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

Eye irritation

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

947-377-6

IUPAC Name:

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

Test material form:

solid: particulate/powder

Remarks:

off-white powder (as reported in the description of the substance)

Details on test material:

Name Boswellia serrata extract (as reported in the report)
Other Name BOSWELLIA SERRATA POUDRE
Batch no. LS210317
Appearance off-white powder
Composition 100% Boswellia serrata extract
CAS No. 97952-72-2
EINECS-No. 308-366-6
Production date Mar. 2017
Expiry date Mar. 2019
Storage Room Temperature (20 ± 5°C), keep away from light/ humidity/ -under inert gas

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.

Test animals / tissue source

Species:

cattle

Strain:

other: bovine cattle (Bos taurus)

Test system

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.

Results and discussion

In vitro

Results

Results 1

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

Results 2

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).