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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

Name: Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction
Batch number: 06/1234
EC number: 948-256-0
Appearance: Amber crystal
Purity: 100% UVCB
Expiry date: 29 February 2020
Storage conditions: Refrigerated (2-8°C), protected from light and humidity (stored in a tightly closed container)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Method

Target gene:

In addition to histidine or tryptophan mutation, each strain has additional mutations, which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The presence of rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction.

The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Citoxlab Hungary Ltd. according to Ames et al. [1] and Maron and Ames [2]. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.

Test concentrations with justification for top dose:

In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
Precipitate/slight precipitate was detected on the plates in both Salmonella typhimurium strains with and/or without metabolic activation at 5000 and 2500 μg/plate concentrations in the preliminary experiment.
No inhibitory or toxic effects of the test item were detected in the preliminary experiment.

Based on the results of the preliminary experiment, the examined test concentrations in the Assays 1-2 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.

Vehicle / solvent:

Due to the better biocompatibility, DMSO was selected as vehicle for the study.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

2 groups: DMSO and distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine (NPD), for TA98, without activation; 2-aminoanthracene for all salmonella strains and E. coli, with activation

Details on test system and experimental conditions:

--> ASSAY 1

Assay 1 followed the standard plate incorporation procedure. Bacteria (cultured in Nutrient Broth) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.

Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).

The content of the tubes:
top agar 2000µL
vehicle or test item formulation (or reference controls) 50µL
overnight culture of test strain 100µL
phosphate buffer (pH 7.4) or S9 mix 500µL

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.



--> ASSAY 2


Assay 2 followed the standard pre-incubation procedure, since no biologically relevant increase in the number of revertant colonies was observed in the Assay 1.

Bacteria (cultured in Nutrient Broth) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
 
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture (Section 5.3.6.) and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.

After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.

Evaluation criteria:

Criteria for Validity:

The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

According to the guidelines [5][6][7][8], statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
 

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: S. typhimurium strains and E. coli WP2

Remarks:

ASSAY 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: S. typhimurium strains and E. coli WP2

Remarks:

ASSAY 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the Assay 1, the plate incorporation method, in the Assay 2, the pre-incubation method was used. The Assays 1-2 were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain. The assays were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.

Based on the results of the preliminary experiment, the examined test concentrations in the Assays 1-2 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.

In the main assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

Precipitate was detected on the plates in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration in Assay 1. Precipitate / slight precipitate was detected on the plates in all examined bacterial strains with and without metabolic activation at 5000 and 1581 μg/plate concentrations in Assay 2.

Inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development) was observed in the Assay 2 in all examined bacterial strains with and/or without metabolic activation at 5000 μg/plate concentration.

In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 strain at 5000 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.67). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.

In Assay 2 (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 158.1 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.27). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
 

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in

Applicant's summary and conclusion

Conclusions:

The test item Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction (Batch Number: 06/1234) had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test, Assay 1 (Plate Incorporation Method) and Assay 2 (Pre-Incubation Method).

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

The test item Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction (Batch Number: 06/1234) had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.