Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation/corrosion

In vitro skin irritation (OECD 439, GLP):

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with GR-87-7596 compared to the negative control tissues was 101%. Since the mean relative tissue viability for GR-87-7596 was above 50% GR-87-7596 is considered to be non-irritant.

 

Eye irritation

-In vitro eye irritation (OECD 437, GLP):

GR-87-7596 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.5 after 10 minutes of treatment.

In conclusion, since GR-87-7596 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 19 to March 26, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
Test system: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 18-EKIN-012
This model is a three-dimensional human epidermis model, which consists of adult humanderived 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.

Source: SkinEthic Laboratories, Lyon, France

Experimental design:
- Test for the Interference of the Test Item with the MTT endpoint:
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

- Test for Color Interference by the Test Item:
GR-87-7596 was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the color interference, 10 μL of GR-87-7596 was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a color check was performed.

- Test for Reduction of MTT by the test item:
GR-87-7596 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 25 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, 25 μL sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed.

- Test system set up:
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 21 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

- MTT medium:
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/mL in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL).

- Environmental conditions:
All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 58 - 84%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.8 – 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

- Application/Treatment of the Test Item:
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five μL of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

- Cell Viability Measurement:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 68 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

- Calculation of Cell Viability:
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) (well with solvent) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- 25 µL of undiluted test item
- 25 µL PBS for negative control
- 25 µL 5% SDS for positive control
Duration of treatment / exposure:
15 ± 0.5 minutes at room temperature
Duration of post-treatment incubation (if applicable):
42 hours at 37°C
Number of replicates:
Triplicate tissues for test item, negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 +/- 0.5 minutes exposure period and 42 hour post incubation period
Value:
101
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:
GR-87-7596 was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that GR-87-7596 did not interact with the MTT endpoint.

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 GR-87-7596 compared to the negative control tissues was 101%. Since the mean relative tissue viability for GR-87-7596 was above 50% GR-87-7596 is considered to be non-irritant.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 13%. The absolute mean OD570 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 ≤ 11%, indicating that the test system functioned properly.
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, GR-87-7596 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified.
Executive summary:

The objective of this study was to evaluate GR-87-7596 for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)).

The possible skin irritation potential of GR-87-7596 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 4 of GR-87-7596 was a yellow liquid. GR-87-7596 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 GR-87-7596 compared to the negative control tissues was 101%. Since the mean relative tissue viability for GR-87-7596 was above 50% after 15 ± 0.5 minutes treatment GR-87-7596 is considered to be non-irritant.

The positive control had a mean cell viability of 13% 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 ≤ 11%, indicating that the test system functioned properly.

In conclusion, GR-87-7596 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes
Species:
other: Bovine eyes from young cattle
Details on test animals or tissues and environmental conditions:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize 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.

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.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 microliters of either the negative control, positive control (Ethanol) or test item
Duration of treatment / exposure:
10 +/- 1 minutes at 32 +/- 1°C
Duration of post- treatment incubation (in vitro):
120 +/- 10 minutes at 32 +/- 1°C.
Number of animals or in vitro replicates:
Total: 9 corneas - 3 corneas/group for test item, negative and positive controls
Details on study design:
- Preparation of Corneas:
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.

- Cornea selection and opacity reading:
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

- Treatment of corneas and opacity measurements:
The medium from the anterior compartment was removed and 750 microliters 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.

- Opacity measurement:
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to:
𝑂𝑝𝑎𝑐𝑖𝑡𝑦 = (((I0/I)-0.9894)/0.0251)
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading.
The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

- Application of Sodium Fluorescein:
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1°C.

- Permeability determinations:
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.
Irritation parameter:
in vitro irritation score
Run / experiment:
10 +/- 1 minutes at 32 +/- 1 °C of exposure and 120 +/- 10 minutes at 32 +/- 1 °C of post-treatment incubation
Value:
0.5
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:
GR-87-7596 was tested neat.

The individual in vitro irritancy scores for the negative controls ranged from 2.5 to 3.8. The individual positive control in vitro irritancy scores ranged from 60 to 69.The corneas treated with the positive control item were turbid after the 10 minutes of treatment.
The corneas treated with GR-87-7596 showed opacity values ranging from -0.5 to 2.4 and permeability values ranging from -0.040 to -0.011. The corneas were translucent after the 10 minutes of treatment with GR-87-7596. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.1 to 2.2 after 10 minutes of treatment with GR-87-7596.

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 64 and 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.
GR-87-7596 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.5 after 10 minutes of treatment.
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, since GR-87-7596 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of GR-87-7596 as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea 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 GR-87-7596 was tested through topical application for 10 minutes.

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

Batch 4 of GR-87-7596 was a yellow liquid. 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 64 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.

GR-87-7596 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.5 after 10 minutes of treatment.

In conclusion, since GR-87-7596 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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

Additional information

Skin Irritation


The study was performed to assess the ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model / EPISKIN-SMTM), in accordance with the GLP principles and following the OECD guideline 439 (In vitro Skin Irritation: Reconstructed Human Epidermis Test Method, (adopted 28 July 2015) and also the EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In vitro Skin Irritation: Reconstructed Human Epidermis Model Test ".


The test substance 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 the test substance compared to the negative control tissues was 101%. Since the mean relative tissue viability for the test substance was above 50% after 15 ± 0.5 minutes treatment the test substance is considered to be non-irritant. The positive control had a mean cell viability of 13% 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 ≤ 11%, indicating that the test system functioned properly. In conclusion, the test substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified.


Eye Irritation


The objective of this study was to evaluate the eye hazard potential of the test substance as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test), in accordance with the GLP principles and following the 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, (adopted October 09, 2017).


The eye damage of the test substance was tested through topical application for 10 minutes. 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 64 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. The test substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.5 after 10 minutes of treatment. In conclusion, since the test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Justification for classification or non-classification

Under the experimental conditions of this in vitro skin irritation study, the test item Scentaurus juicy (GR-87 -7596) is not classified as skin irritant according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS (result > 50%).

 

According to the in vitro eye irritation study and under the experimental conditions reported, Scentaurus juicy (GR-87 -7596) is not corrosive/irritating to the eye according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (IVIS ≤ 3 ).

Categories Display