Registration Dossier
Registration Dossier
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EC number: 289-995-2 | CAS number: 90063-37-9 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Lavandula angustifolia, Labiatae.
Endpoint summary
Administrative data
Description of key information
In an in vitro skin irritation study (OECD 439) test item was not irritant to skin ( Read Across GLP, Rel. K1)
In an in vitro eye irritation study (OECD 492) test item was considered irritant to the eyes (Read Across GLP, Rel K1)
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 06-18 January 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted according to OECD test Guideline No. 439. Read-across substance
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please see section 13 for justification)] - Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 05 March 2015
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Linalool consortium / EE 86453
- Physical state: Clear pale yellow liquid
- Date of receipt: 22 December 2015
- Expiration date of the lot/batch: December 2018
- Purity test date: 16 December 2015
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature, protected from light and under nitrogen atmosphere - Test system:
- human skin model
- Source species:
- human
- Cell type:
- other: human reconstructed epidermis (tissues) reconstructed from normal human epidermal keratinocytes
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Species: Human reconstructed epidermis (tissues)
Supplier: Episkin, Lyon, France.
Selection: At receipt, the pH (colour of the agar medium) and temperature indicators were checked to ensure the good quality of the tissues before use.
Storage conditions: At receipt, the living Episkin™ tissues were kept at room temperature in their packaging until required.
Description: The Episkin™ model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra-structure and is functionally equivalent to human in vivo epidermis.
PRELIMINARY TESTS
Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.
Test for direct MTT reduction with the test item: 10 μL of the test item were added to 2 mL of a 0.3 mg/mL freshly prepared MTT solution; a negative control was tested concurrently by adding 10 μL of water for injections to 2 mL of a 0.3 mg/mL freshly prepared MTT solution; both mixtures were incubated in darkness at 37 °C for 3 hours (± 5 minutes). Then the colour of the solutions obtained was evaluated.
Test for the detection of the colouring potential of the test item: The intrinsic colour or the ability of the test item to become coloured in contact with water (simulating a tissue humid environment) was evaluated by adding 10 μL of test item to 90 μL of water for injections in a transparent recipient. After 1 hour (± 3 minutes) of mixing, the presence and intensity of the coloration was evaluated.
MAIN TEST
Pre-incubation of the tissues on their day of arrival (Day 0): A volume of 2 mL of pre-warmed (at 37 °C) maintenance medium was added to 3 wells of a 12-well plates (one plate per item). Then, each Episkin™ tissue was transferred into the maintenance medium pre-filled wells (three tissues per plate). The plates were incubated at 37 °C, 5% CO2 in a humidified incubator for at least 24 hours.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The exposure of the tissues to the test and control items was performed at room temperature for 15 minutes (± 1 minute)
- Temperature of post-treatment incubation: Tissues were incubated at 37 °C, 5% CO2 in a humidified incubator for 42 hours
REMOVAL OF TEST MATERIAL AND CONTROLS
- At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D-PBS to gently remove any residual test or control items. The rinsed tissues were transferred to wells containing 2 mL of maintenance medium in each well and the plates were incubated at 37 °C, 5% CO2 in a humidified incubator for 42 hours.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of MTT solution (0.3 mg/mL)
- Incubation time: On Day 3, following the 42 h post-exposure incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h at 37 °C, 5 % CO2 in a humidified incubator. At the end of the MTT incubation period, tissues were incubated with 500 μL of acidic isopropanol. Then, to extract the formazan (reduced MTT) from the MTT-loaded tissues, each tube was stored after vortexing at +5°C and protected from light until Day 6 of the experiment.
- Optical density measurements (Day 6): At the end of the formazan extraction period, the optical density was measured at 570 nm using a plate reader.
NUMBER OF REPLICATE TISSUES: Triplicate tissues for test item, negative and positive controls
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
Classification of irritation potential is based upon relative mean tissue viability following the 15 - minute exposure period followed by the 42 - hour post-exposure incubation period
Relative mean tissue viability is ≤50%: Irritant
Relative mean tissue viability is >50%: Non-Irritant - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL of the test item was applied to the epidermis surface
- Concentration (if solution): undiluted
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL Dulbecco’s Phosphate-Buffered Saline (D-PBS)
- Concentration (if solution): Negative control was prepared by diluting D-PBS 10X to 1X in water for injections.
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution
- Concentration (if solution): SDS was diluted in water for injection to 5% (w/v). - Duration of treatment / exposure:
- Triplicate tissues were treated with the test item for an exposure period of 15 minutes (± 1 minute).
- Duration of post-treatment incubation (if applicable):
- At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5 % CO2 in a humidified incubator for 42 h.
- Number of replicates:
- One 12-well plate was used for each test and control items: one plate for the test item, one plate for the negative control and one plate for the positive control. Each item was applied onto triplicate tissues.
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 15 minute exposure period and 42 h post-exposure incubation period
- Value:
- 70
- 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:
- PRELIMINARY TESTS
Test for direct MTT reduction with the test item: The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on water-killed tissues in parallel to the main test.
Test for the detection of the colouring potential of the test item: During this test, as the water solution containing the test item did not change colour, the test item was found not to have a colouring potential. As a result, no additional controls were used in parallel to the main test.
MAIN TEST
- Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 70% with a standard deviation of 7% as assessed by the MTT assay. As the mean viability was > 50% after the MTT reduction, the results met the criteria for a non-irritant response.
- Evaluation of the colouration of tissues at the end of the MTT incubation period: All test item-treated tissues appeared blue which was considered indicative for viable tissues.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes - Interpretation of results:
- GHS criteria not met
- Conclusions:
- Under the experimental conditions of this study, the test item is considered to be non-irritant to skin. According to the results of this study, the classification of the test item should be: not classified (Directive 67/548/EEC) and no category (Regulation (EC) No. 1272/2008).
- Executive summary:
An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.
Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Test item, SAUGE SCLAREE ESS. / CLARY SAGE OIL was applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 h at 37 °C, 5 % CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100 % (reference viability).
In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.
All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.
All test item-treated tissues appeared blue which was considered indicative for viable tissues. Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 70% with a standard deviation of 7% as assessed by the MTT assay. As the mean viability was > 50% after the MTT reduction, the results met the criteria for a non-irritant response.
Under the experimental conditions of this study, the test item is considered to be non-irritant to skin. According to the results of this study, the classification of the test item should be: not classified (Directive 67/548/EEC) and no category (Regulation (EC) No. 1272/2008).
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 06-18 January 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted according to OECD test Guideline No. 439. Read-across substance
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please see section 13 for justification)] - Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 05 March 2015
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- other: human reconstructed epidermis (tissues) reconstructed from normal human epidermal keratinocytes
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 15 minute exposure period and 42 h post-exposure incubation period
- Value:
- 70
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Under the experimental conditions of this study, the test item is considered to be non-irritant to skin. According to the results of this study, the classification of the test item should be: no category (Regulation (EC) No. 1272/2008). Therefore it can be concluded that Lavender oil is not classified under Regulation 1272/2008.
- Executive summary:
An in vitro skin irritation study was performed, on Clary sage oil according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.
Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Test item, SAUGE SCLAREE ESS. / CLARY SAGE OIL was applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 h at 37 °C, 5 % CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100 % (reference viability).
In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.
All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.
All test item-treated tissues appeared blue which was considered indicative for viable tissues. Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 70% with a standard deviation of 7% as assessed by the MTT assay. As the mean viability was > 50% after the MTT reduction, the results met the criteria for a non-irritant response.
Under the experimental conditions of this study, Clary sage is considered to be non-irritant to skin. According to the results of this study, the classification of the test item should be: no category (Regulation (EC) No. 1272/2008). Therefore it can be concluded that Lavender oil is not classified under Regulation 1272/2008.
Referenceopen allclose all
Table 7.3.1/1: Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls
Group |
Tissue No. |
OD measurements |
Mean ODblank |
cOD |
Mean cOD |
Viability (%)
|
||
1st |
2nd |
1st |
2nd |
|||||
Negative control |
1 |
0.610 |
0.620 |
0.037 |
0.573 |
0.583 |
0.578 |
89 |
2 |
0.740 |
0.759 |
0.703 |
0.722 |
0.712 |
110 |
||
3 |
0.689 |
0.693 |
0.652 |
0.656 |
0.654 |
101 |
||
Positive control |
1 |
0.106 |
0.108 |
0.037 |
0.069 |
0.071 |
0.070 |
11 |
2 |
0.119 |
0.116 |
0.082 |
0.079 |
0.080 |
12 |
||
3 |
0.125 |
0.118 |
0.088 |
0.081 |
0.084 |
13 |
||
Test item |
1 |
0.485 |
0.511 |
0.037 |
0.448 |
0.474 |
0.461 |
71 |
2 |
0.428 |
0.455 |
0.391 |
0.418 |
0.404 |
62 |
||
3 |
0.525 |
0.544 |
0.488 |
0.507 |
0.497 |
77 |
||
Table 7.3.1/2: Mean tissue viability and Standard Deviations for the test item, the negative and positive controls
Group |
cOD |
Viability (%) |
||
Mean |
SD |
Mean |
SD |
|
Negative control |
0.648 |
0.067 |
100 |
10 |
Positive control |
0.078 |
0.007 |
12 |
1 |
Test item |
0.454 |
0.047 |
70 |
7 |
OD = optical density
cOD = blank corrected optical density
SD = Standard Deviation
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 19 January to 04 August 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted in compliance with OECD Guideline No. 492. Read-across substance.
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please see section 13 for justification)] - Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- according 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)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 05 March 2015
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 150095
- Physical state: Transparent liquid
- Dates of receipt: 15 January 2016 (used for the preliminary and invalidated first main test); 09 February 2016 (used for the validated first main test); 23 May 2016 (used for the second main test); 04 July 2016 (used from the third main test)
- Expiration date of the lot/batch: 30 September 2018
- Purity test date: 11 January 2016
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in the refrigerator set at 5 °C, protected from light, under nitrogen atmosphere - Species:
- other: Reconstructed Human cornea-like epithelium (tissues).
- Details on test animals or tissues and environmental conditions:
- Species: Reconstructed Human cornea-like epithelium (tissues).
Supplier: MatTek, Bratislava, Slovak Republic.
Selection: At receipt, the tissues were inspected for obvious defects as they could have been rejected based on blistering, excess fluid or air bubbles below the tissue insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
Storage conditions: At receipt, the living EpiOcular™ tissues were stored as described in the Preincubation of the tissues, on their day of arrival. - 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): 50 μL
- Concentration (if solution): Undiiluted - Duration of treatment / exposure:
- During each main test, the test item and both negative and positive controls were applied topically on duplicate tissues and incubated at 37 °C for 30 minutes (± 2 minutes).
- Duration of post- treatment incubation (in vitro):
- At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes (± 2 minutes) at room temperature to remove any remaining test item absorbed into the tissue, blotted on absorbent material, and then incubated for another 2 hours (± 15 minutes) at 37 °C, 5% CO2 in a humidified incubator.
- Number of animals or in vitro replicates:
- Test item, negative and positive controls were applied on duplicate tissues.
- Details on study design:
- - RhCE tissue construct used, including batch number: The EpiOcularTM (OCL-200, OCL-212) model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinized epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotypic 3D cornea-like model. The 3D tissue consists of highly organized cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue. The EpiOcular tissues were used within 72 hours of their production. Batch numbers 237 00; 23711 and 23722 were used for the first, second and third main tests, respectively.
Preliminary tests: Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its coloring potential.
- Test for direct MTT reduction with the test item: 50 μL of the test item were added to 1 mL of a 1.0 mg/mL freshly prepared MTT solution; a negative control was tested concurrently by adding 50 μL of sterile deionized water to 1 mL of a 1.0 mg/mL freshly prepared MTT solution. Both mixtures were incubated in darkness at 37 °C for 3 hours (± 10 minutes) and color of the solutions obtained was evaluated.
- Test for the detection of the coloring potential of the test item: The maximum amount of test item, 50 μL was added to: (i) 1 mL of water and incubated for at least 1 hour in the dark at 37 °C, 5% CO2 and (ii) 2 mL of isopropanol, incubated in a 6-well plate and placed on an orbital plate shaker for 2 to 3 hours at room temperature. After that, the presence and intensity of the coloration were evaluated.
Main test:
- Doses of test chemical and control substances used: 50 μL
- Pre-incubation of the tissues: On the day of treatment for the first main test or on the day before treatment for the second and third main tests, tissues were equilibrated (in the 24-well shipping container) to room temperature for at least 15 minutes. The tissue inserts were transferred aseptically into the 6-well plate and pre-incubated at 37 °C, 5% CO2 in a humidified incubator for 1 hour. After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24 h) at 37 °C, 5% CO2 in a humidified incubator.
Treatment of tissues
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: During each main test, the test item and both negative and positive controls were applied topically on duplicate tissues and incubated at 37 °C for 30 minutes (± 2 minutes). At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes (± 2 minutes) at room temperature to remove any remaining test item absorbed into the tissue, blotted on absorbent material, and then incubated for another 2 hours (± 15 minutes) at 37 °C, 5% CO2 in a humidified incubator.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): One 6-well plate was used for the first test item-treated tissues. Positive and negative controls were placed on separate 6-well plates (one plate for each).Test item, negative and positive controls were applied on duplicate tissues.
- MTT viability assay: Following the post-treatment incubation, the cell viability was assessed by means of the colorimetric MTT reduction assay. Tissues were incubated with MTT solution in 24-well plates for 3 hours (± 10 minutes) at 37 °C, 5% CO2 in a humidified incubator. At the end of the 3-hour incubation period, tissues were blotted on absorbent paper and the degree of MTT staining was evaluated. For the test item, negative and positive control-treated tissues, the inserts were transferred to new wells of the 24-well plate containing 2 mL of isopropanol per well. Formazan extraction was performed overnight at 2-8 °C and protected from light.
- Optical Density measurements: At the end of the formazan extraction period, the plates were placed under orbital shaking at room temperature for at least 15 minutes before using them. Then, tissues (test item, negative and positive control treated tissues) were pierced. The extract solution was mixed and two 200 μL aliquots were transferred to the pre-labeled 96-well plate. For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 μL of isopropanol only was used as the blank. The OD was measured at 570 nm using a plate reader.
Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability). - Irritation parameter:
- other: relative mean viability of the tissues
- Run / experiment:
- First main test
- Value:
- 64
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Remarks:
- mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.
- Irritation parameter:
- other: relative mean viability of the tissues
- Run / experiment:
- Second main test
- Value:
- 53
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Remarks:
- non concordant results were obtained in the two first main tests, it was ag reed with the Sponsor to perform a third main test.
- Irritation parameter:
- other: relative mean viability of the tissues
- Run / experiment:
- Third main test
- Value:
- 59
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Remarks:
- mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissue led to non-concordant classification, this was considered as a borderline result.
- Other effects / acceptance of results:
- PRELIMINARY TESTS
Test for direct MTT reduction with the test item: The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on freeze-dead tissues in parallel to the main test.
Test for the detection of the coloring potential of the test item: During this test, as both water and isopropanol solutions containing the test item did not change color, the test item was found not to have a coloring potential. As a result, no additional controls were used in parallel to the main test.
MAIN TESTS
Evaluation of the coloration of tissues at the end of the MTT incubation period: All test item-treated tissues appeared blue/white which was considered to be indicative of semi-viable tissue.
Evaluation of the MTT results:
First main test: The relative mean viability of the tissues treated with the test item was 64% with a difference of 1% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response. However, since a mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.
Second main test: The relative mean viability of the tissues treated with the test item was 53% with a difference of 3% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However, since non concordant results were obtained in the two first main tests, it was agreed with the Sponsor to perform a third main test.
Third main test: The relative mean viability of the tissues treated with the test item was 59% with a difference of 13% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However since mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissue led to non-concordant classification, this was considered as a borderline result.
Two out of three main tests gave irritant responses (with one of them with borderline results, i.e. mean % viability equal to 60 ± 5%). When tissues (from all main tests) are considered individually: 50% gave irritant responses (i.e. below 60% viability) and 50% non-irritant response. Even if two out of three main tests gave borderline results, a fourth main test would not be necessary. When all main tests are considered together, the mean %viability tends to borderline results with positive classification (irritant), indeed a significant decrease of viability is obtained. As a result and under our experimental conditions, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium.
ACCEPTANCE OF RESULTS:
- All of the acceptance criteria for the negative and positive controls were fulfilled, therefore each main test was considered as valid. - Interpretation of results:
- Category 2 (irritating to eyes) based on GHS criteria
- Conclusions:
- Under the experimental conditions of this study and based on results of three independent and validated main tests, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium. According to the results of this study, the classification of the test item should be: Category 1/category 2 (GHS 2013) and category 1 (H318)/category 2 (H319) (Regulation (EC) No. 1272/2008).
- Executive summary:
An in vitro eye irritation teston the EpiOcularTM cornea epithelial modelwas performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test item.
Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its coloring potential. Following the preliminary tests, the eye irritation potential of the test item was assessed in main tests. During each main test, the test item and both negative and positive controls were applied topically on duplicate tissues and incubated at 37 °C for 30 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature to remove any remaining test item absorbed into the tissue, blotted on absorbent material, and then incubated for another 2 hours at 37 °C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).
In the preliminary tests, the test item was found not to have direct MTT reducing properties or coloring potential.
First main test: The relative mean viability of the tissues treated with the test item was 64% with a difference of 1% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response. However, since a mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.
Second main test: The relative mean viability of the tissues treated with the test item was 53% with a difference of 3% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However, since non-concordant results were obtained in the two first main tests, it was agreed with the Sponsor to perform a third main test.
Third main test: The relative mean viability of the tissues treated with the test item was 59% with a difference of 13% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However since mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissues led to non-concordant classification, this was considered as a borderline result.
Two out of three main tests gave irritant responses (with one of them with borderline results,i.e.mean % viability equal to 60 ± 5%). When tissues (from all main tests) are considered individually: 50% gave irritant responses (i.e.below 60% viability) and 50% non-irritant response. Even if two out of three main tests gave borderline results, a fourth main test would not be necessary. When all main tests are considered together, the mean % viability tends to borderline results with positive classification (irritant), indeed a significant decrease of viability is obtained. As a result and under our experimental conditions, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium.
All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.
Under the experimental conditions of this study and based on results of three independent and validated main tests, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium. According to the results of this study, the classification of the test item should be: Category 1/category 2 (GHS 2013) and category 1 (H318)/category 2 (H319) (Regulation (EC) No. 1272/2008).
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 19 January to 04 August 2016
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please see section 13 for justification)] - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- Qualifier:
- according 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)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 05 March 2015
- Species:
- other: Reconstructed Human cornea-like epithelium (tissues).
- Vehicle:
- unchanged (no vehicle)
- Irritation parameter:
- other: relative mean viability of the tissues
- Run / experiment:
- First main test
- Value:
- 64
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Remarks:
- mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.
- Irritation parameter:
- other: relative mean viability of the tissues
- Run / experiment:
- Second main test
- Value:
- 53
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Remarks:
- non concordant results were obtained in the two first main tests, it was ag reed with the Sponsor to perform a third main test.
- Irritation parameter:
- other: relative mean viability of the tissues
- Run / experiment:
- Third main test
- Value:
- 59
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Remarks:
- mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissue led to non-concordant classification, this was considered as a borderline result.
- Interpretation of results:
- Category 2 (irritating to eyes) based on GHS criteria
- Conclusions:
- Under the experimental conditions of this study and based on results of three independent and validated main tests, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium. According to the results of this study, the classification of the test item should be: Category 1/category 2 (GHS 2013) and category 1 (H318)/category 2 (H319) (Regulation (EC) No. 1272/2008).
- Executive summary:
An in vitro eye irritation teston the EpiOcularTM cornea epithelial modelwas performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test item.
Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its coloring potential. Following the preliminary tests, the eye irritation potential of the test item was assessed in main tests. During each main test, the test item and both negative and positive controls were applied topically on duplicate tissues and incubated at 37 °C for 30 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature to remove any remaining test item absorbed into the tissue, blotted on absorbent material, and then incubated for another 2 hours at 37 °C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).
In the preliminary tests, the test item was found not to have direct MTT reducing properties or coloring potential.
First main test: The relative mean viability of the tissues treated with the test item was 64% with a difference of 1% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response. However, since a mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.
Second main test: The relative mean viability of the tissues treated with the test item was 53% with a difference of 3% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However, since non-concordant results were obtained in the two first main tests, it was agreed with the Sponsor to perform a third main test.
Third main test: The relative mean viability of the tissues treated with the test item was 59% with a difference of 13% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However since mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissues led to non-concordant classification, this was considered as a borderline result.
Two out of three main tests gave irritant responses (with one of them with borderline results,i.e.mean % viability equal to 60 ± 5%). When tissues (from all main tests) are considered individually: 50% gave irritant responses (i.e.below 60% viability) and 50% non-irritant response. Even if two out of three main tests gave borderline results, a fourth main test would not be necessary. When all main tests are considered together, the mean % viability tends to borderline results with positive classification (irritant), indeed a significant decrease of viability is obtained. As a result and under our experimental conditions, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium.
All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.
Under the experimental conditions of this study and based on results of three independent and validated main tests, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium. According to the results of this study, the classification of the test item should be: Category 1/category 2 (GHS 2013) and category 1 (H318)/category 2 (H319) (Regulation (EC) No. 1272/2008).
Referenceopen allclose all
Table 7.3.2/1: Main tests: Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls
Group |
Exposure duration |
Tissue No. |
OD570 nm measurements |
|
Mean blank |
cOD570 nm measurements |
|
Mean cOD570 nm |
Viability (%) |
1st |
2nd |
1st |
2nd |
||||||
First main test |
|||||||||
Negative control |
30 min |
1 |
1.847 |
1.850 |
0.037 |
1.810 |
1.813 |
1.812 |
96 |
2 |
2.001 |
1.993 |
1.964 |
1.956 |
1.960 |
104 |
|||
Positive control |
30 min |
1 |
0.686 |
0.695 |
0.037 |
0.649 |
0.658 |
0.654 |
35 |
2 |
0.774 |
0.780 |
0.737 |
0.743 |
0.740 |
39 |
|||
Test item |
30 min |
1 |
1.245 |
1.236 |
0.038 |
1.207 |
1.198 |
1.202 |
64 |
2 |
1.261 |
1.255 |
1.223 |
1.217 |
1.220 |
65 |
|||
Second main test |
|||||||||
Negative control |
30 min |
1 |
1.992 |
1.936 |
0.041 |
1.952 |
1.896 |
1.924 |
95 |
2 |
2.154 |
2.172 |
2.114 |
2.132 |
2.123 |
105 |
|||
Positive control |
30 min |
1 |
0.947 |
0.953 |
0.041 |
0.907 |
0.913 |
0.910 |
45 |
2 |
0.775 |
0.773 |
0.735 |
0.733 |
0.734 |
36 |
|||
Test item |
30 min |
1 |
1.145 |
1.143 |
0.037 |
1.108 |
1.106 |
1.107 |
55 |
2 |
1.091 |
1.084 |
1.054 |
1.047 |
1.050 |
52 |
|||
Third main test |
|||||||||
Negative control |
30 min |
1 |
1.992 |
1.990 |
0.043 |
1.949 |
1.947 |
1.948 |
102 |
2 |
1.909 |
1.924 |
1.866 |
1.881 |
1.873 |
98 |
|||
Positive control |
30 min |
1 |
0.679 |
0.677 |
0.043 |
0.636 |
0.634 |
0.635 |
33 |
2 |
0.631 |
0.632 |
0.588 |
0.589 |
0.588 |
31 |
|||
Test item |
30 min |
1 |
1.040 |
1.039 |
0.042 |
0.998 |
0.997 |
0.997 |
52 |
2 |
1.290 |
1.289 |
1.248 |
1.247 |
1.247 |
65 |
|||
Table 7.3.2/2: Main tests - Mean tissue viability and standard deviations for the test item, the negative and positive controls
Group |
Exposure duration |
cOD570 nm |
Viability (%) |
|||
Mean |
SD |
Mean |
SD |
Difference (%) |
||
First main test |
||||||
Negative control |
30 min |
1.886 |
0.105 |
100 |
6 |
8 |
Positive control |
30 min |
0.697 |
0.061 |
37 |
3 |
5 |
Test item |
30 min |
1.211 |
0.012 |
64 |
1 |
1 |
Second main test |
||||||
Negative control |
30 min |
2.023 |
0.141 |
100 |
7 |
10 |
Positive control |
30 min |
0.822 |
0.124 |
41 |
6 |
9 |
Test item |
30 min |
1.079 |
0.040 |
53 |
2 |
3 |
Third main test |
||||||
Negative control |
30 min |
1.911 |
0.053 |
100 |
3 |
4 |
Positive control |
30 min |
0.612 |
0.033 |
32 |
2 |
2 |
Test item |
30 min |
1.122 |
0.177 |
59 |
9 |
13 |
OD = optical density
cOD = blank corrected optical density
SD = standard deviation
Table 7.3.2/1: Main tests: Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls
Group |
Exposure duration |
Tissue No. |
OD570 nm measurements |
|
Mean blank |
cOD570 nm measurements |
|
Mean cOD570 nm |
Viability (%) |
1st |
2nd |
1st |
2nd |
||||||
First main test |
|||||||||
Negative control |
30 min |
1 |
1.847 |
1.850 |
0.037 |
1.810 |
1.813 |
1.812 |
96 |
2 |
2.001 |
1.993 |
1.964 |
1.956 |
1.960 |
104 |
|||
Positive control |
30 min |
1 |
0.686 |
0.695 |
0.037 |
0.649 |
0.658 |
0.654 |
35 |
2 |
0.774 |
0.780 |
0.737 |
0.743 |
0.740 |
39 |
|||
Test item |
30 min |
1 |
1.245 |
1.236 |
0.038 |
1.207 |
1.198 |
1.202 |
64 |
2 |
1.261 |
1.255 |
1.223 |
1.217 |
1.220 |
65 |
|||
Second main test |
|||||||||
Negative control |
30 min |
1 |
1.992 |
1.936 |
0.041 |
1.952 |
1.896 |
1.924 |
95 |
2 |
2.154 |
2.172 |
2.114 |
2.132 |
2.123 |
105 |
|||
Positive control |
30 min |
1 |
0.947 |
0.953 |
0.041 |
0.907 |
0.913 |
0.910 |
45 |
2 |
0.775 |
0.773 |
0.735 |
0.733 |
0.734 |
36 |
|||
Test item |
30 min |
1 |
1.145 |
1.143 |
0.037 |
1.108 |
1.106 |
1.107 |
55 |
2 |
1.091 |
1.084 |
1.054 |
1.047 |
1.050 |
52 |
|||
Third main test |
|||||||||
Negative control |
30 min |
1 |
1.992 |
1.990 |
0.043 |
1.949 |
1.947 |
1.948 |
102 |
2 |
1.909 |
1.924 |
1.866 |
1.881 |
1.873 |
98 |
|||
Positive control |
30 min |
1 |
0.679 |
0.677 |
0.043 |
0.636 |
0.634 |
0.635 |
33 |
2 |
0.631 |
0.632 |
0.588 |
0.589 |
0.588 |
31 |
|||
Test item |
30 min |
1 |
1.040 |
1.039 |
0.042 |
0.998 |
0.997 |
0.997 |
52 |
2 |
1.290 |
1.289 |
1.248 |
1.247 |
1.247 |
65 |
|||
Table 7.3.2/2: Main tests - Mean tissue viability and standard deviations for the test item, the negative and positive controls
Group |
Exposure duration |
cOD570 nm |
Viability (%) |
|||
Mean |
SD |
Mean |
SD |
Difference (%) |
||
First main test |
||||||
Negative control |
30 min |
1.886 |
0.105 |
100 |
6 |
8 |
Positive control |
30 min |
0.697 |
0.061 |
37 |
3 |
5 |
Test item |
30 min |
1.211 |
0.012 |
64 |
1 |
1 |
Second main test |
||||||
Negative control |
30 min |
2.023 |
0.141 |
100 |
7 |
10 |
Positive control |
30 min |
0.822 |
0.124 |
41 |
6 |
9 |
Test item |
30 min |
1.079 |
0.040 |
53 |
2 |
3 |
Third main test |
||||||
Negative control |
30 min |
1.911 |
0.053 |
100 |
3 |
4 |
Positive control |
30 min |
0.612 |
0.033 |
32 |
2 |
2 |
Test item |
30 min |
1.122 |
0.177 |
59 |
9 |
13 |
OD = optical density
cOD = blank corrected optical density
SD = standard deviation
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Skin irritation:
An in vitro skin irritation study was performed, on Clary sage oil according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN TM reconstructed human epidermis model.
Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Test item, SAUGE SCLAREE ESS. / CLARY SAGE OIL was applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 h at 37 °C, 5 % CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100 % (reference viability).
In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.
All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.
All test item-treated tissues appeared blue which was considered indicative for viable tissues. Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 70% with a standard deviation of 7% as assessed by the MTT assay. As the mean viability was > 50% after the MTT reduction, the results met the criteria for a non-irritant response.
Under the experimental conditions of this study, Clary sage is considered to be non-irritant to skin. According to the results of this study, the classification of the test item should be: no category (Regulation (EC) No. 1272/2008). Therefore it can be concluded that Lavender oil is not classified under Regulation 1272/2008.
Eye irritation:
An in vitro eye irritation test on the EpiOcular TM cornea epithelial model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test item.
- First main test: The relative mean viability of the tissues treated with the test item was 64% with a difference of 1% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response. However, since a mean % viability equal to 60 ± 5% is considered as a borderline result, it was agreed with the Sponsor to perform a second main test.
- Second main test: The relative mean viability of the tissues treated with the test item was 53% with a difference of 3% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However, since non-concordant results were obtained in the two first main tests, it was agreed with the Sponsor to perform a third main test.
- Third main test: The relative mean viability of the tissues treated with the test item was 59% with a difference of 13% between duplicate tissues. As the mean viability was < 60% after the MTT reduction, the results met the criteria for an irritant response. However since mean % viability was equal to 60 ± 5% and since viability results obtained for each individual tissues led to non-concordant classification, this was considered as a borderline result.
Two out of three main tests gave irritant responses (with one of them with borderline results,i.e.mean % viability equal to 60 ± 5%). When tissues (from all main tests) are considered individually: 50% gave irritant responses (i.e.below 60% viability) and 50% non-irritant response. Even if two out of three main tests gave borderline results, a fourth main test would not be necessary. When all main tests are considered together, the mean % viability tends to borderline results with positive classification (irritant), indeed a significant decrease of viability is obtained. As a result and under our experimental conditions, the test item is considered to be irritating to Reconstructed human Cornea-like Epithelium.
Justification for classification or non-classification
Self-classification:
Based on the available information (OECD 439 test), the substance is not classified for skin irritation/ corrosion according to the Regulation (EC) No. 1272/2008.
Based on the available information, the substance should be classified as eye irritant Category 2 (H319: Causes serious eye irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP). The substance is not expected to be classified as H318 (Causes serious eye damage), as it is not irritant to the skin.
No data was available regarding respiratory irritation.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
