treated with 30 µL of the test material for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 µl of solution A to serve as negative controls and triplicate tissues were treated with 30 µL of 1% w/v SDS to serve as positive controls. The plates were incubated at 37°C, 5% CO2 in air during the exposure time. Rinsing consisted of initially rinsing each tissue insert using DPBS. Rinsing was achieved by filling and emptying tissue Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 µl of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing, the tissues (two per group) were transferred to a pre-labelled 24-well plate designated ‘MTT Loading plate’ containing 300 µL of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37°C, 5% CO2 in air. At the end of the incubation period, the tissues were visually examined and the degree of MTT staining evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labelled 24-well plate designated ‘MTT extraction plate’ containing 0.75 mL of Isopropanol in each of a sufficient number of wells. An extra 0.75 mL of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminum foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue. At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of Isopropanol alone was added to three wells designated as ‘blanks’. The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader.

- Justification for the use of a different negative control than ultrapure H2O (if applicable): "Solution A" : used as supplied ; composition per litre : Na2HPO4 0.142 g/L; Glucose 1.802 g/L ; HEPES 7.149 g/L ; KCl 0.224 g/L ; NaCl 7.597 g/L ; Two tissues were used for the control group. This solution was provided in accordance with the test kit manufacturer instructions.
- Justification for the use of a different positive control than neat methyl acetate (if applicable): Not applicable. Sodium Dodecyl Sulphate (SDS) 1%w/v was used as the positive control rather than Methyl Acetate ; this was a suitable PC according to the test method at the time. Does not impact the interpretation of the study.
- Description of any modifications to the test procedure: Not applicable.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable):
Colour-interference: was not checked. However the test item is colourless and it can be considered that colour-interference is not applicable.
Direct reduction of MTT: was checked. The test item was checked for possible direct MTT reduction before the definitive test. 30 µL of test item was added to a 0.5 mg/mL MTT solution and incubated at room temperature in the dark for 60 minutes. Untreated MTT solution was used as a control. If the MTT solution turned black/purple, the test material was presumed to have reduced the MTT. If the MTT solution colour turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay. Conclusion: the test item did not directly interact with MTT and therefore considered was not a potential direct MTT reducer. As a result: NSMTT, NSCliving and NSCkilled were not required.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): Duplicate.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm
- Description of the method used to quantify MTT formazan: Athos 2001 microplate reader – spectrophotometrically (at 540 nm).
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): Not applicable.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: 60% viability cut off, according to the manufacturer instructions. However, later applications of the method indicate to utilise the 50% cut off per the OECD TG 492 and GHS system.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: The absolute mean OD540 of the negative control tissues were marginally outside the OECD TG 492, acceptability range of OD > 1.0 and < 2.5 : at 0.938 and 0.963. The difference may be due to the use of “solution A” as NC rather than later proscribed Ca2+/Mg2+-free DPBS. The NC was tested
concurrently. The difference in viability was < 10% between replicates. The Positive Control gave a response within an expected PC range mean = 39% (positive) tissue range : 24 – 53%. The difference in viability was > 20% between replicates. Therefore the results of the assay should be interpreted with additional caution and not as a standalone assay.
- Complete supporting information for the specific RhCE tissue construct used: Attached to the full study report.
- Reference to historical data of the RhCE tissue construct: Full historic control data was not available in the study report.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The test assay is (post study) validated. The test laboratory had previously demonstrated technical proficiency and this information is in the public domain.
- Positive and negative control means and acceptance ranges based on historical data: See above.
- Acceptable variability between tissue replicates for positive and negative controls: < 10 % in NC. >20% in PC. The results of the assay should however be interpreted with additional caution and not as a standalone assay.
- Acceptable variability between tissue replicates for the test chemical: Yes.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Mean tissue viability (% of negative control)

Remarks:

n=2

Run / experiment:

mean (n=2) - test item

Value:

85

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The absolute mean OD540 of the negative control tissues were marginally outside the OECD TG 492, acceptability range of OD > 1.0 and < 2.5 : at 0.938 and 0.963. The difference may be due to the use of “solution A” as NC

Positive controls validity:

other:

Remarks:

Positive Control gave a response within an expected PC range mean = 39% (positive) tissue range : 24 – 53%. The difference in viability was > 20% between replicates. See Remarks.

Remarks on result:

other: - Acceptable variability between tissue replicates for positive and negative controls: < 10 % in NC. >20% in PC. The results of the assay should however be interpreted with additional caution and not as a standalone assay.

Remarks:

See positive control results

Results 2

Irritation parameter:

other: Mean tissue viability (% of negative control)

Remarks:

n=2

Run / experiment:

mean (n=2) - positive control

Value:

39

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The absolute mean OD540 of the negative control tissues were marginally outside the OECD TG 492, acceptability range of OD > 1.0 and < 2.5 : at 0.938 and 0.963. The difference may be due to the use of “solution A” as NC

Positive controls validity:

other: Positive Control gave a response within an expected PC range mean = 39% (positive) tissue range : 24 – 53%. The difference in viability was > 20% between replicates. See Remarks.

Remarks on result:

positive indication of irritation

Remarks:

- Acceptable variability between tissue replicates for positive and negative controls: < 10 % in NC. >20% in PC. The results of the assay should however be interpreted with additional caution and not as a standalone assay.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None. The test item tissue was considered viable (MTT uptake visual evaluation). The NC test tissues were similarly viable. The PC indicated blue/white tissue therefore only: semi-viable

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test assay is (post study) validated. The test laboratory had previously demonstrated technical proficiency and this information is in the public domain.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Although the absolute mean OD540 of the negative control tissues were marginally outside the OECD TG 492, acceptability range of OD > 1.0 and < 2.5 : at 0.938 and 0.963. The difference may be due to the use of “solution A” as NC rather than later proscribed Ca2+/Mg2+-free DPBS. The NC was tested concurrently. The difference in viability was < 10% between replicates.
- Acceptance criteria met for positive control: No. Tissue variability was >20% in PC. The results of the assay should be interpreted with additional caution and not as a standalone assay.
- Range of historical values if different from the ones specified in the test guideline: See 'details on study design' for further information. See further comments above.

Applicant's summary and conclusion

Interpretation of results:

other: Using the results of the study as a standalone assay: under the GHS criteria “no prediction can be made” ; the test item is suggested to have "limited" potential for eye irritation as part of weight of evidence.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, as a standalone assay: under the GHS criteria “no prediction can be made” ; the test item is suggested to have "limited" potential for eye irritation as part of weight of evidence. The test item had a relative mean viability was > 60% and therefore the test item was considered potentially not a serious eye irritant or corrosive. However, under the modern OECD TG 492 guidelines, the absolute mean OD540 of the negative control tissues were marginally outside the acceptability range of OD > 1.0 and < 2.5 at OD540 : 0.938 and 0.963. Additionally, the 1%w/v SDS positive control, gave a mean viability 39% (positive prediction) with a tissue range : 24 – 53%. The difference in viability was > 20% between replicates. Additionally, the test exposure was 10 minutes rather than the modern OECD TG 492 guideline specified 30 minutes. Therefore the results of the assay should be interpreted with additional caution and not as a standalone assay.

Executive summary:

The study was performed to assess the eye irritancy potential of the test item using the SkinEthic HCE EIT model in accordance with GLP. Duplicate tissues were treated with 30 µL of the test material for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Duplicate tissues were treated with 30 µL of solution A to serve as negative controls and duplicate tissues were treated with 30 µL of 1% w/v SDS to serve as positive controls. “Solution A" was used as supplied ; composition per litre : Na2HPO4 0.142 g/L; Glucose 1.802 g/L ; HEPES 7.149 g/L ; KCl 0.224 g/L ; NaCl 7.597 g/L. This solution was provided in accordance with the test kit manufacturer instructions. The plates were incubated at 37°C, 5% CO2 in air during the exposure time. Rinsing consisted of initially rinsing each tissue insert using DPBS. Rinsing was achieved by filling and emptying tissue Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 µL of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing, the tissues (two per group) were transferred to a pre-labelled 24-well plate designated ‘MTT Loading plate’ containing 300 µL of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 37°C, 5% CO2 in air. At the end of the incubation period, the tissues were visually examined and the degree of MTT staining evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labelled 24-well plate designated ‘MTT extraction plate’ containing 0.75 mL of Isopropanol in each of a sufficient number of wells. An extra 0.75 mL of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminium foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue. At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 µL tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of Isopropanol alone was added to three wells designated as ‘blanks’. The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD540) using the Anthos 2001 microplate reader. In pre-testing the test item did not directly interact with MTT and therefore was a direct MTT reducer. The relative mean tissue viability obtained after 10 minutes treatment with the test item compared to the negative control tissues was 85%. The positive control had a mean cell viability of 39% after 10 minutes exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the test considered acceptable. Under the modern OECD TG 492 guidelines, the absolute mean OD540 of the negative control tissues were marginally outside the acceptability range of OD > 1.0 and < 2.5 at OD540 : 0.938 and 0.963. The difference in viability was < 10% between replicates. The positive control gave a response within an expected range, mean = 39% (positive prediction) with a tissue range : 24 – 53%. The difference in viability was > 20% between replicates. Under the conditions of this study, under the GHS criteria “no prediction can be made” ; the test item is suggested to have "limited" potential for serious eye irritation as part of weight of evidence. The test item had a relative mean viability was > 60% and therefore the test item was considered potentially not a serious eye irritant or corrosive. However, under the modern OECD TG 492 guidelines, the absolute mean OD540 of the negative control tissues were marginally outside the acceptability range of OD > 1.0 and < 2.5 at OD540 : 0.938 and 0.963. Additionally, the 1%w/v SDS positive control, gave a mean viability 39% (positive prediction) with a tissue range : 24 – 53%. The difference in viability was > 20% between replicates. Additionally, the test exposure was 10 minutes rather than the modern OECD TG 492 guideline specified 30 minutes. Therefore the results of the assay should be interpreted with additional caution and not as a standalone assay.