Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction

EC Number:

948-256-0

IUPAC Name:

Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction

Specific details on test material used for the study:

Identification: Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction
Batch (Lot) Number: 06/1234
Expiry date: 31 May 2020
Physical Description: Amber crystal
Purity/Composition: 100% UVCB
Storage Conditions: In refrigerator (2-8°C) protected from light
Additional information
Test Facility test item number: 210638/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Stability at higher temperatures: Not indicated
Chemical name (IUPAC, synonym or trade name): Resinoid of Boswellia serrata (Burseraceae) – exudate with hexane and elimination of the solvent to obtain resinoid
CAS number: 97952-72-2

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approximately 10 weeks old)
- Weight at study initiation: 18.9 to 25.7g
- Housing: On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.

- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

- Water (e.g. ad libitum): Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.


ENVIRONMENTAL CONDITIONS
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22 to 23°C with an actual daily mean relative humidity of 39 to 47%. The value that was outside the targeted range occurred one day and was without a noticeable effect on the clinical condition of the animals or on the outcome of the study. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25%, 10%, 5%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TEST
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test item concentrations were tested; a 25% and 50% concentration. The highest concentration was the highest concentration that could be prepared homogeneously.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. Ears were cleaned prior to ear thickness measurements on Days 3 and 6.
Animals were sacrificed after the final observation.


MAIN STUDY
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

I/ Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
 

II/ Excision of the Nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were euthanized according to laboratories Standard Operating Procedures. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

III/ Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

IV/ Radioactivity Measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

other: For both scientific and animal welfare reasons, no concurrent positive control group were included in the study.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

>= 0.9 - <= 1.1

Test group / Remarks:

0% (control)

Results 2

Key result

Parameter:

SI

Value:

>= 0.6 - <= 1

Test group / Remarks:

5% group

Results 3

Key result

Parameter:

SI

Value:

ca. 0.9

Test group / Remarks:

10% group

Results 4

Key result

Parameter:

SI

Value:

>= 0.8 - <= 2.4

Test group / Remarks:

25% group

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Executive summary:

The skin sensitization of the test item was assessed according to the OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay). Since there was no indication that the test item elicits a SI≥3 when tested up to 25%, Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction was considered not to be a skin sensitizer.