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Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
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

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 resultsopen allclose all
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.