Essential oil of Dipterocarpus (Dipterocarpaceae) obtained from the resin tapped from Dipterocarpus trees by steam distillation (gurjunene quality)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 Apr 2018 - 18 Jun 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Essential oil of Gurjun obtained from the resin tapped from Dipterocarpus trees (Dipterocarpaceae) by steam distillation (Gurjunene quality)

EC Number:

947-839-7

IUPAC Name:

Essential oil of Gurjun obtained from the resin tapped from Dipterocarpus trees (Dipterocarpaceae) by steam distillation (Gurjunene quality)

Test material form:

liquid

Details on test material:

Name in used in report: Gurjun Balsam oil (Gurjunene)
Test facility number 209144/A

Method

Target gene:

His, Trp

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:

S9

Test concentrations with justification for top dose:

Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512, 1600 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: It has been identified as a suitable solvent

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation) and preincubation
- Cell density at seeding (if applicable): 10^9 cells/mL

DURATION
- Preincubation period: 30 ± 2 minutes by 70 rpm at 37 ± 1°C
- Exposure duration: 48 ± 4 h

SELECTION AGENT (mutation assays): Histidine and tryptophan absence

METHODS OF COLONY COUNTING: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually.

DETERMINATION OF CYTOTOXICITY : Reduction of the bacterial background lawn

Rationale for test conditions:

According to the OECD guideline. Furthermore performing both the pre-incubation and the plate incorporation assay provide more information about the possible mutagenicity of the test item.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No statistical hypothesis testing was done.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Direct Plate Assay

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Direct Plate Assay

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Direct Plate Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Direct Plate Assay

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

3.4 fold increases

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Direct Plate Assay

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

2 fold increase

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Direct Plate Assay

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

3.4 fold increase

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Direct Plate Assay

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

1.9 fold increase

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

highest concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Direct Plate Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Pre-Incubation Assay

Metabolic activation:

without

Genotoxicity:

not determined

Remarks:

Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Pre-Incubation Assay

Metabolic activation:

with

Genotoxicity:

not determined

Remarks:

Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 11

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Pre-Incubation Assay

Metabolic activation:

without

Genotoxicity:

not determined

Remarks:

Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 12

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Pre-incubation assay

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

2.9 fold increase

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 13

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Pre-Incubation Assay

Metabolic activation:

without

Genotoxicity:

not determined

Remarks:

Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 14

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Pre-Incubation Assay

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

2.1 fold increase

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 15

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Pre-Incubation Assay

Metabolic activation:

without

Genotoxicity:

not determined

Remarks:

Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 16

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Pre-Incubation Assay

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 17

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Pre-incubation Assay

Metabolic activation:

without

Genotoxicity:

not determined

Remarks:

Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation is distinguished by a noticeable precipitate on the plate, however the precipitate does not influence automated counting of the plates.

RANGE-FINDING/SCREENING STUDIES: Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512,1600 and 5000 μg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) - refer to any other information

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: reduction in the background lawn
- Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.

Any other information on results incl. tables

Historical Control Data of the Solvent Control

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 29	3 – 27	3 – 20	3 – 23	8 - 41	8 – 55	61 – 176	60 - 160	10 – 59	9 - 67
Mean	10	11	6	6	16	22	110	106	26	33
SD	3	4	2	3	5	7	17	20	6	8
n	2458	2426	2402	2352	2416	2458	2473	2398	2237	2217

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between May 2016 and May 2018. 

Historical Control Data of the Positive Control Items

	TA1535		TA1537		TA98
S9-mix	-	+	-	+	-	+
Range	128 – 1530	73 – 1206	58 – 1407	54 – 1051	365 – 1978	250 – 1977
Mean	901	239	817	340	1355	903
SD	174	115	354	160	230	357
n	2400	2296	2051	2337	2357	2367

 

	TA100		WP2uvrA
S9-mix	-	+	-	+
Range	439 – 1993	408 - 2379	93 – 1958	111 - 1359
Mean	905	1249	1059	444
SD	163	371	506	144
n	2402	2354	2153	2232

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between May 2016 and May 2018. 

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Gurjun Balsam Oil (Gurjunene) is mutagenic in the Salmonella typhimurium reverse mutation assay in the tester strains TA100 and TA98, in the absence of S9.
The test item is not mutagenic in the tester strains TA1535 and TA1537, in the Salmonella typhimurium reverse mutation assay or in the Escherichia coli reverse mutation assay, with or without S9.
Although the test item induces a significant increase in the tester strain TA98 in the presence of S9, the increase is below three (3) times the concurrent control. It therefore does not fulfil the acceptability criterium, and is thus not considered mutagenic. The 1.9- and 2.1-fold increases observed in the TA100 strain, in the presence of S9 is considered equivocal, as the level is lower or very close to the two (2) times concurrent control acceptability criterium.

Executive summary:

The potential of Gurjun Balsam Oil (Gurjunene) and/or its metabolites to induce reverse mutations at the histidine locus was tested in a OECD TG 471, Ames study. Mutagenicity was determined in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

 

First mutation assay (direct plate assay): based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 17 to 5000 µg/plate in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. 

- In the absence of S9-mix, the test item induced up to 3.4-fold increases in the number of revertant colonies compared to the solvent control in the tester strains TA100 and TA98. T

- In the presence of S9-mix, the test item induced up to 1.9-, 1.7- and 2.0-fold increases in the number of revertant colonies compared to the solvent control in the tester strains TA100, TA1535 and TA98, respectively.

 

In the second mutation experiment (pre-incubation assay): the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. 

- In the absence of S9-mix, no increases in the number of revertant colonies were observed at any of the dose levels tested in all tester strains. However, due to cytotoxicity in the absence of S9-mix,the number of revertant colonies could not be determined.

- In the presence of S9-mix, the test item induced up to 2.9- and 2.1-fold increases in the number of revertant colonies compared to the solvent control in the tester strains TA98 and TA100, respectively. Both increases were above the laboratory historical control data range.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Overall assessment of the results:

It is concluded that Gurjun Balsam Oil (Gurjunene) is mutagenic in the Salmonella typhimurium reverse mutation assay in the tester strains TA100 and TA98, in the absence of S9.

The test item is not mutagenic in the tester strains TA1535 and TA1537, in the Salmonella typhimurium reverse mutation assay or in the Escherichia coli reverse mutation assay, with or without S9.

Although the test item induces a significant increase in the tester strain TA98 in the presence of S9, the increase is below three (3) times the concurrent control. It therefore does not fulfil the acceptability criterium, and is thus not considered mutagenic. The 1.9- and 2.1-fold increases observed in the TA100 strain, in the presence of S9 is considered equivocal, as the level is lower or very close to the two (2) times concurrent control acceptability criterium.