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Description of key information

Eye Irritation

Evaluation of the eye Hazard potential of FRESKOMENTHE using the bovine Corneal Opacity and Permeability test (BCOP) dated on 2016 and perfomed according to the OECD Guideline No. 437, has determined that the FRESKOMENTHE IVIS is > 3 ≤ 55, and no prediction on the classification can be made.

The QSAR Toolbox 3.4.0.17 prediction ( Database version: 3.8.8/3.1.2) dated on 2017 on Freskomenthe shows the eye irritant potential of FRESKOMENTHE is negative with very strong confidence.

Skin irritation

In Vitro Skin Irritation Test With Freskomenthe Using A Human Skin Model dated on 2016 and performed accoridng to the OECD guideline No. 439 shows that the FRESKOMENTHE is irritant to skin in the test conditions.

In vitro Skin Corrosion Test with FRESKOMENTHE using a Human Skin Model dated on 2017 and performed accoridng to the OECD guideline No. 431 shows that the FRESKOMENTHE is not corrosive to skin in the test conditions.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 April 2016 to 18 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Remarks:
There were no deviations from standard operating procedures that affected the integrity of the study.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name (as stated in the report): Freskomenthe
Batch: VE00411894
Expiration date: February 11, 2018
Test system:
human skin model
Source species:
human
Cell type:
other: 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.
Cell source:
other: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-015,)
Source strain:
other: SkinEthic Laboratories, Lyon, France
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:
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-015, See APPENDIX 4).
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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Undiluted FRESKOMENTHE was applied (25 μl)
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hour
Number of replicates:
1
Irritation / corrosion parameter:
% tissue viability
Remarks:
Mean tissue viability (percentage of control)
Value:
8
Negative controls validity:
valid
Remarks:
100% Mean tissue viability (percentage of control)
Positive controls validity:
valid
Remarks:
27% Mean tissue viability (percentage of control)
Other effects / acceptance of results:
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 27%.
The absolute mean OD(570) 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 19%, indicating that the test system functioned properly.

The mean tissue viability obtained after 15 ± 0.5 minutes treatment with FRESKOMENTHE was compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with FRESKOMENTHE compared to the negative control tissues was 8%. Since the mean relative tissue viability for FRESKOMENTHE was below 50% it is considered to be irritant.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Finally, it is concluded that this test is valid and that FRESKOMENTHE is irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

In vitro skin irritation test with FRESKOMENTHE using a human skin model.

This report describes the ability of FRESKOMENTHE to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of FRESKOMENTHE was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch VE00411894 of FRESKOMENTHE was a clear colourless liquid. FRESKOMENTHE was applied undiluted (25 μl), directly on top of the skin tissue for 15 ± 0.5 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 item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with FRESKOMENTHE compared to the negative control tissues was 8%. Since the mean relative tissue viability for FRESKOMENTHE was below 50% after 15 ± 0.5 minutes treatment it is considered to be irritant.

The positive control had a mean cell viability of 27% after 15 ± 0.5 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 19%, indicating that the test system functioned properly (study plan deviation 1).

Finally, it is concluded that this test is valid and that FRESKOMENTHE is irritant in the in vitro skin irritation test under the experimental conditions described in this report

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May 2017 to 12 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Qualifier:
according to
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".
Principles of method if other than guideline:
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name (as stated in the report): Freskomenthe
Batch: VE00426966
Expiration date: 19 February 2018
Test system:
human skin model
Source species:
human
Cell type:
other: human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
Cell source:
other: EpiDerm Skin Model (EPI-200, Lot no.: 25789 kit I and J)
Source strain:
other: MatTek Corporation, Ashland MA, U.S.A.
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Remarks:
The liquid test item was applied undiluted (50 μl) directly on top of the tissue.
Details on test system:
EpiDerm Skin Model (EPI-200, Lot no.: 25789 kit I and J,
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 μl
Duration of treatment / exposure:
3 hours
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
after 3-minute treatment
Value:
121
Negative controls validity:
valid
Remarks:
the negative control tissues was within the acceptance limits
Positive controls validity:
valid
Remarks:
9.1% tissue viability 1-hour exposure
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
after 1-hour treatments
Value:
68
Other effects / acceptance of results:
The positive control had a mean relative tissue viability of 9.1% after the 1-hour exposure.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit <= 2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates of the negative control was <= 30%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 121% and 68%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate FRESKOMENTHE for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The

possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch VE00426966 of the test item was a clear colourless liquid. The test item was applied undiluted (50 μl) on top of the skin tissue.

The positive control had a mean relative tissue viability of 9.1% after the 1-hour exposure.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit <=2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates of the negative control was

<= 30%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 121% and 68%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
Principles of method if other than guideline:
There were no deviations from standard operating procedures that affected the integrity of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name (as stated in the report): Freskomenthe
Batch: VE00411894
Expiration date: February 11, 2018
Species:
other: Bovine
Details on test animals or tissues and environmental conditions:
Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
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.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
Vehicle:
unchanged (no vehicle)
Remarks:
The test item was tested neat.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Undiluted FRESKOMENTHE
Duration of treatment / exposure:
The test item was tested undiluted
Details on study design:
The medium from the anterior compartment was removed and 750 μl of either the negative control, positive control (Ethanol) or test item 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 item 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 (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item 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.
Irritation parameter:
cornea opacity score
Remarks:
mean in vitro irritancy score after 10 minutes of treatment
Value:
8.4
Negative controls validity:
valid
Remarks:
0.9
Positive controls validity:
valid
Remarks:
46.2

The individual in vitro irritancy scores for the negative controls ranged from 0.7 to 1.1. The individual positive control in vitro irritancy scores ranged from 41 to 55 for Ethanol. The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with FRESKOMENTHE showed opacity values ranging from 5.9 to 9.2 and permeability values ranging from 0.028 to 0.173. The corneas were clear after the 10 minutes of treatment with FRESKOMENTHE. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 6.7 to 9.9 after 10 minutes of treatment with FRESKOMENTHE.

Interpretation of results:
GHS criteria not met
Conclusions:
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 (Ethanol) was 46 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
FRESKOMENTHE induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 8.4 after 10 minutes of treatment.
Since FRESKOMENTHE induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.
Executive summary:

Evaluation of the eye hazard potential of FRESKOMENTHE using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of FRESKOMENTHE was tested through topical application for 10 minutes.

The study procedures described in this report were based on the most recent OECD guideline.

Batch VE00411894 of FRESKOMENTHE was a colourless liquid with a purity of 99.1%. The test item was applied as it is (750 μl) directly on top of the corneas.

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 (Ethanol) was 46 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

FRESKOMENTHE induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 8.4 after 10 minutes of treatment.

Since FRESKOMENTHE induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Endpoint:
eye irritation: in vivo
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
QSAR Toolbox 3.4.0.17

2. MODEL (incl. version number)
Database version: 3.8.8/3.1.2

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CCC(C)C1CCCCC1=O
Reason / purpose:
(Q)SAR model reporting (QMRF)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
Experimental data taken from the OECD [Q]SAR Toolbox
Deviations:
not specified
Principles of method if other than guideline:
[Q]SAR prediction for skin corrosion detrmination using the OECD [Q]SAR Toolbox.
GLP compliance:
not specified
Specific details on test material used for the study:
Chemical name(s):
cyclohexanone, 2-(1-methylpropyl)-
2-sec-butylcyclohexan-1-one
2-(butan-2-yl)cyclohexanone
cyclohexanone, 2-sec-butyl-
2-sec.-butylcyclohexanone (cyclohexanone, 2-(1-methylpropyl)-)
cyclohexanone, 2-sec-butyl- (7ci,8ci)
CAS number:14765-30-1
SMILES: CCC(C)C1CCCCC1=O
Species:
other: QSAR
Strain:
not specified
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: Taken from the QSAR
Score:
0
Max. score:
4
Reversibility:
not specified

Eye Irritation:

The prediction is based on 16 neighbours' values, 7 of them equal to prediction.

Prediction confidence is measured by the p-value = 2.19E-15 (very strong confidence).

Conclusions:
QSAR:
The target chemical FALLS within applicability domain.
Negative in an in vivo test system conducted to OECD 405 guidance on high klimisch experimental data.
Very high confidence in the result as indicated by the p-value.
The prediction is based on 16 neighbours' values, 7 of them equal to prediction.
Prediction confidence is measured by the p-value = 2.19E-15 (very strong confidence)
Executive summary:

QSAR prediction on Freskomenthe is negative for eye irritation in rabbits with very strong confidence.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Freskomenthe has been determined to be a non eye irritant substance according to the test conditions; Nevertheless, this substance has shown a skin irritant potential.

Freskomenthe meets the criteria to be classified Category 2 (irritant) based on GHS classification criteria.