Extract of Boswellia serrata (Burseraceae) obtained from exudate by solvent extraction

Endpoint summary

Description of key information

- Skin irritation/corrosion: not classified (OECD 439, GLP, Rel. 1)

- Eye irritation: not classified (OECD 437, GLP, Rel. 1)

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:

02 May - 01 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 439 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 23 July 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

27 April 2017

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Used as supplied

Test system:

human skin model

Source species:

other: RECONSTRUCTED HUMAN EPIDERMIS

Cell type:

non-transformed keratinocytes

Cell source:

other: foreskin

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 0.50 cm² reconstructed epidermis (Episkin SA, RHE/S/17 Batch No. 17-RHE-063) were received on 30 May 2017.
- On the same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA) during 2 hours and 25 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA).

TREATMENT
- The test item was applied during 42 minutes at room temperature, at the dose of 16 mg, to 5 living skin models, previously moistened with 10 µL of distilled water.
- In the same experimental conditions, a positive control (5% SDS) and a negative control (DPBS) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermises, during all the treatment period, the test item was recovered with a nylon mesh provided by Episkin SA.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the nylon mesh was removed and the human epidermises were washed with 25 x 1 mL of DPBS. The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues. They were incubated for a 42-hour post-treatment incubation-period in fresh medium at 37°C, 5% CO2. Then, the epidermis were put in contact with the MTT solution, except the two tissues for the non-specific colour control which were placed in the maintenance medium for 3 hours at 37°C, 5% CO2.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The cell viability was quantified by the measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD were proportional to the number of living cells.
- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the OD (Optical Density) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).
- The OD of MTT extract was measured in triplicate. The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

VIABILITY CALCULATION:
- The results were expressed as a viability percentage compared with the negative control: viability % = (mean OD test item / mean OD negative control) * 100
- As the test item was identified as potentially causing colour interferences: True viability % = [(OD of living tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT) / OD of living tissues exposed to negative control] x 100
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1.

PREDICTION MODEL / DECISION CRITERIA
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:
- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >50%.
- The test item is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is "non-corrosive". In accordance with Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.
- The test item is considered to be irritant or corrosive to skin if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤50% and in absence of information on a skin corrosion test. In accordance with the Regulation (CE) No.1272/2008 and in absence of information on a skin corrosion test, the item has to be classified in Category 2 "Irritant" or in Category 1 "Corrosive". The corresponding hazard statement is respectively, "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger".

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

42 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours post-incubation period at 37°C, 5% CO2

Number of replicates:

5 living human skin models including 2 for colour controls

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

main test (duration of exposure: 42 minutes)

Value:

95.9

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 interaction with MTT:
A white suspension with test item on top of the MTT solution was observed after 3 hours of incubation between 36.9°C and 37.6°C, 5% CO2. Therefore, there was no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
In water, a white suspension with test item on top of the MTT solution was obtained after 1 hour of incubation between 36.9°C and 37.6°C, 5% CO2.
In isopropanol, a homogeneous yellow suspension was obtained after 2 hours of incubation at room temperature. Since a change of coloration was observed in isopropanol, a spectral analysis was done in the isopropanol supernatant.
- Spectral analysis of the test item in isopropanol:
The mean of the corrected OD (blank subtracted) was 0.09 in isopropanol which is higher than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). Therefore, the test item was identified as causing colour interference with the viability assay and two viable control tissues were added to the study which underwent the entire testing procedure but were incubated with medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSC living) control.


MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues was 95.9%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate).
- The mean percent tissue viabilities obtained with the negative controls and positive controls were within the range of historical data and therefore validated the experiment.

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

	Skin tissues	OD	Mean OD/disc (#)	Mean OD/product	Viability %	Mean viability %	Standard deviation (SD)
Negative control	1	0.965	0.966	0.903	106.9	100.0	6.2
		0.980
		0.954
	2	0.887	0.887		98.2
		0.873
		0.901
	3	0.860	0.857		94.9
		0.852
		0.859
Positive control	1	0.013	0.012	0.011	1.3	1.3	0.1
		0.011
		0.013
	2	0.011	0.010		1.1
		0.009
		0.010
	3	0.013	0.012		1.3
		0.012
		0.013
Test item	1	0.931	0.929	0.865	102.8	95.8	8.6
		0.953
		0.904
	2	0.796	0.778		86.1
		0.804
		0.735
	3	0.970	0.888		98.3
		0.928
		0.767
Test item (NSC control)	1	-0.002	-0.002	-0.001	-0.2	-0.1	0.2
		-0.003
		-0.002
	2	-0.001	0.000		0.0
		-0.001
		-0.003
Test item (corrected)						95.9

 #: mean of 3 values (triplicate of the same extract)

OD: optical density

 

Acceptability criteria:

- SD ≤ 18%;

- Negative control: OD value of the 3 replicates in the range ≥0.8 and ≤3.0

OD was measured after a 1:2 dilution of the formazan extracts in isopropanol: the acceptability criteria should be in the range >=0.4 and =<1.5 for the negative control.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.

Executive summary:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test substance.

The test item was applied during 42 minutes, at the dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) previously moistened with 10 µL of distilled water. The treatment of the epidermis was followed by a rinse with 25 mL of DPBS and a 42 hours post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Additionally, 2 living Human skin model surfaces were treated in the same manner but they were incubated in culture medium instead of MTT solution in order to generate nonspecific living colour controls.

The mean corrected percent viability of the treated tissues was 95.9%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate). The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validated the experiment.

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.

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:

09 May - 06 July 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study performed according to OECD Guideline 431 with minor deviation: mean OD490 nm of the vehicle control in one of the two runs was higher than the historical mean values. This deviation was considered not to have compromised the validity or integrity of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

dated 26 July 2013

Deviations:

yes

Remarks:

mean OD490 nm of the vehicle control in one of the two runs was higher than the historical mean value

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

02 November 2016

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item formulation was prepared within 4 hours of use and stored at room temperature until use. Moreover, and since the test item formulation was brownish homogeneous suspension, it was maintained under magnetic stirring at least 15 minutes before treatment and then during use.

Species:

cattle

Strain:

other: bovine cattle (Bos taurus)

Vehicle:

other: paraffin oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL (± 8 μL)
- Concentration (if solution): 20% (w/v) in paraffin oil

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 μL (± 8 μL)

Duration of treatment / exposure:

4 hours (± 5 minutes)

Duration of post- treatment incubation (in vitro):

90 minutes (± 5 minutes)

Number of animals or in vitro replicates:

Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
- Bovine eyes were obtained from freshly slaughtered cattle (age: typically 5-8 months old) at the abattoir EVA, Saint-Pierre-sur-Dives, France. The eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.
- Upon arrival at CiToxLAB France, the tissues surrounding the eyeball of selected corneas were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared. The corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 μg/mL final) at room temperature. The corneas were used within a maximum of 24 hours.
- In both experiments, corneas obtained from freshly slaughtered calves were mounted in corneal holders [OPKIT (polypropylene), diameter 18 mm, ref. ED 4004 SB (MC2, Clermont-Ferrand, France)]. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C (± 1°C).
- At the end of the pre-incubation period, the medium was removed from both chambers of the holder using a metal gavage tube attached to a vacuum pump to ensure complete evacuation. They were refilled with fresh cMEM (previously heated to +32°C), starting with the posterior chamber and taking care that no air bubbles were present. The chambers were re-sealed and the corneas were examined macroscopically through the holder to detect the presence of any defects. Then, the opacity of the cornea was measured to obtain OPT0. Corneas that showed any macroscopic defect or an OPT0 value over 7 were discarded.

QUALITY CHECK OF THE ISOLATED CORNEAS: A careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.

NUMBER OF REPLICATES: Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

APPLICATION DOSE AND EXPOSURE TIME: The test item, formulated at 20% in paraffin oil, was evaluated in these two experiments using a treatment time of 4 hours and using the open-chamber method.

TREATMENT METHOD: The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea. The test item [750 μL (± 8 μL)], formulated at 20% in paraffin oil, was applied for a treatment time of 4 hours (± 5 minutes) using the open-chamber method. Vehicle and positive controls were applied using the same treatment time and the closed-chamber treatment method.
The treatment time of each series of three corneas was carefully measured with a chronometer, starting from the beginning of treatment of the first cornea of each series. Then each further operation (rinsing, measurement, etc) was carried out in the same order for the three corneas of each series.
After application of the items, the holders were incubated vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at +32°C (± 1°C), for the selected treatment time.

RINSING OF THE CORNEAS: On completion of the treatment period, the test item formulation was removed from the front opening of the anterior chamber (open-chamber method) and the epithelium was rinsed as follows:
- any test item formulation adhering to the walls of the anterior chamber was removed using a pipette of heated cMEM (+32°C),
- the corneas were rinsed four times with pre-warmed cMEM containing phenol red (i.e. until the test item formulation had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.
The rinsing efficiency was visually confirmed by observing the transparency and the colour changing of the rinsing medium (containing phenol red). Despite the rinsing performed, residual test item was noted on one cornea (No. 64) during the first experiment, and on all the test item-treated corneas during the second one.
The anterior chamber was refilled with fresh pre-warmed cMEM without phenol red. The front cover was replaced. Care was taken to make sure that no air bubbles were present within the holders (by ensuring that each chamber was filled to overflowing with pre-warmed cMEM).
Following the 4-hour treatment and the rinsing step, the medium of both chambers was renewed with pre-warmed cMEM (+32°C) and the second opacity measurement (OPT2) was performed directly.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. Corneal opacity was determined by reading each holder in the right hand chamber of the calibrated opacitometer, versus an empty holder (without cMEM, cornea and glasses), placed in the left hand chamber.
- Corneal permeability: After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution. As the test item is a non-surfactant solid, the concentration of the fluorescein solution was 5 mg/mL. Before each use, the fluorescein solution was validated. For this purpose, the solution of fluorescein was diluted in cMEM in order to obtain a 5 μg/mL solution and the Optical Density at a wavelength of 490 nm (OD490 nm) of this dilution was measured. For both experiments, as the values obtained were between 0.850 and 0.940, the fluorescein solution was validated.
For each series of three corneas, a chronometer started from the fluorescein solution application time of the first cornea of the series. The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes).
At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank).
- Macroscopic examination: After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium. Then, the corneas were discarded.

SCORING SYSTEM: The In Vitro Irritancy Score (IVIS) was determined from the opacity and permeability measurements, as described below.
- Opacity: The change in opacity value of each individual cornea treated with test item, negative control or positive control was calculated by subtracting the initial base-line opacity measurement (OPT0) from the post-treatment opacity reading (OPT2). The average change in opacity for the corneas treated with the vehicle control was calculated and this value was subtracted from the change in opacity for each cornea treated with test item or positive control to obtain a corrected opacity value (cOPT). When the average change in opacity for the corneas treated with the negative/vehicle control was negative, it is considered equal to 0. The mean cOPT value of each series of three corneas was calculated from the individual corrected opacity values.
- Permeability: The corrected OD490 nm (cOD490 nm) value (i.e. permeability) for each cornea treated by test item or positive control was calculated by subtracting the average vehicle control cornea OD490 nm value from the original OD490 nm value of each cornea. When the average vehicle control cornea OD490 nm value was negative, it is considered equal to 0. The mean cOD490 nm value of each series of three corneas was calculated from the individual cOD490 nm values.
- In Vitro Irritancy Score calculation: The following formula was used to determine the In Vitro Irritancy Score (IVIS):
IVIS = cOPT + (15 x cOD490 nm)
The IVIS was calculated for each test item and positive control cornea. The mean IVIS for each series of three corneas was calculated from the individual scores.

ACCEPTANCE CRITERIA:For the validation of an experiment, the following criteria had to be fulfilled:
- the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two standard deviations of the historical mean,
- the mean opacity and mean OD490 nm of the vehicle control corneas should be less than the established upper limit of historical mean

DECISION CRITERIA: The IVIS cut-off values for identifying the test item as inducing serious eye damage (UN GHS Category 1) or as not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are the following:
- If the test item induces an IVIS ≤ 3: No category
- If the test item induces an 3 < IVIS ≤ 55: No prediction can be made
- If the test item induces an IVIS > 55: Category 1
Two experiments composed of three corneas were performed to unequivocally classify the test item.

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

experiment 1

Value:

4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

experiment 2

Value:

1

Vehicle controls validity:

not valid

Remarks:

0.059 instead of ≤ 0.043 (historical value); considered not to have compromised the validity or integrity of the study

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

MACROSCOPIC EXAMINATIONS:
- Fluorescein fixation and/or residual amount of test item were observed on two of the three test item-treated corneas in the first experiment and in all three corneas in the second experiment.
- In both experiments, no notable opaque spots or irregularities were observed on vehicle control corneas, and opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

IN VITRO IRRITANCY SCORE:
First experiment:
- The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 4. Individual IVIS values of test item-treated corneas were: 1, 1 and 11. Refer Table 7.3.2/1 for details.
- Two of the three corneas gave discordant predictions from the mean of all three corneas since the third corneas gave a higher IVIS principally due to the opacity value. As residual test item was noted over these corneas after the rinsing step, it was therefore not possible to determine if the high corneal opacity value noted for these corneas was due to the test item reacting with cell structures (i.e. protein precipitation, etc.) or only to these residuals amounts of test item which stuck to the cornea. Moreover, during this assay, results could be considered as borderline between "no category" and "no prediction can be made" since the mean IVIS was 4. In this context, the result in the first testing experiment was considered borderline and a second experiment was performed.

Second experiment:
- The mean IVIS of the test item-treated corneas was: 1. Individual IVIS values of test item-treated corneas were: 2, 0 and 1. Refer Table 7.3.2/2 for details.

On the basis of the two experiments performed as part of this study, five out of the six test item-treated corneas gave a mean IVIS < 3, and the only IVIS > 3 could be related to a high corneal opacity value likely due to representative residual amounts of test item which stuck to the corresponding cornea. Residual amounts of test item were also noted on each test item-treated cornea in the second experiment, but amounts were not graded, and no significant increase in opacity was noted, suggesting only very few amount of test item remained onto these corneas.
As a consequence, based on the concordant results obtained on five out of six corneas during these two experiments, no additional experiment was performed and the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: In first experiment, opacity and permeability (OD490 nm) were within the historical range. During the second experiment, mean OD490 nm of the vehicle control corneas was found at 0.059 instead of ≤ 0.043 (historical value). The cOD490 nm values of positive control and test item are calculated by subtracting each individual OD490 nm by the mean OD490 nm of the vehicle control (i.e. 0.059). Consequently, this unusually high mean OD490 nm of the vehicle control corneas could have potentially led to an over-correction of the cOD490 nm of the test item and positive control, and therefore possibly lower IVIS values. Individual IVIS were re-calculated for the test item and positive control without applying mean vehicle control OD490 nm value correction and obtained IVIS values still fell within the same IVIS range (i.e. no change in conclusion). The mean IVIS of the positive control was still found within the historical data range, and results of test item-treated corneas remained roughly unchanged (i.e. recalculated mean IVIS = 2, with individual IVIS = 3, 1 and 1, for the three corneas, respectively), despite no concordant classifications based on mean IVIS were obtained at completion of the first and second experiments, no additional experiment was performed as noted in the study plan. It was indeed judged obvious that concordant results IVIS obtained from five out of six corneas was sufficient for conclusion and that no further testing was required. This deviation was considered not to have compromised the validity or integrity of the study.
- Acceptance criteria met for positive control: IVIS values for positive control were within the range of historical values during both experiments.
- Range of historical values if different from the ones specified in the test guideline: IVIS for positive control: 113-169; opacity ≤ 4 and permeability (OD490 nm) ≤ 0.043 for negative control (4-hour treatment); refer image of Table 7.3.2/3 for details.

Table 7.3.2/1: First experiment – Results

 

Calibration of the opacitometer	Before OPT0	After OPT0	Before OPT2	After OPT2
Calibrator No. 1	75	76	75	74
Calibrator No. 2	146	np	np	np
Calibrator No. 3	218	np	np	np

 

Group	Holder	Opacity					Permeability			IVIS
		OPT0	OPT2	OPT2-OPT0	Neg correction	cOPT	OD490 nm	Neg correction	cOD 490 nm
Vehicle control	1	1	0	-1	0	-	0.031	0.031	-	-
	2	1	0	-1	0	-	0.023	0.023	-	-
	3	1	1	0	0	-	0.016	0.016	-	-
	Mean	-	-	-	0.0	-	-	0.023	-	-
	SD	-	-	-	0.0	-	-	0.008	-	-
Test item	1	0	0	0	0	0	0.084	0.084	0.061	1
	2	1	1	0	0	0	0.113	0.113	0.090	1
	3	2	11	9	9	9	0.142	0.142	0.119	11
	Mean	-	-	-	-	3.0	-	-	0.090	4
	SD	-	-	-	-	5.2	-	-	0.029	5.6
Positive control	1	0	110	110	110	110	3.264	3.264	3.241	159
	2	1	123	122	122	122	3.228	3.228	3.205	170
	3	2	114	112	112	112	3.192	3.192	3.169	160
	Mean	-	-	-	-	114.7	-	-	3.205	163
	SD	-	-	-	-	6.4	-	-	0.036	6.4

 

 

Table 7.3.2/2: Second experiment – Results

 

Calibration of the opacitometer	Before OPT0	After OPT0	Before OPT2	After OPT2
Calibrator No. 1	75	76	75	76
Calibrator No. 2	149	np	np	np
Calibrator No. 3	221	np	np	np

 

Group	Holder	Opacity					Permeability			IVIS
		OPT0	OPT2	OPT2-OPT0	Neg correction	cOPT	OD490 nm	Neg correction	cOD 490 nm
Vehicle control	1	2	1	-1	0	-	0.065	0.065	-	-
	2	2	2	0	0	-	0.087	0.087	-	-
	3	2	1	-1	0	-	0.024	0.024	-	-
	Mean	-	-	-	0.0	-	-	0.059	-	-
	SD	-	-	-	0.0	-	-	0.032	-	-
Test item	1	1	3	2	2	2	0.053	0.053	0.000	2
	2	2	2	0	0	0	0.048	0.048	0.000	0
	3	3	3	0	0	0	0.093	0.093	0.034	1
	Mean	-	-	-	-	0.7	-	-	0.011	1
	SD	-	-	-	-	1.2	-	-	0.020	1.0
Positive control	1	1	118	117	117	117	3.192	3.192	3.133	164
	2	2	112	110	110	110	3.240	3.240	3.181	158
	3	3	116	113	113	113	3.320	3.320	3.261	162
	Mean	-	-	-	-	113.3	-	-	3.192	161
	SD	-	-	-	-	3.5	-	-	0.065	3.2

np: not performed

OD: optical density

cOD: optical density corrected by mean OD value of vehicle control

cOPT: corneal opacity corrected by mean opacity value of vehicle control

OPT0: corneal opacity before treatment

OPT2: corneal opacity after treatment

Neg correction: all individual values below 0 are set at 0

SD: standard deviation

IVIS: In Vitro Irritation Score

Vehicle control: Paraffin oil

Positive control: 20% imidazole solution in 0.9% NaCl

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item, the test item was identified as not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Executive summary:

An ex vivo eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on bovine corneas.

The test item was evaluated in two experiments. In both experiments, corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C. Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

Before treatments, a first opacity measurement was performed on each cornea using an opacitometer.

The test item, formulated at 20% in paraffin oil, was evaluated in these two experiments using a treatment time of 4 hours and using the open-chamber method. Vehicle and positive controls were applied using the same treatment time and the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.

A second opacity measurement was then performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

With one exception during the second experiment (mean OD490 nm of the vehicle control), all acceptance criteria were fulfilled during both experiments. The study was therefore considered as valid.

In first experiment, fluorescein fixation and/or residual amount of test item were observed on two of the three test item-treated corneas. No notable opaque spots or irregularities were observed on the remaining cornea. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 4.

Individual IVIS values of test item-treated corneas were: 1, 1 and 11. Two of the three corneas gave discordant predictions from the mean of all three corneas since the third corneas gave a higher IVIS principally due to the opacity value. As residual test item was noted over these corneas after the rinsing step, it was therefore not possible to determine if the high corneal opacity value noted for these corneas was due to the test item reacting with cell structures (i.e. protein precipitation, etc.) or only to these residuals amounts of test item which stuck to the cornea. Moreover, during this assay, results could be considered as borderline between "no category" and "no prediction can be made" since the mean IVIS was 4. In this context, the result in the first testing experiment was considered borderline and a second experiment was performed.

In the second experiment, fluorescein fixation and residual test item were observed on all the corneas treated with the test item. The mean IVIS of the test item-treated corneas was: 1. Individual IVIS values of test item-treated corneas were: 2, 0 and 1.

On the basis of the two experiments performed as part of this study, five out of the six test item-treated corneas gave a mean IVIS < 3, and the only IVIS > 3 could be related to a high corneal opacity value likely due to representative residual amounts of test item which stuck to the corresponding cornea. Residual amounts of test item were also noted on each test item-treated cornea in the second experiment, but amounts were not graded, and no significant increase in opacity was noted, suggesting only very few amount of test item remained onto these corneas.

As a consequence, based on the concordant results obtained on five out of six corneas during these two experiments, no additional experiment was performed and the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Under the experimental conditions of this study, the test item, the test item was identified as not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test substance.

The test item was applied during 42 minutes, at the dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) previously moistened with 10 µL of distilled water. The treatment of the epidermis was followed by a rinse with 25 mL of DPBS and a 42 hours post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Additionally, 2 living Human skin model surfaces were treated in the same manner but they were incubated in culture medium instead of MTT solution in order to generate nonspecific living colour controls.

The mean corrected percent viability of the treated tissues was 95.9%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate). The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validated the experiment.

Eye irritation/ corrosion:

An ex vivo eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on bovine corneas.

The test item was evaluated in two experiments. In both experiments, corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C. Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

Before treatments, a first opacity measurement was performed on each cornea using an opacitometer.

The test item, formulated at 20% in paraffin oil, was evaluated in these two experiments using a treatment time of 4 hours and using the open-chamber method. Vehicle and positive controls were applied using the same treatment time and the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.

A second opacity measurement was then performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

With one exception during the second experiment (mean OD490 nm of the vehicle control), all acceptance criteria were fulfilled during both experiments. The study was therefore considered as valid.

In first experiment, fluorescein fixation and/or residual amount of test item were observed on two of the three test item-treated corneas. No notable opaque spots or irregularities were observed on the remaining cornea. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 4.

Individual IVIS values of test item-treated corneas were: 1, 1 and 11. Two of the three corneas gave discordant predictions from the mean of all three corneas since the third corneas gave a higher IVIS principally due to the opacity value. As residual test item was noted over these corneas after the rinsing step, it was therefore not possible to determine if the high corneal opacity value noted for these corneas was due to the test item reacting with cell structures (i.e. protein precipitation, etc.) or only to these residuals amounts of test item which stuck to the cornea. Moreover, during this assay, results could be considered as borderline between "no category" and "no prediction can be made" since the mean IVIS was 4. In this context, the result in the first testing experiment was considered borderline and a second experiment was performed.

In the second experiment, fluorescein fixation and residual test item were observed on all the corneas treated with the test item. The mean IVIS of the test item-treated corneas was: 1. Individual IVIS values of test item-treated corneas were: 2, 0 and 1.

On the basis of the two experiments performed as part of this study, five out of the six test item-treated corneas gave a mean IVIS < 3, and the only IVIS > 3 could be related to a high corneal opacity value likely due to representative residual amounts of test item which stuck to the corresponding cornea. Residual amounts of test item were also noted on each test item-treated cornea in the second experiment, but amounts were not graded, and no significant increase in opacity was noted, suggesting only very few amount of test item remained onto these corneas.

As a consequence, based on the concordant results obtained on five out of six corneas during these two experiments, no additional experiment was performed and the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

According to results from OECD 439 study, the registered substance should not be classified according to Regulation EC N° 1272/2008 (CLP) and GHS.

According to the results of OECD TG 437, the registered substance should not be classified according to Regulation EC N° 1272/2008 (CLP) and GHS.